CA2261740A1 - Apparatus and method for real-time measurement of cellular response - Google Patents

Abstract

An apparatus and method for real-time measurement of a cellular response of a test compound or series of test compounds (303) on a flowing suspension of cells (349), in which a homogeneous suspension of each member of a series of cell types (349) is combined with a concentration of a test compound (303), directed through a detection zone (355), and a cellular response of the living cells is measured in real time as the cells in the test mixture are flowing through the detection zone (355). The apparatus may be used in automated screening of libraries of compounds, and is capable of real-time variation of concentrations of test and standard compounds and generation of dose/response profiles within a short timespan.

Description

W O ~ 3~ PCT~US97/13602 APPARATUS AND METHOD FOR REAL-TIME MEASUREMENT OF C'l ~ Vl4D RESPONSE

FIELD OF THE INVENTION
This invention relatss generaily to an apparatus for screening and the pharmacological profiling of 5 compounds modulatin~ a cellular physiological response. This invention also relates to devices for rapid assessment of the properties of compounds that modulate the activities of cell surface receptors and ion channels. More specifically, this invention relates to methods and apparatus for detecting, evaluating and characterizing the ability and potency of substances to act as agonists or antagonists against receptors and ion channels localized on a cell surface membrane.
BACKGROUND INFORMATION
Biological cells contain receptor molecules located on their external membrane. The function of these receptors is to "sense" the cell environment and supply the cell with an input signal about any changes in the environment. In ~' ~oli-. organisms such cell environment is comprised of the neighboring cells and the function 15 of the receptor is to aUow cells to communicate with each other directly Ithe paracrine regulatory system~ or indirectly Ithe endocrine regulatory systeml thus achieving harmonized response of a tissue, organ or a whole organism. In prokaryotic cells, the surface localized receptors provide a means for detecting extracellular environment.
Having received such a signal, neurotransmitters, hormones, chemoattractant or chemorepellant substances 20 for example, the surface localized receptors transmit this information about extracellular environment into the cell through specific intracellular pdth~ a,s in such a way that the cell responds in the specific fashion to accommodate these changes. When there is an altered supply of the external signal molecules or an altered activity of the cell surface molecules, the cell response would be abnormal causing malfunctioning of a tissue or an organ.
In eucariotic cells, receptor moleculûs determine the selective response of the cell. Each type of receptor 25 can interact only with a specific set of ligand molecules. For example, adrenergic receptors interact with adrenaline and noradrenaline, cholinergic receptors interact with acetylcholine, serotoninergic receptors interact with 5-hydroxytriptamine, dopamineergic with DOPA and so on. The cells derived from the different tissues invariably express specific sets of tissue receptors. Different types of receptors are connected to different signal transduction path...~.
For example, nicotinic choliner~ic receptor, upon binding acetylcholine molecule, directly activates sodium channel 30 (Claudio et al., 1987, is incorporatedherein by ,Lfo" q' ~-protein coupled receptors activate enzymes of second messenger pathways, for example, adenylate cyclase or phospholipase C with subsequent ~IK~I of cAMP or phosphoinositide cascades ~Oivecha and Itvine, 1995, is inc~ d herein by l-ife.. ~' Receptor tyrosine kinases activate cascade of MEKIMAPK kinases leading to cell difl~.~"ti~ r and proliferation ( Marshall, 1995 and Herskowitz, 1995, are incorporated herein by reference)]~ Cytokine receptors activate ~IAKISTAT cascade which in 35 turn can regulate other pati.. u~ as well as activate gene llans~ iu" ~Hill & Treisman, 1995, is inc l alOd hsrein by referencel.

W O 98~'~3~9 PCT~US97/13602 Together with the receptors, the cell surface membrane carries ion pumps, ion transporters and ion channels.
These molecular assemblies work in concert to maintain intracellular ion homeostasis. Any changes in the activity of these systems would cause a shift in the intracellular c- Irai Jns of ions and cons, :1~ to the cell metabolic response.
lon pumps act to maintain transmembrane ion gradients utilizing ATP as a source of energy. The examples of the ion pumps are: Na+lK+-ATPase maintaining transmembrane gradient of sodium and potassium ions, Ca2+
ATPase maintaining transmembrane gradient of calcium ions and H+-ATPase maintaining transmembrane ~qradient of protons.
Ion ll. - pcrl~,s use the El~ -' Pmical energy of transmembrane gradients of one ion species to maintain 10 gradients of other ion counterpart. For example, the Na+llCa2+ f ' -ng uses the chemical potential of the sodium gradient directed inward to pump out calcium ions against their chemical potential.
Ion channels, upon aLli.~I;on, allow for the ions to move across the cell membrane in ~rr .'; e with their electrochemical potential. There are two main types of ion channels: voltage operated and li~ l-yptP~I Voltage operated channels are activated to the open state upon changes in transmembrane electric potential. Sodium 15 channels in the neuronal axon or L type calcium channels in neu~ junctions exemplify this kind of channel.
Lig ~ g~tPd channels are activated to the open state upon binding a certain ligand with the chen ~L~lU~ part of their molecules. The classical example of lig 1.DetPd channels is nicotinic cholinergic receptor which, at the same time, is the sodium channel.
There are numerous methods for detecting ligandlreceptor in: ,r1 The most conventional are methods 20 where the affinity of a receptor to a substance of interest is measured in, ' lig~ ' binding assays. In these assays, one measures specific binding of a reference ra~ Bd ligand molecule in the presence and in the absence of different r - dl' ~ of the compound of interest. The chdla.,lL.i~li" inhibition parameter of the specific bindiny of the reference radiolabeled ligand with the compound of interest, IC50, is taken as a measure of the affinity of the receptor to this compound (Weiland & Molinoff, 1981 and Swillens et all., l995, are ~ rrrdlLd herein by 25 reference). Recent advances in microchip sensor technology made it possible to measure direct interactions of a receptor molecule with a compound of interest in real time. This method allows for determination of both association and dissociation rate constants with subsequent calculation of the affinity parameter (F gerstam et al., 1992, is incorporated herein by ~erl, r ~) While being very precise and ~ , these methods do not allow to distinguish between agonist and - gc : activity of the ~ pOL ~
The type of biological activity of the compounds, agonist or antagonist, may be determined in the cell based assays. In the methods described in Harpold & Brust, 1995, which is ~~r~,o,alcd herein by laf~ , cells culldll;,lL~lLd with a receptor gene and reporter gene construct, are used to provide means for 1~ retion of agonist and a ~ potential pharmaceutical compounds. These methods are inconvenient because they require very laborious manipulations with gene l~ ioil procedures, are highly time c- ~, and use artificially modified 35 cells. Besides, to prove that the agonistic effect of a particular compound is r~ !d to the stimulation of a r W O~J'~ PCTrUS97/13602 transfected receptor, several control experiments with a positive and negative control cell lines should be performed as well.
Most closely related to the methods of this invention are the methods described in Parce et al., 1994, which is incorporated herein by l~fL.b -e~ These prior art methods use natural cells and are based on registering the 5 natural cell responses, such as the rate of metabolic acidification, to the biolwJically active compounds. The disadvantage of the prior art is low throughput speed, each measurement point taking about three minutes. Another disadvantage of the prior art is the use of cells immobilized on the internal surface of the measuring microflow chamber. This leads to the necessity of using separate silicon sensors, or cover slips, with the cells adherent to them for each corlr dt' point of the agonist or afit 1f~ t, for the receptors that undergo d ~nriti~tion upon binding 10 to the ayonist molecule. This results in high variability of the experimental results.
Ionized calcium, unlike other intracellular ion events, e.g. changes in the intraceilular concentrations of protons, sodium, magnesium, or potassium, serves as the most common element in different signal transduction pdt;..d~a of the cells ranging from bacteria to specialized neurons IClaPham, 1995, is inrr pr dlLd herein by ,~fe" e) There are two major pools which supply signal transduction pdti..dj5 in the cell with the calcium ions, 15 oxtracellular space and the endoplasmic reticulum. There are several mechanisms to introduce small bursts of calcium into cytosol for signal transduction.
Both excitable and ~ ~' cells have on their plasma membrane predominantly tWG receptor classes, G-protein coupled :~LI~ li..., receptors IGPCSR) and the receptor tyrosine kinases IRTK), that control calcium entry into cell cytoplasm. Both 6PCSR and RTK receptors activate phospholipase C to convert phosphatidylinositol into inositol(1,4,5)-l~i, h~ e llnsP3) and diacylglicerol. InsP3 acts as an intracellular second messenger and activates specialized receptor that spans the endoplasmic reticular membrane. The aL~i.dliun of this receptor triggers release of calcium ions from the endoplasmic reticulum ~Berridge, l9g3, is ;"c..,p~ al~d herein by l~.f~ lf ~' The calcium ions can also enter the c~l F'- of excitable and nL " " cell from extracellular environment through specialized voltage-independent Ca2+ selective channels triggered by specific ligands. In r,or, ' ' cells, hyperpolarization of the plasma cell membrane also enhances entry of calcium ions through passive transmembrane diffusion along the electric potential. For example, opening of potassium channels brings the membrane potential to more negative values inside the cell, thus facilitating Ca2+ entry across the plasma membrane. Excitable cells contain ~LItl~r '' re ~ent Ca2+ channels on their plasma membrane, which, upon membrane depolarization, open for a short period of time and allow inflow of Ca2+ from external media into cytoplasm. The endoplasmic reticulum membrane as well as plasma membrane of the excitable cells contains InsP3 receptors and Ca2+ sensitive ryanodine receptors IRYR) releasing Ca2+ from intracellular stores upon membrane receptor triggered phospholipase C acti.dl or depolarization-induced short burst of Ca2+ entry into cell cytoplasm from extracellular media ,~e,~r,~
It is well E~ r~ that G-protein coupled serpentine receptors initiate Ca2+ mobilization through the ? 'i~_ of phospholipase C~ ~Sternweis and Smrcka, 1992, is r dtLd herein by ,~f~,.c; ) whereas tyrosine kinase receptors activate phospholipase Cy with subsequent intracellular Ca2+ mobilization ~Berridge & Irvine, 1989, is:l~D,~ordtLdherein by ,Lf~

WO !~X~05J5~ PCT/US97/13602 There are many plasma membrane G protein coupled serpentine receptors, tyrosine kinase growth factor receptors and voltage and ligand re~ulated channels known to initiate intracellular Ca2+ mobilization.
Ca2+ plays an essential role in many functional p,~ ac of a cell. For ei~ample, Ca2~ affects the cell cycle (Means, 1994, is incorporated herein by lufe,u ~) and activates specific transcription factors ~Sheng et al., 5 1991, is h~LG~ ;IdlUd herein by ~U.I ce' Scores of receptors and ion channels use the Ca2+ signal to initiate events as basic as cell motility, c :, c , secretion, division etc..
Increases in cytosolic and, consequently, in nuclear c5nc~ I~aIion of the Ca2+ can also be a cell death promoting signal. For example, prolonged increase in free Ca2+ activates degradation, ~ es in programmed cell death, apoptosis, activates '~ ~ ;rr that cleave DNA and degrade cell chromatin, promotes DNA digestion by direct 10 stimulation of . ~ 'c ' --s, or indirectly by activation of Ca2+ .' r~ Id l p,.Utl.a;Scs~ l~hl~, h; ~PCPs and phospholipases, resultin~ in a loss of chromatin structural integrity INicotera et all., 1994, is incorporated herein by ,~.f~..ur-e) A ~ lr of intracellular fi ~ c: calcium indicators (Cr~"l~ et all., 1985, is incGr~vral~d herein by r~ ..ce) made it possible for intracellular ~ - aii~n of free calcium to be measured directly in the 15 living cell. Thus the ability to register changes in intracellular calcium c~ .ai .. provide the means for monitoring effects of different compounds useful in treating various diseases, whose action is thought to be a result of an interaction with membrane receptors and ion channels.
With the advent of combinatorial chemistry approaches to identify pharmacologically useful compounds, it is increasingly evident that there is a need for methods and q,, a~ bs capable of performing automated 20 l~halaLIu~iLdl ~n of pharmacological profiles and corresponding potencies of the compounds in synthesized combinatorial libraries. This would enable the rapid screening of a large number of compounds in the combinatorial library the identification of those compounds which have biological activity, and the chaldcl."i~dli~... of those compounds in terms of potency, affinity and :..,I~,.,Ii.;~y.
It is an object of this invention to provide methods for screening and the quantitative chalaLIL.i~dtion of 25 potentially pharmacologically effective compounds that specifically interact with and modulate the activity of cell membrane receptors, ion pumps and ion channels using living cells.
It is an additional object of this invention to provide methods capable of chala~,lu.i~ing an affinity of the active compounds to the binding sites of the cell.
It is another additional obiect of this invention to provide methods to distinguish between agonistic and 30 antagonistic activity of the compounds.
It is yet another additional object of this invention to provide methods to determine the nature of the receptor, ion channel or ion pump entity which is sensitive to the active compounds d ~D~..id during the screening process.
It is yet another additional obiect of this invention to provide methods to chalal,ll..iLu cell receptor pattern 35 for patticular cell source tissue.

WO ~ 5,3~ PCT/US97/13602 lt is yet another additional object of this invention to perform each of the above methods on each member of a series of cell types.
It is yet another additional object of the invention to determine the pattern of cell surface receptors expressed in one or more cell types.
It is yet another additional object of the invention to confirm that a test compound influences the activity of a particular receptor.
It is yet an additional object of the invention to determine the activity of a ~qiven receptor in a variety of cell types in which it is expressed.
It is a specific object of this invention to provide an apparatus for fulfillment of the objectives above.
It is yet another additional object of this invention to provide an apparatus for fulfillment of each of the -b; : .~s above for each member of a series of cell types.
At least some of these and other -b;!c~;. s are adl' L~scd by the various embodiments of the invention disclosed herein.
SUMMARY OF THE INVENTION
The present invention odl' e~S~,S the above and other needs by providing a method and c ,., ding appr dl~l.,.,s which allows the automated chdldcl~ dlion of pharmacological profiles and corresponding potencies of compounds in synthesized combinatorial libraries. This enables the rapid screening of a large number of compounds in the combinatorial library, the identification of those compounds which have biological activity, and the Lhd(aLt~ _'- of those compounds in terms of potency, affinity and s~ li.ity.
A variety of effects caused by the compounds to be screened may be detected and quantitatively chalaLI~.iLcd according to the present invention. P~ , these effects include but are not limited to changes in intracellular c~c I,di Jr of ionized calcium, cAMP or pH, transmembrane potential and other physiological and biochemical Lhà,aLlo,i~liL~ of living cell which can be measured by a variety of c .. ~il ' means, for example using specific fluorescent, luminescent or color developing dyes.
The present invention also includes methods of screening for agonist or antagonist activity of drugs, methods of chdldct~ ;..g their potency profiles, methods of identifying the receptor expression pattern of cell membrane ("receptor fingerprinting") and methods of determining toxicity profiles for the compounds. In these methods, a steady flow of cells is mixed with flows of the compound and a standard substance. The effects of the compound alone and in mixture with the standard substance are measured and provide the means for pharmacological profiling of the compounds, drug screening and cell receptor pattern cha,acll,.i.;.lg.
In a preferred embodiment of the invention, the compounds to be screened and standard agonist and antagonist substances are organized in a 96-well plate format, or other regular two dimensional array, such as a 48 - well and 24-well plate format or an array of test tubes. In another preferred embodiment of the invention, the non adherent cells are grown in a r ~ ~ ,, of freely flowing cells by growing them in an 1~ p~ ,le cell cultivating system.

WO ~ PCT/US97tl3602 ln another preferred embodiment of the invention, the naturally adherent cells which need attachment to a surface for their growth, are grown in the i,, Ur ial~ cell cultivating system containing commercially available micro spherical beads to which the celts adhere during the growth.
In yet another preferred embodiment of the invention, the naturally adherent cells which need attachment 5 to â surface for their growth, are grown in the cell culture flasks with a subsequent detachment of the cells from the flask bottom with an , F ~F i~l~ detaching reagent.
In accordance with the present invention, either eukariotic or prokariotic cells can be used. The cells can be llnnale~ d with â gene coding to express a receptor of interest, for example, an orphaned receptor. In addition, the variety of compounds having ' ' ~6 -'Iy relevant activity may be used including but not limited to 10 neurotransmitters, hormones, toxins, receptor aCli~di . ~ and inhibitors, ion channels and ion pump modulators, irritants andlor drugs.
The cells grown in acs~r' with the preferred embodiments described above, are mixed with an appropriate 1ll Jrl t dye, for example FURA 2AM for me ~ ~ Is of c~zce lldl - of intracellular calcium or BCECF-AM for measurements of intracellular pH, and are incubated in the 3~ idtd conditions to allow the dye 15 to penetrate into the cell. The cells loaded with a dye are supplied to the apparatus. In the apparatus, the cells are successively mixed with a solutions of the compounds to be tested.
One aspect of the present invention is a method for ' til~; ~, compounds having biological activity, comprising the steps of: (a) combining a homogeneous pr of living cells with a test compound having an unknown cellular effect to form a test mixture, (b) directing the test mixture through a detection zone; and (c) 20 measuring a cellular response of the ,~ ~ ' d cells to the test compound as the test mixture is flowing through the detection zone. The method will often include the additional steps of: ld) combining a homogeneous , r'r of the cells with a standard compound having a known effect on the cellular response of the cells to form a standard mixture; le) directing the standard mixture through the detection zone; and (f) measuring the cellular response of the cells to the standard compound. In one embodiment, the standard compound and the test compound are 25 simultaneously mixed with the cells in the combining steps, and the measuring step detects the known effect or an alteration of the known effect. The standard , ' can be an agonist or antagonist of the cellular response.
In one mode of operation, steps (a) and (d) are performed simultaneously; steps (b) and (e) are performed simultaneously; and steps (c) and (f) are performed simul af '~ using a single s , ~ of the cells. In another mode of DF di' , steps (a), (b), and Ic) are performed first, and then steps (d), (e), and (f) are performed, wherein 30 the test compound is added together with the standard compound in step (d). If the cellular response is detected in step ~c) to indicate that the test compound is active to generate the response, and the standard compound is an : 6 t, then a decrease in the cell response from step (c) to step (f) is indicative that the test compound is an agonist of the known effect. If the cellular response is not detected in step Ic), indicating that the test compound is not active to generate the response, and the standard compound is an agonist, then an alteration of the known 35 effect detected in step (f) is indicative that the test compound is an -: gc-: of the know effect. r,~rer ~, the method is F Iml d aut ti~ under the direction of a programmable computer on a plurality of test compounds . .

W O 98/05959 PCTrUS97113602 and a plurality of standard compounds, and a ~ cce ~ series of antagonists are automatically added as the standard compound in step Id) if the cellular response is detected in step (c) to indicate that the test compound is active to generate the cellular response, whereby a decrease in the ceOular response detected in step (f) is indicative that the test compound is an agonist of the known effect; and a series of agonists are automatically added as the standard compound in step (d) when the cellular response is not detected in step (c), whereby an alteration of the known effect detected in step (f) is indicative that the test compound is an antagonist of the known effect.
In one embodiment of the method, if step If l indicates that the compound is an agonist of the known effect, then the method includes automatically determining the - ildi d ~r d !n of agonist activity of the test compound by repeating steps ~a), (b), and ~c), and Id), (e), and (f) while varying the concentration of the test compound and the standard compound and recording resultant changes in the cellular response; and if step (f) indicates that the compound is an v :, then the method includes ~ at~ determining the concentration dependence of inhibition of the cellular response in the presence of the agonistic standard compound by repeating steps (d), (e), and (f) while varying the ~ at of the test compound and the standard compound and recording resultant changes in the cellular response. Qr~ the method also comprises the step, when step (f) indicates ~5 that the s , ~ ' is an antagonist, of: (9) automatically determining the c2z~ llai d, ' e of cell response a.,li. -Dr by repeating steps (d), (e), and (f) for a zero concentration of the test compound while varying the s- - ~,ation of the standard compound and recording resultant changes in cellular response, and then repeating this step (g) for different c~nre ~ of the test compound. The method may further include the step when step (f) indicates that the compound is an agonist, of: (h) automatically determining the c~ lion d, n~ - of cell response a~li.dliun by repeating steps (d), (e), and (f3 for a zero c~nc~ !~dlion of the standard s pc ' while varying the ~r lalion of the test compound and recording resultant changes in cellular response, and then repeating this step (h) for different cr 1 ~ Is of the compound. Variation of the s c dtion of the test compound andlor the standard compound can be done continuously or in a stepwise manner. One preferred step includes graphically displaying the recorded changes in the cellular response.
The cellular response can be any desired cellular response susceptible of being measured or detected as the cells flow past a detector in s , - It can be c.~ d by analyzing the cells thnm 1~..3 or the medium in which the cells are sl pr ' ' Cellular 1~ .es can be measured, for example, from a change in intracellular ion ccrc !.a~ n such as calcium, magnesium, proton, sodium, or potassium. In one embodiment, the ion is detected using an intracellular dye such as a visible andlor lluu,~;sc~..l dye.
In another embodiment, a method comprises the steps of: (a) combining a hr rg ous suspension of living cells with a test compound having an unknown cellular effect to form a test mixture, (b) directing the test mixture through a detection zone; (c) ~ b~y a cellular response of the s ~Spr '~ d cells to the test compound as the test - mixture is flowing through the detection zone; and repeating steps (a)-(c) on each cell type of a series of cell types to be tested so as to measure the effect of said test com, ~ on each cell type of said series of cell types. In this embodiment, the method may further comprise the steps of: combining a hc ~ DLrs:, r of each of said cell types in said series of cell types with a standard compound having a known effect on said cellular response of said cells to form a series of standard mixtures; directing said series of standard mixtures through the detection zone;
and measuring the cellular response of each of said cell types to said standard compound. The standard compound may be an agonist or l v ~ict of said cellular response.
The invention also includes an apparatus for automatically measuring the effect of a plurality of test 5 compounds on living cells, comprising: a test compound sampler for sequentially providin~q samples of multiple test compounds, a cell , -; input for providing a h- r~ .. of living cells, a mixing zone, coupled to the test compound sampler, for receiving the samples of the test compounds from the test compound sampler, and receiving the - , r r. of living cells from the cell s--r ~r. input and mixing each test compound with the - " of living cells; and a detector, coupled to the mixing zone, for measuring a cellular response of the , ' ' cells to each test compound. The apparatus may additionally include a standard compound sampler, coupled to the mixing zone, for providing a sample of a standard compound having a known effect on the cellular response of the - , I ' cells, wherein the mixing zone receives the sample of the standard compound from the standard compound sampler and mixes the standard compound with the - , ' ' cells and the detector measures the cellular response of the suspended cells to the standard compound. In one embodiment, the mixing zone 15 simultaneously mixes the test compound and the standard compound with the suspended cells and the detector detects the known effect or an alteration of the known effect. The appr dtl..~ may also include a first gradient device, coupled to the test compound sampler, for a: P'l~ adjusting the c~ - ~.. Ievel of the test compound transferred to the mixing zone from the test compound sampler; and a second gradient device, coupled to the standard compound sampler, for automatically adjusting the s~ al Ievel of the standard compound 20 transferred to the mixing zone from the standard compound sampler. The 3l, _ dll.l:l may further include a switching valve, coupled to the first and second gradient devices at an input of the switching valve and coupled to the mixing zone at an output of the switching valve, for s~ ly switching the flow of a cr ~Id~iC of the test compound or a cn ~ i of the standard compound or both to the mixing zone where the test compound andlor the standard compound is then mixed with the suspension of cells. In addition, the apparatus may include a calibration 25 unit, coupled to the switching valve, wherein the switching valve also s~,5~li.c'~ switches the flow of a calibration solution provided by the calibration unit into the mixing zone where the calibration solution is mixed with the suspension of cells.
The reaction time of the cells with the various test and standard compounds may be controlled through use of various lengths of reaction developing lines coupled to the output of the mixing zone, for receiving a mixture of 30 the cell -, mixed with either the test compound, the standard compound or the calibration solution, and providing a flow path for the mixture such that there is adequate time for the ~ i~ cells to react with the test compound, the standard compound or the calibration solution, wherein the reaction d ..', ~, lines is further coupled to the input of the detector which receives the mixture from the reaction d~.r'(r ~ lines. In one preferred embodiment, the detector detects changes in intracellular ion sor~f t.~: - Preferred ions are described above.
The ~ r dl~.~ may additionally include a controller, coupled to the first and second gradient devices, the test compound sampler, the standard s pc ~ sampler and the switching valve, for controlling their rF di- , and .9.
a computer, coupled to the controller, for sending command signals to the controller in accordancs with a software program implemented by the computer, wherein the computer is also coupled to the detector in order to send and rscsive cell response measurement signals to and from the detector. In order to automate the apparatus further, the test compound sampler can be an automated robotic sampler capable of selecting a specified test compound from 5 a library of test compounds. A controller can be coupled to the test compound sampler, for controlling the operation of the test compound sampler, using the computer, coupled to the controller, for sending command signals to the controller in ac d e with a software program implemented by the computer, thereby controlling the selection and retrisval of test compounds by the test compound sampler from the test compound library.
The apparatus can direct flow through the various elements under either positive or negative pressure.
10 Thus, one embodiment includes a gradient pump having an input and an output, coupled to the test compound sampler, for adjusting the c Ildl level of the test compound I,ar,~ d to the mixing zone from the test compound sampler, wherein the test compound sampler comprises a first intake nozzle for rscsiving the specified test compound, a second intake nozzle for receiving a buffer solution; and wherein the gradient pump is coupled to the first and second intake nozzles and receives specified c- .l~ai ~ of the test compound by adjusting the amount 15 of test compound and buffer solution received by the first and second intake nozzles, I. ~mLF . ~v~ wherein the buffer solution is a diluting agent of the test compound. This embodiment may also include a standard compound sampler for providing a sample of a standard compound to the mixing zone. The standard compound sampler is preferably an automated robotic sampler capable of selecting a specified standard compound from a library of standard compounds. The 3" d~ may also include a second gradient pump having an input and an output, coupled to the 20 standard compound sampler, for adjusting the c c~ level of the standard compound providsd to the mixing zone from the standard compound sampler, wherein the standard - , d sampler [ , ;;.~s a third intake nozzle for receiving the specified standard compound, a fourth intake nozzle for receiving a buffer solution; and wherein the second gradient pump is coupled to the third and fourth intake nozzles and receives specHied c~ e ~ of the standard compound by adjusting the amount of standard compound and buffer solution received by the third and 25 fourth intake nozzles, respectively, wherein the buffer solution is a diluting agent of the standard compound. The apparatus may further comprise a second mixing zone coupled to the outputs of the first and second gradient pumps, for receiving and mixing the specified c Il ~~ of the specified test compound and the specified standard compound, such that the output of the second mixing zone is provided to the first mixing zone. Another element that can be included is a calibration unit for providing a calibration solution; and a switching valve, having a first 30 input coupled to the second mixing zone, a second input coupled to the calibration unit, and an output coupled to the first mixing zone, for switching between the flow of either a compound mixture from the second mixing zone or the calibration solution from the calibration unit and then providing the flow to the first mixing zone where it may be mixed with the cell s , ~r F~ y, the calibration unit comprises a calibration maximum solution which provides for maximal cell response when mixed with the cell , )n, a calibration minimum solution which 35 provides for minimal cell response when mixed with the cell s ~pe- ~r a diverting valve having a first input coupled to the calibration maximum solution and a second input coupled to the calibration minimum solution, for switching W O ~ S~ PCTrUS97/13602 between the flow of either the calibration maximum solution or calibration minimum solution; and a pump, coupled to the output of the diverting valve and an input of the switching valve, for pumping either the calibration maximum or calibration minimum solution from the diverting valve into the switching valve. The positive pressure version of the app,~dlus can also include a second pump, coupled to an input of the first mixing zone, for pumping the 5 suspension of cells from the cell suspension input into the first mixing zone. Reaction developing lines, having an input coupled to an output of the first mixing zone and an output coupled to an input of the detector, for providing a flow path and a reaction time delay for a mixture received from the first mixing zone and for providing the mixture to the detector, can also be included.
In a preferred embodiment, as above, the 3" dlllS can include a controller, coupled to the first and second 10 ~radient pumps, the test compound sampler, the standard compound sampler and the switching valve, the first and second mixing zones, the first and second pumps and the diverting valve for controlling their DF~ dlian, and a computer, coupled to the controller, for sending command signals to the controller in a - ; -e with a software program im~ ented by the computer, wherein the computer is also coupled to the detector in order to send and receive cell response measurement signals to and from the detector.
The apparatus of the present invention can also be run under negative pressure by utilizing a pump, coupled to the output of the detector, for providing negative pressure to the apparatus, a proportionating valve, coupled to the test compound sampler, for adjusting the CCl di- Ievel of the test compound l~ II,d to the mixing zone from the test compound sampler, wherein the test compound sampler further comprises a first intake nozzle for receiving the specified test compound, a second intake nozzle for receiving a buffer solution; and the, ~ r~ i', oting 20 valve receives specified r1nc ll ~ of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles"~, r liv~'y, wherein the buffer solution is a diluting agent of the test compound. The negative pressure apparatus can also include an automated standard compound sampler capable of selecting a specified standard compound from a library of standard compounds, the standard compound sampler including a third intake nozzle for receiving the specified standard compound and a fourth intake nozzle for 25 receiving a buffer solution; and a second r ~ pr-i ating valve, coupled to the third and fourth intake nozzles, for receiving specified ccc~ ~ of the standard compound by adjustiny the amount of standard compound and buffer solution received by the third and fourth intake nozzles, ~ , wherein the buffer solution is a diluting agent of the standard npr I In one version, the au~,~,al,.s includes a first priming vah~e, coupled to the output of the first proportionating valve, for receiving the specified cc ~ ',di' r of the test compound and providing the 30 test compound to the mixing zone, and a second priming valve, coupled to the output of the second, ~pD i ~ling valve, for receiving the specified c ',dliun of the standard compound and providing the standard compound to the mixing zone. A calibration unit can be included as described r 1~ as can reaction d~,.. ' F g lines, robotic input, and computer control. The cell , -- input may comprise a cell , reservoir, a buffer reservoir, a third diverting valve, having a first input coupled to the cell L r ' reservoir and a second input coupled to 35 the buffer reservoir, for adjusting the r~ r ~, ~ of the cell suspension, wherein the buffer is a diluting agent of the cell ~ , s and a fourth priming valve, coupled to the output of the third diverting valve, for receiving the W O 98/05959 PCTrUS97/13602 cell ~ ;~ n mixture from the third diverting valve and providing this mixture to the second mixing zone. In one embodiment, the a~lr alL~ further comprises a plurality of cell suspension l~3a.~. a.
Another aspect of the present invention is a method of Lhalac~ ;"g the receptors present in a cell comprising the steps of (a) combining a ~ pr- - of cells with a test agent known to influence the activity of a 5 particular receptor to form a test mixture; (b) directing said test mixture through a detection zone; (c) measuring a cellular response of said - ~prm - of living cells to said test agent as said test mixture is flowing through said detection zone wherein a response to a test agent indicates that said cell expresses a receptor known to respond to said test agent; and Id) repeating steps (a)-(c) with a series of test agents until the effects of each test agent has been measured. The test agent may comprise an agonist, an antagonist, or a mixture of an antagonist and an 10 agonist. In one embodiment, this method further comprises repeating steps (a)-(d) on a series of different cell types to determine the receptors expressed by each cell type.
Another aspect of the invention is a method of confirming that a test compound has an effect on the activity of a receptor comprising the steps of contacting a negative control cell type which lacks said receptor with said test agent to form a negative control mixture; directing said negative control mixture through a detection zone;
15 measuring the cellular response of said -, n~ of living cells to said test compound as said negative control mixture is flowing through said detection zone; contacting cells of the same cell type as the negative control which have been engineered or induced to express said recep~or with said test agent to form a test mixture; directing said test mixture through a detection zone; measuring the cellular response of the cells in the test mixture to said test a~ent, whereby a difference in the measured response of said cells in said test mixture relative to the measured 20 response of said negative control cells indicates that said test agent has an effect on the activity of said receptor.
The test agent may comprise an agonist, an - ~9 :, or a mixture of an agonist and an antagonist.
Another aspect of the present invention is a method of determining the activity of one or more receptors in a series of cell types comprising (a) combining a .- , lr of living cells comprising a member of said series of cell types with an agent known to influence the activity of a particular receptor to form a test mixture; (b) directing 25 the test mixture through a detection zone; (c) measuring the cellular response of said , of living cells to said test agent; (d) repeating steps (a)-(c) on each member of the series of cell types until the effect of said test agent has been measured in each cell type of said series. The test agent may comprise a known receptor agonist, a known receptor antagonist a mixture of a known agonist and a known antagonist, a compound whose activity is unknown, or a mixture of a compound whose activity is unknown and a compound which is a known agonist or a 30 known antagonist.
Certain preferred embodiments of the present invention are discussed below in more detail in c~
with the drawings and the detailed description of the preferred embodiments. These preferred embodiments do not limit the scope or nature of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

WO ~ ,3,3~ PCT/US97/13602 ~l2 Fig. 1 is a block-diagram of one embodiment of the combinatorial screening apparatus of the present inventiùn.
Fig. 2 is a block-diagram of a positive pressure fluidic system which may be used in a combinatorial screening a~J~Jaldlu~ of the present invention.
Fig. 3 is a block diagram of one embodiment of a preferred system which may be used in a combinatorial screening 1": dtL~ of the present invention.
Fig. 4 represents a simplified algorithm of a screening mode which may be utilized in a combinatorial screening 3\pr dtl.~ of the present invention.
Fig. 5 ,., eso..ts a simplified algorithm for a potency mode which may be utilized in a combinatorial 10 screening 3"~cdllof the present invention.
Fig. 6 is a flow diagram of a preferred primary mode operation which may be implemented by a combinatorial screening apparatus of the present invention. Fig. 6 comprises the combination of Figs. 6a-6d Fig. 7 is a flow diagram of a preferred screening mode operation which may be implemented by a combinatorial screening apparatus of the present invention. Fig. 7 comprises the combination of Figs. 7a-g.
Fig. 8 is a flow diagram of a preferred potency mode operation which may be implemented by a combinatorial screening apparatus of the present invention. Fig. 8 comprises the combination of Figs. 8a-e.
Fig. 9 l~ s~ the experimental results of ET-1 dose :', n~ t Ca2+ mobilization in the presence of BO-123.
Fig. 10 r,, t~ t5 the semi logarithmic l~dr,~ lni r of the data of the Fig.9.
Fig. 11 ,l, G~ t5 the experimental results of dose d, e ~ t inhibition of ET-1 induced Ca2+ mobilization in the TE-671 cells with BQ-123.
Fig. 12le, ~ lstheexperimentalresultsofET1 dosed ,3m'~ ICa2+ mobilizationinTE671 cellswith the subs, t inhibition with the B~ 123 Fig.13 I., t3.,..1~ a simplified algorithm for cell mapping mode which may be utilized in a combinatorial 25 screening apparatus of the present invention Fig. 14 ll r 1~ U5 a simplified algorithm for functional chaldclcH~dt of orphaned receptors mode which may be utilized in a combinatorial screening apparatus of the present invention.Fig. 15 ,~, e~,.ls a flow diagram of a preferred cell mapping mode operation which may be implemented by a combinatorial screening 3~ r- dU..~ of the present invention. Fig. 15 comprises the combination of Figs. 15a-15f Fig. 16 ", es~.. ls a flow diagram of a preferred orphaned receptor determination mode operation which may be implemented by a combinatorial screening ~r' dt~ Of the present invention. Fig. 16 comprises the combination of Figs. 16a-16gf DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides for real time, continuous monitoring and detection of the rh~ C;~ ~1 or pharmacr~, ' effect of a test ~ , ' on a series of cell types or on a single cell type. In its simplest - r WO ~8~ 5~33 PCT/US97/13602 ~13 embodiment, the present invention comprises a method and apparatus for continuously contacting a single cell suspension or a series of cell s, ~ ~ each of which contains a single cell type included in the series of cell types to be tested, with a predetermined conca..l~atio., of at least one potentially active compound, preferably with predetermined concentrations of at least two active compounds. Then, intracellular changes that occur in response 5 to contact between the cells and the active compounds are c ~ measured as the , I containing the cell type to be tested or a member of the series of cell types to be tested and the test compounds pass a detector.
It is contemplated that the present invention will be of major value in high-throughput screening; e.g., in screening a large number of candidate compounds for activity against one or more cell types. It has particular value, for example, in screening synthetic or natural product libraries for active compounds.
In one preferred embodiment, a test compound, a standard compound, and a cell suspension containing the cell type to be tested or a member of the series of cell types to be tested are continuously mixed together and, after an incubation period, are passed by a detector that measures the cerr ~-ai in the cells or in the intracellular medium of at least one analyte. In one embodiment, the c~ ~ : at )r of the test compound andlor the standard compound is varied over time to generate r, 'r~ pr- ,Q curves as output from the detector.
It is preferred that the 1,~, al~ of the present invention is under the control of a computer or other programmable controller. The controller can ~ ~Ll~ monitor the results of each step of the process, and can automatically alter the testing paradigm in response to those results.
The incubation period after mixing of the compound or compounds and the cells can adva ~1ge~ cly be controlled by passing that mixture through a length of tubing li"g the mixing zone with the detector. The 20 incubation period will thus be determined by the flow rate and the length of the tubing. Incubation periods can vary by several orders of magnitude, depending on the particular analyte and the resultant reaction time. For example, the incubation period could be as little as one second or a fraction of a second for rapid or short-lived rh~ ainal responses, or as long as several minutes or even hours.
The analyte can be any analyte that is readily d~ttct''l by detectors andlor detL.,IIlllL' mictry 25 combinations. Thus, various ion or El~lr~l~le co ~.dlions, colorimetric changes, optical density changes, flu . e ~e luminescence, pH, gas r ~ ~ ' , and the like are all readily adaptable for use in the present method and apparatus.
In detecting ion or El~ e changes, calorimetric or fluorescent dyes are one particularly preferred embodiment. For example, calcium ion is detectable by such probes as Fura 2, Indo 1, Fura Red or Uuin-2, sodium 30 ions by SBFL, proton ions by BCECF, SNAFL, DM-NERF, magnesium ions by Mag Fura 2 or Mag Fura-5, chloride ions by SPO, SPA or MOAE. All these dyes are ~Q .... I 'Iy available, for example, from Molecular Probes, Inc., Oregon.
FIG. 1 illustrates a schematic configuration of a preferred embodiment of an apparatus of the present invention. The preferred embodiment consists of a test compound sampler 101, for receiving and holding a sample of a compound to be tested; a s ld d~ sampler 103, for receiving and holding a sample of a standard solution le.g., 35 a buffer andlor a known agonist or -: Jrlh ) to be mixed with the compound to be tested; a r1 - ~,al gradient device 105 com~ d to compound sampler 101 for controlling the flow volume of the compound to be tested; a .. .. . .

W O ~81'~3,;~ PCTAUS97/13602 concentration gradient device 107,: ~.1 ' to standards sampler 103, for controlling the flow volume of the standard solution; a switching valve 111 for receiving a compoundlstandard solution mixture or a calibration solution and for directing them to the mixing zone 112 for mixing one of these solutions with cell , - containing a cell type to be tested or a member of a series of cell types to be tested; at least one cell suspension reservoir 113 land, where a series of cell types is to be examined, a plurality of cell " r I~SOr~ ~ each of which contains a cell suspension of one of the cell types in the series to be examined); a c " ai Jr unit 117, for supplying calibration solutions to the switching valve 111 so that the calibration solutions may be mixed with the cell suspension in predetermined ratios; a reaction developing lines unit 115, for receiving the cell suspensions mixed with either the compoundlstandard solution mixture or the calibration solution and for transferring this mixture to a detector 119; and a drain 121 for receiving and draining the mixture from detector 119 afterthe cell response has been detected and measured by detector 119. A controller 109 is coupled to the test compound sampler 101, the standards sampler 103, gradient devices 105 and 107 and switching valve 111. The controller controls the above devices by receiving command signals from a computer 123 which in turn gcnc.dt~s, sends and receives signals in accordance with a software program 125. The mixing zone 112 can constitute a chamber, a tube, a series of baffles, or any other structure in which mixing can occur.
After a compound andlor standard is mixed with cells in mixing zone 112, which is readily commercially available and in the preferred embodiment is the static mixer 1251345 (Bio Rad), this mixture is then sent through reaction developing lines 115 SO that it may be mixed more thoroughly and provided with enough time for its reagents to thoroughly react with one another. The detector 119, is c e ll,d to the reaction developing lines unit 115, and receives the celllcompoundl~: '~ d mixture from the reaction developing lines 115.
In a preferred embodiment, each of the samplers 101 and 103 include a sipper nozzle which may be positioned into a vial containing a ,l, li.~ compound or standard solution. These samplers are well-known in the art and are readily available commercial products, e.g. AIS 300 Autosampler, Catalog # 15006330, Scientific Measurement Systems, Inc., Colorado. For handling multiple samples, c~r,..".i -' automated or robotic equipment ~not shown) may be used to supply the apparatus with the samples. Banks of different samples, arranged in 48 or 96 well plate format for example, can easily be accommodated by such automated sampling equipment.
The gradient devices 105 and 107 are also readily available commercial devices, e.g., GP40 Gradient Pump ~Dionex). Depending on the mode of action, the " pcrding c~ ,alion gradient devices 105 and 107 can prepare either discrete CCILe~IIai-~~Or continuous gradients of cGncL..;,: l~ of the compound to be tested or the standard s~ b 1- ~agonist or antagonistl by diluting them with buffer. For example, if a r-: ~: curve of cell response versus compound c~ c .lldl )~ in the presence of a standard substance is desired, the computer 123 will instruct the controller 109 to control the gradient devices 105 and 107 in such a way that continuous gradients of r. pt ~i~ compound and a predetermined constant c ~ 1 - I, dlion of standard solution are provided to switching valve 111. All~.llali.~ely, if a continuous curve of cell response versus standard solution cc -- I~: in the presence of a compound is desired, the computer 123 will instruct the controller 109 to control the gradient devices 105 and WO ~ ,5J53 PCT/US97113602 107 such a way that continuous ~radients of the ~ c ~ standard solution and a predetermined constant concentration of the ~, e li.~ compound are provided to switching valve 111.
The calibration unit 117, connected to the switching valvE 111, supplies calibration solutions needed to calibrate the output signal, MAX and MIN, of the apparatus. In a preferred embodiment, this calibration unit consists 5 of a diverting valve which is a readily available commercial device, e.g,. the SV3 2 Diverter Valve, ~Bio-Rad). The diverter valve alternates supply of two calibration solutions to the switching valve 111. The switching valve 111 combines outflows from the gradient devices 105 and 1 D7 or, altL, Ilali.. '~, from the calibration unit 117 with a cell flow from a cell ~r, reservoir 113 (or from one of a plurality of cell , Ib_O~e' S 113 each of which contains a cell type in a series of cell types to be examined) and directs the mixed flow into one of the reaction ~ r ~, lines 115. The switching valve 111 is of a type which is well known in the industry and in a preferred embodiment is the 3-way microvalve 4 8 900 manufactured by General Valve Corp. The cell ~ ~spr- reservoir(s) 113 are also of a type which is well known in the art and in a preferred embodiment is a regular glass beaker.
Cells are maintained in suspension pending i~ ,d~.liu. into the device by simple low shear stirring.
The reaction developing lines 115 are typically tubes made from a non-corrosive material, having a specified diameter, through which the above mentioned mixture of cell suspension, compound and standard solution may flow.
For example, pe'~clh~. e, rv'~l -P~: e or p '~I~I,allLu:ceth~; P tubing can be used. Tubing to which cells and other reagents will not stick is particularly preferred. The diameter of the tubin~q is a matter of choice. Capillary tubing having an inner diameter of from about 0.2mm to about 2 mm is particularly advantageous, because it allows the use of very small sample sizes.
This tubing is typically set in a winding configuration so that as the mixture flows through it, the mixture is thoroughly agitated and mixed. Even if the mixture is well mixed before introduction into the tubing, a spiral or wound configuration allows long tubing lengths in a compact area. These reaction developing lines 115 are commercially available and in one embodiment may be formed by Teflon tubing.
The mixture of a cell suspension containing a cell type to be tested or a cell type included in a series of cell types to be tested and compoundlstandard solution, also referred to as "reaction r ;jlr ~ enters the deteclor s flow through optical cell or other detection zone with a time delay or incubation period determined by the lenath of the reaction developing line 115. Once the reaction SL~ r reaches detector 119, the detector 119 can measure the cell suspension response to the specified concentration of the test compound. In a preferred embodiment, the detector 119 measures a fl en~ re signal from the calcium sensitive dye, FURA 2, spectral chalal,t~ ,s of which depend on a concL,.;,ai - of the intracellular ionized calcium, in order to determine the level of cell activity. In order to make this PP I :, the detector 119 " "al~'y irradiates the reaction ;l ~
passing throuqh a flow through optical quvette, with the light of the ~ a~ Ih~ 340 nm and 380 nm and measures flu . e ee intensity at 540nm. The ratio ~R) of the ll~ e5 ce: :en ti~s l~ d at 540 nm upon eJlcitdtR
at 340 nm and 380 nm". pc 5.~, is Ir r :led to the computer 123. The computer 123 calculates the 35 con"~,l, of the intracellular ionized calcium in accord~ -e with the following equation which is well known in the art:

W O9$1~3ga~ PCT~US97/13602 ~16-ICa2+l - Kd IR Rmin)lRmax - R~ (Ifllb) where Kd is the dissociation constant of calciumlFURA 2 complex and Rmjn and RmaX are the ratios obtained in the presence of the calibration solutions MIN and MAX, lu pc li~ , and If and Ib are the P .3~ intensities measured at 540 nm upon e,-~itai at 380 nm in the presence of caîibration solutions MIN and MAX respectiveîy.
A computer 123, - - d to the detector 119, dictates its operation. The computer 123 is also connected to or is a part of the controller 109 which controls the first and second gradient devices 105 and 107, switching valve 111 as well as test compound sampler 101 and standard sampler 103 in accordance with a software 125 implemented within the computer 123. The detector 119 is capable of measuring the particular desired signal, whatever its origin. An optical detector 119, may be a spectrophotometer, spectrofluorometer, or a luminometer, 10 each of which have a flow-through optical cell. these devices are well known in the art and are commercially available. For example, one embodiment of the apparatus of the present invention may use an AMlNC0-Bowman Series 2 Luminescence ~pe :~u~luorometer IFa-256, Sp ~,l Instruments, Inc.). If the detector is a direct ion measuring device, it can, for example, comprise a pH sensor or an ion selective ~I~.,t~ Sodium, calcium, and potassium detectors are examples of such devices. Detectors of this type are commercially available. All of these 15 devices can be CGIlIlu'' ' by means of a computer 123 during the data acquisition process.
The 3~ pa dlUS of the present invention is contemplated in two different specific embodiments. A positive pressure system may be created with piston pumps or other suitable pumps supplying reagents under positive pressure. Alt~.na~ 'y, a negative pressure system may be created with a peristaltic pump or other suitable pump drawing reagents through the system. The negative pressure system is the simplest embodiment, because a large 20 number of input sources can be driven by a single d~ ", pump.
Fig. 2 illustrates a s~ . configuration of one . .br ' t of a positive pressure fluidic system. In a preferred embodiment, this system operates automatically under the control of a programmable controller, as will be explained in more detail below. This embodiment includes an autosampler 201 which holds one or more compounds to be tested 203 and buffer 205 which may be mixed with the compound 203 in order to provide various 25 c tl3'' of the compound 203; intake nozzles, or ports, 207 and 209 for receiving a compound and buffer, respectively and delivering the same to a gradient pump 211 which controls the concentration level and flow of a test compound 203 into a mixing zone 227; an autosampler 213 which holds one or more standards (i.e., antagonists 215 andlor agonists 217) and a buffer 219 which may be mixed with the standard 215 or 217, in order to provide various cor,cL,.I,ai ~ of the standard; intake nozzles, or ports, 221 and 223, for receiving a standard and buffer, 30 respectively, and providing the same to a gradient pump 225 which controls the cu~.c~.,t, Ievel and flow of a standard solution lagonist or anl J. t) into the mixing zone 227. Each of the gradient pumps, 211 and 225, ,~ pr 'i~ ~y, may o('- : ~6 ~ have two inlet tubing lines and one outlet tubing line, the outlet tubes being r l~d to each other through the mixing zone 227. A controller 1109 of Fig. 1~ which is not shown in Fig. 2 controls the operation of autosamplers 201 and 213 and gradient pumps 211 and 225 by supplying remote signals 35 to :~tal'~ , the pumping. In a preferred embodiment, gradient pumps 211 and 225 may be the GP 40 Gradient Pump 'aLlu,~d by Dionex.

~17-The mixing zone 227 receives the compound solution flowing through gradient pump 211 and, optionally, a standard solution flowing through the gradient pump 225, and mixes the compound and standard substances together. A diverting valve 235 alternates the supply of either the calibration solution for m~rfmal response 233 or the calibration solution for minimal response 237. The diverting valve 235 preferably includes two inlet tubes, one tube for each of the two different calibration solutions 233, 237, and one outlet tube c- ILd to a pump 231.
The diverting valve 235 is well-known in the art and can be implemented by the SV-3 Diverter Valve (BioRad). The controller 1109 of Fig.1) which is not shown in Fig.2, sends signals to the diverter valve 235 which switches intake ports to connect one calibration solution 233 or another, 237, to the intake of the pump 231. The pump 231 for supplying calibration solutions to the fluidic system is also shown in Fig. 2. The pump 231 receives either the calibration max. solution 233 or the calibration min. solution 237 from diverting valve 235 and then pumps the received calibration solution to switching valve 229. The outlet tube of pump 231 is c- ~LI d to an input of the switching valve 229. The pump 231 is advantageously a standard piston pump which is well-known in the art. In a preferred embodiment, the pump 231 is the series 1350 Soft Start Pump IBioRad). The switching valve 229 alternates the supply of either the compoundl: ~' d mixture or one of the calibration solutions to a mixing zone 239. A pump 241 supplies cells from a cell suspension reservoir 243 containing a cell type to be tested (or from one of a plurality of cell , . reservoirs 243 each containing a cell type included in a sr~ries of cell types to be examined) to the mixing zone 239. The pump 241 receives cells from the cell suspension reservoir 243 from an inlet tube and pumps the received Gells through an outlet tube to one intake of the mixing zone 239. The comp ~ d solutions or calibration solutions come through another intake of mixing zone 239. Both mixing zones 227 and 239, are well known in the art and in a preferred embodiment may be implemented by the Static Mixer, 125-1345 ~BioRad). The pump 241 can be a standard piston pump which is well-known in the art. In a preferred embodiment, the pump 241 is the series 1350 Soft-Start Pump IBioPad). The mixture of cells and compoun~Jc 3~ ' dsolution or cells and a calibration solution is then fed from mixing zone 239 to reaction developing lines 245. The length of these lines, combined with the flow rate, determines the incubation period; i.e., the time elapsed from the point where the cells are mixed with the compoundlstandard mixture or the calibration mixture to the point of reaching detector 247. The detector 247 then measures the amount of cell response due to the compoundi~: -' d mixture or the calibration solution. After the cell response has been measured by detector 247, the mixture is then drained from the detector via drain 249.
Fig. 3 illustrates a .~ jr configuration of a preferred embodiment of the negative pressure fluidic system. As shown in Fig. 3, one preferred embodiment includes an autosampler 301 which holds one or more compounds INj) 303 and a buffer 305, the autosampler 301 further including two intake nozzles, or ports, 307 and 309, for receiving the compound INj) 303 and buffer 305, l~pecIi..,l~, a proportionating valve 311 for preparing - dilutions of the compound 303 with the buffer 305 in predetermined or specified ~ ~r~rliu.,;, and delivering this mixture to a priming valve 313; an autosampler 315 which holds standard all~ t! IMj) 317 agonists ~Lk) 319 and the buffer 305, the autosampler 315 further including two intake nozzles 323 and 325 for receiving the standard and the buffer"., :t .al~, a pr~ r ~ valve 327 for preparing dilutions of the standard substance 317 or , . ~ . . .

W O 98/05959 PCTrUS97/13602 319 with buffer 305 in predetermined or specified proportions and delivering this mixture to a priming valve 329.
The proportionating valves 311 and 327 may be of any type which is wellknown in the art. In a preferred embodiment these valves 311 and 327 are the series 4 miniature solenoid type valves 14 8-900 General Valve Corp.) working in a proportioning ~ mode. Similarly, priming valves 313 and 329 may be of any "normally opened"
type which is well known in the art and, in a preferred embodiment, are the series 4 miniature solenoid type valves t4 39-900) m ~aci oJ by General Valve Corp.
A mixing zone 331 mixes a compound solution received from priming valve 313 and a standard solution received from priming valve 329. The first mixing zone 331, as with the other mixing zones discussed herein, can constitute a simple "Y" c ~,., a chamber having a diameter several times that of the tubing; a length of tubing;
10 a serpentine, baffled chamber; a chamber containing a mechanical rotating mixer; or any other suitable structure.
Typically, the method will involve very small ~ of liquid te.g., a full c ~,di - gradient run can be accomplished with as little as 0.3 ml of sample~. Thus, the volumes to be mixed are small and the mixing zone should have small internal volume and high mixing efficacy. These types of mixers are well known in the art and in a preferred embodiment may be implemented by the Visco Jet~ Micro-Mixer l#TCMA0120113T) manufactured by 15 The Lee Company.
A diverting valve 335 alternates the supply of either a calibration solution for ma~r~m~/ response 337 or a calibration solution for mini~nal response 339. The diverting valve 335 includes two inlet tubes for receiving the two different calibration solutions and one outlet tube cr ' to another diverting valve 333. The diverting valve 333 can have two inlet tubes, one for receiving a compoundlstandard solution mixture and the other for receiving one 20 of the calibration solutions 337 or 339. The diverting valve 333 then alternates the supply of either the compoundlstandard mixture or one of the calibration solutions through an outlet tube to a priming valve 341 which then delivers the received fluid to a mixing zone 343. A third diverting valve 347 e" "ales the supply of either cells from a cell suspension reservoir 349 containing a cell type to be tested (or from one of a plurality of cell ~ ' )r each containing a cell type included in a series of cell types to be tested) or the buffer solution 305 25 through an outlet tubing 346 to a priming valve 345 which then delivers either the cells or buffer to the mixing zone 343 where it is mixed with either a compoundlstandard mixture or one of the calibration solutions received from priming valve 341. The diverting valves 333, 335 and 347 may be of any type which is well known in the art, for example, a SV-3 Diverter Valve (BioRad). This mixture is then sent to reaction developing lines 353 which determine the time elapsed from the point where the cells are mixed with the compoundlstandard mixture or one of the 30 calibration solutions to the point where the mixture reaches the detector optical cell 355. As explained above, the reaction dc.~, C lines 353 and the detector 355 may be of any type which are well-known in the art.
A Fe i ' pump 357, used as a negative pressure pump, can 3d~ 9e_ ly supply the rere~srry pressure required to make the various solutions and mixtures described above flow through the various valves 1311, 313, 327, 329, 333, 335, 341, 345 and 347) mixing zones 1331 and 343~, the reaction d~.' r- ~ lines 353, the 35 detector 355, and finally to the drain 359. Suitable peristaltic pumps 357 are well-known in the industry and in a prefsrred embodiment may be implemented by a EP-1 Econo Pump IBioRad). The pumps, valves, detectors, and T

other active components of the system are r l~f~ 'r under the automated control of a programmable controller, as explained in more detail below.
The lrF- dlUS in the present invention ~ fO~ has two primary modes of action, a screening mode and a potency mode.
Fig. 4 shows an algorithm that may be used in the invention to detect cell response during the screening mode. First, a celllcompound mixture is provided to a detector (step 401). Next, the apparatus drtermines if the compound, upon contact with the cells, triggers any cell response (step 4031. There are two possibilities: either the compound does not produce any response (N0), or it induces the cell response (YES). Cell response is determined by monitoring the signal from the detector for the particular analyte being detected. The control system first calibrates to establish a baseline and a maximal response, and any signal from the detector falling between those values is considered to be a positive response.
If the compound causes no cell response, (A~O, as measured by the monitoring part of the apparatus, the cells are ~ e ~I~ and automatically brought into contact with a mixture of the compound and the standard suhstances from a, .,.1.,t~,."dned set of agonist solutions ~step 407). Each solution in the set contains one or more ingredients that initiates cell response in the absence of the test compound, e.g., through the stimulation of a known cell receptor, ion pump or ion channel molecules. Next, a determination is made as to whether a cell response normally triggered with a particular agonist is suppressed by the particular compound (step 411). The apparatus will keep repeating an admixture of different standard agonist substances with the compound until it detects that the cell response triggered with a particular standard agonist is suppressed in the presence of the compound, or until all agonists available to the machine have been tested. If in step 411, it is determined that a cell response normally triggered with a particular agonist is su~,a,.,ss. d by the particular compound (YES), that compound is cat ~ iL~d as an antagonist to the receptor Ri (step 415). After this happens, the instrument is switched over to the potency mode of action if ~1~l l.,d by the software managing the apparatus' performance (step 417).
If the contact of the cells with the compound does initiate the cell response, (Y~S), as measured by the monitoring part of the appalal. s, the cells are automatically brought into contact with s r- ,i~ mixtures of the compound and the standard substance from a predetermined set of ani g ~t solutions (step 405~. Each solution in the antagonist set contains one or more ingredients that block the cell response initiated by at least one known agonist through the stimulation of a known cell receptor, ion pump or ion channel molecules. Next, a determination is made as to whether a cell response triggered with the compound is suppressed in the presence of a particular standard all; gr- ~t (step 409). The apparatus will keep repeating an admixture of different standard ~ gc substances with the compound until it detects that the cell response triggered with the compound is suppressed in the presence of particular standard a..: 9- :, or until all the v - ~ts have been tested. If it is determined in - step 409, that a cell response triggered with the compound is suppressed by the particular antagonist (YES), that compound will be r,l,aldcl~ .,d as an agonist to the receptor Ri (step 413). After this happens, the instrument is 35 switched over to the potency mode of action if ;,~ d by the software managing the 3" - aluS' F ~L.I
(step 417).

WO !>~ 5~ PCTIUS97/13602 By using known sets of standard agonist and antagonist substances to different receptors, it is possible to screen the compounds against several receptor types and subtypes for spe~ifiLi~l, and !e ~i~ity. When a series of cell types is to be tested, the process can be repeated for each of the cell types included in the series of cell types to be tested in order to evaluate the compounds' activities in a number of cell types.
For example, for the endothelin receptor, stimulation of which is indicated by an increase in intraceUular alion of ioni~ed calcium, the following receptor subtype specific . lqgr ts may be used: BQ-123, BQ 788, BQ 153, BQ-485, BMS-182874 PD 151, 242, and the following receptor subtype specific agonists may be used:
endothelin-1, endothelin 2, and endothelin 3. Sarafotoxin S6c, IRL 1620, Bû 3020.
For calcium channels, there are sets of channel type specific agonists and antagonists which can be used 10 in a preferred embodiment. For example, agonists of intracellular calcium channels are: Ins 11,4,5)P3, Ryanodine, Caffeine, Heparine, F~.l ' ' ale, and their 9~ ~t~ are: De~ 'te, Ruthenium Red and high ~c tldP of Ryanodine In another preferred embodiment, the screening mode is used for the Lha,aLl~ dlion of the cell receptor pattern, commonly known as "receptor ~ i ting". In ar~ ~, with this em'~ , a cell type to be tested 15 or a series of cell types are screened against standard substances from a predetermined set of agonist solutions.
Each solution in the set contains one or more ingredients that initiates cell response through the stimulation of a known cell receptor. In this way, the patterns of receptor expression in two or more cell types may be evaluated.
When one or more of the cell types respond to the particular agonist Sl' i -e, the instrument is switched over to the potency mode of action if instructed by the software managing the apparatus' performance.
Fig. 5 shows one algorithm that may be used in the invention in the preferred potency mode. The first step in the potency mode is to determine whether a particular compound has been c~: 9L iLcd as an agonist or antagonist Istep 501). The apparatus then prepares -- linlln~S r- lce lld~: gradients of either the standard substance or the compound being tested.
If the compound has been r,ai g i.~d as an agonist for one or more of the cell types to be tested during 25 the screening mode, the 7Pr al~ will measure and register the 1~ e : dti~. dependence of the cell response in the responding cells ~step 503). During step 503, the apparatus will generate continuous experimental ? ~;, " ~
curves for the â~.li...i 1~ compound. From these curves, one can calculate the potency of the compound in terms, for example, of EC50 ~the effective c - ~Ir~ of an activator which causes 50% of the maximal stimulatory response of the cDIlc)~tPp 505). This calculation can be ~ ~I l.. d using well known in the art curve fitting 30 software. In a preferred embodiment this software may be in,' t~d by the graphical software PRISM, man 'a~ d by GraphPad, Inc.
If the compound is determined to be an l 19 I for one or more of the cell types to be tested during the screening mode, the apparatus will measure and register the c~r :làlion dependence of cell response inhibition in these cell types in the presence of a standard agonist ~step 507). During step 507, the ql ~ dll~ will generate 35 continuous experimental inhibition curves for the - ;70l)r I compound taken at constant c ~ llalions of the stanr!ard agonist 5''; Cr From these curves, one can calculate the potency of the compound in terms, for W O 98/05959 PCT~US97/13602 ~2l-example, of IC50 llhe effective concentration of a blocker which causes 50% of the maximal inhibitory response cslls) Istep 509). The calculation of the lCsO valves can also be implemented in a preferred embodiment with the PRISM
software package.
If the compound is determined to be an antagonist for onev or more of the cell types to be tested, the apparatus will also measure and register the cor~ , ' ne of the cell response in these cell types to a standard agonist substance in the presence of several c nr ~,a: ~ of the antagonist compound Istep 511).
During step 511, the apparatus will generate a series of continuous experimental activation curves for the standard agonist substance taken at different discrete ~ d; ~ of the antagonist compound. The apparatus will then calculate the affinity and the potency of the compound in terms, for example, of PA2 values and determine whether the v : is competitive or non-competitive IsteP 513). The PA2 value is proportional to a negative logarithm of the binding constant of a ligandlreceptor complex and is a measure of the affinity of the ligand to the receptor:
the bigger PA2 value, the higher the compound's affinity to the receptor. Practically, PA2 value can be calculated from the shift of the acli.di ~ curves in the presence of different e ~ai' . of the a Ee I compound and can be implemented by the formula:
PA2 LoglR 1) -Log B, where R is a ratio of e,, ~: c '.~ -e of the standard agonist b~: nle measured both in the presence of discrete concentration IB) of the antagonist compound and without the antagonist. In a preferred embodiment the equipotent CG e t~ of the standard agonist substance can be found from the PRISM software avli..:' curves. With the above sets of experimental curves, one can evaluate the potency and the pharmac~1~v -' profile for thev compound under investigation in ace~ d with Cheng Prusoff ICheng & Prusoff, 1973, inc I nlvd herein by .ef~v..n~ ) Gaddum (Gaddum, 1957, incorporated herein by ~vfe.~ e) or Schild lArunlakshana & Schild, 1959, incorporated herein by le~ ce) analyses.
A detailed description of the operation of the negative pressure fluidics system of Figure 3 is given below.

The process is started when a computer prompts an operator to chose a mode of opevration from a choice of three modes: system priming mode, screening mode and potency profiling mode. In the presently preferred top-level process, the first choice is to perform system priming. Referring to Figure 3, before the priming process starts, the operator is prompted to place the nozzles of intake ports 309, 325 and 351 into a reservoir 305 filled with a buffer, the no~les of the intake ports 336 and 338 into ~evv~ s 337 and 339, respectively, filled with 30 cor, - 9I v calibration solutions, and the nozzle of the intake port 348 into one of the cell suspension ~ ,v",.
349 filled with a cell types to be tested.
A c~ I;..uvus flow diagram of a preferred system priming process is shown in Figs. 6a 6d for the preferred - negative pressure fluidics system presented in Fig. 3. Referring to Figure 6a, the system priming program begins from a start state 600 and enters state 602 where it sets all parameters to their initial values. These initial parameters include, but are not limited to, the internal initialization of software variables and subroutines.

W O3~1W,33 PCTrUS97/13602 ~22-Once the initial parameters are determined, autosamplers 301 and 302 ~Figure 3), position their corresponding intake nozzles 307 and 323 at "zero" position (Step 604). Each autosampler, in its "zero" position, contains a reservoir 305 filled with a washing buffer. During step 604, the power supply which supplies power to proportioning valves 311 and 327 (Figure 3), to diverting valves 333,335 and 347 (Figure 3), and to priming valves 313, 329, 341 and 345 (Figure 3)is turned off. In the preferred embodiment, the priming valves, 313, 329, 341 and 345, are normally opened in a nonp . .,.~d state loff); the r ~ ing valves, 311 and 327, in the non-powered (off) state, connect their outlets 312 and 328 with Ib;~ ...ti. a ~normally opened" intake ports, 309 and 325;
and in the rJn p . c.Ld ~off) state, the diverting valve 335 connects its common outlet 334 with the "normally opened" intake port 336, diverting valve 333 connects its common outlet 342 with the "normally opened" intake port 332, and diverting valve 347 connects its common outlet 346 with the "normally opened" intake port 348.
After the system has been initialized, pe,i~ldlli., pump 357is started (Step 606) and priming valves 313 and 345 are turned on (Step 608). In the powered state, the priming valves 313 and 345 are closed. This forces the buffer flow through tubing 325, outlet tubing 328of F ~, ~rli~.ning valve 327, priming valve 329, mixing zone 331, intake tubing 332 and outlet tubing 342 of the diverting valve 333, priming valve 341, mixing zone 343, a reaction '~ line 353, detector 355, pump 357 and then into a drain container 359.
Referring again to Fig.1, computer 123 implements software code 125 to provide control signals to a controller 109 which controls the various valves, chambers etc. of the system. This control signal is delayed in order to provide adequate time for liquid to fill the fluidic lines (Step 610). Next, the proportioning valve 327is turned on (Step 612). While proportioniny valve 327is in the powered state, the buffer located at "zero" position of the autosampler 315 will flow through tubing 323, outlet tubing 328 of PI~F liur,ing valve 327, priming valve 329, mixing zone 331, intake tubing 332 and outlet tubing 342of diverting valve 333, priming valve 341, mixing zone 343, a reaction d . '(, 9 line 353, detector 355, pump 357 and then into a drain container 359.
After energizing the, .p 6 ling valve 327, the control signal is delayed again (Step 613) in order to provide the time needed for the buffer to fill out the fluidics system. After the delay in Step 613, the control signal will set the proportioning valve 327 to its off position (Step 614), turn off ~open) priming valve 313 (Step 616) and turn on (close) the priming valve 329 (Step 618). This will fill out fluidic lines 309, 312, 332, 342, priming valve 341, mixing zone 343, a reaction developing line 353, detector 355 and pump 357 during a delay (Step 619) which is needed for the lines to be filled with buffer 305.
Next, the proportioning valve 311is powered ~Step 620) for a time duration controlled by Step 621. During this delay period, the same fluidic lines are being fed with the buffer located at "zero" position of the autosampler 301, coming through the nozzle 307.
After the delay period (Step 621) the control signal simult - L~ly turns off both proportioning valve 311 (Step 622) and priming valve 329 (Step 624) and turns on both diverting valve 335 and diverting valve 333 (Step 626). During a delay period, determined by Step 627, the liquid flow will be directed from intake tubing 338 and outlet tubing 334 of the diverting valve 335 through the intake tubing 340 and the outlet tubing 342 of the diverting valve 333 through the priming valve 341, the mixing zone 343 and further through the reaction developing line 353, r ~23-the detector 355, the pump 357 and then to the drain container 359. Then the control signal turns off diverting valve 335 IStep 628), which allows the same fluidic lines to be fed from intake tubing 336. The length of time required is a function of the length of the different lines involved and the rate of fluid flow through the system, which for any particular system can be readily determined.
After the delay provided by Step 629, the control signal turns off diverting valve 333 IStep 630), turns off loPens) priming valve 345 IStep 632) and turns on (closesl priming valve 341 IStep 634). Under these conditions, the liquid flow will be directed from the intake tubing 348 to outlet tubing 346 of diverting valve 347, through the priming valve 345, the mixing zone 343, the reaction developing line 353, the detector 355, the pump 357 and then to the drain container 359. The time required for the above liquid flow to occur is provided by a delay period in Step 635. After this delay, the control signal turns on diverting valve 347 IStep 636), which allows the same fluidic lines to be filled with the buffer 305 coming from tubing 351, during a delay period provided in Step 637.
After the delay provided by Step 637, the control signal turns off lopens) priming valve 341 IStep 6381, and turns off the diverting valve 347 (Step 640). The control signal then turns off pump 357 IStep 642). Finally, the priming program ends the system priming mode and all components of the fluidics system are filled with liquids IStep 643).
The state of the valves at each step of the program is shown in Fig. 6d. Priming valves 345, 341, 313 and 329 are of a two-way normally opened type. Diverting valves 335, 333 and 347 are of a three-way type with one common outlet port and one each normally opened and normally closed intake ports. P~ r - i n?ting valves 311 and 327 are also of the three-way type similar to diverting valves 335, 333 and 347. The symbols "" and "+"
indicate turned off and turned on state of the valves. The state of all the valves after the priming mode is finished is the same as at the initial state.
After the priming of the system is done, the operator is prompted to start either the screening or potency profiling mode. If screening mode is chosen, the operator is prompted to specify how many compounds are located in the set of compounds to be tested, NmaX~ and how many antagonists, MmaX~ and agonists, LmaX~ solutions are located in the ~., li.e sets of the standards. If potency profiling mode is chosen, the operator is prompted to specify which compounds in a set should be measured.
A continuous flow diagram of the presently preferred screening mode with the negative pressure fluidics system of Fig. 3 is shown on Figs. 7a - 79.
The screening program begins from a start state 700 and enters state 702 where it sets all parameters to their initial values. These initial parameters include, but are not limited to, the internal initialization of software variables and subroutines.
Once the initial parameters are determined, the autosamplers 301 and 315 IFig. 3) position the intake - nozzles 307 and 323 at the cc.,~ "zero" position occupied by a wash buffer reservoir 305 IStep 704), and turns off r ~jlC.i' ing valves 311 and 327, diverting valves 333, 335 and 347, and priming valves 313, 329, 341 and 345 (Step 7061. In a turned off state, priming valves 313, 329, 341 and 345, are normally opened, proportioning valves 311 and 327 connect, ~. reel;~d~ their outlets 312 and 328 with e .., ding "normally ... . . . ..

WO ~ 5, PCT/IUS97/13602 opened" intake ports 309 and 325, diverting valve 335 connects its common outlet 334 with "normally opened"
intake port 336, diverting Yalve 333 connects its common outlet 342 with the "normally opened" intake port 332, and diverting valve 347 connects its common outlet 346 with the "normally opened" intake port 348.
After the system has been initialized, pump 357 is started ~Step 708). This will force the wash buffer from the "zero" p ~; nel' leso~,. s of both autosamplers 301 and 315 to run through nozzles 309 and 325 and outlet ports 312 and 328 of proportioning valves 311 and 327, ~ , priming valves 313 and 329, mixing zone 331, intake port 332 and outlet port 342 of diverting valve 333, priming valve 341, mixing zone 343, a reaction developing line 353, detector 355, pump 357 and then into a drain container 359. In mixing zone 343 this flow is mixed with cell s~ uls which contain a cell type to be tested, from cell i~ '~r reservoir(s) 349 coming 10 from intake tubing 348 and an outlet port 346 of diverting valve 347 through priming valve 345. Thus, the total flow passing mixing zone 343 is a sum of three flows, one of each coming from the proportioning valves 311 and 327 and one coming from diverting valve 347. During this step, both pn.~ i ring valves supply wash buffer 305 from the ,evar~,. s located at "zero" position of each autosampler in such a way that the final mixture consists of one part cell s ~ and two parts wash buffer 305.
After the delay determined in step 709, which is needed for the flow of the mixture of the cell ~
containing the cell type to be tested (or a member of a series of cell types to be tested) and wash buffer 305 to stabilize, detector 355 registers a basal signal produced by the cells alone, BS tStep 710). After the BS signal is registered, it is saved as a reference ISteP 712) and computer 123 (Fig. 1 ) triggers i...,,, ~ tal counter N (Step 714).
The numerical content of the incremental counter N is increased by unity each time it is triggered thus determining the position of the nozzle of intake port 307 of autosampler 301 which samples the sets of compounds 303 to be tested. Next, the numerical content, Nj, of the incremental counter is checked against an entered maximum number of the compounds to be tested, NmaX lSteP 716). If Nj does not exceed NmaX~ autosampler 301 positions nozzle 307 into the compound reservoir located at the Nj position of a rack of compounds 303. Next, r ~ ing valve 311 is switched on to open its "normally closed" intake port 307 to the common output port 312 ~Step 720). During Step 720, tne combined flow in mixing zone 343 and altvr- a,ds in detector 355, is composed of one portion of buffer 305 coming through nozzle 325 of proportioning valve 327, one portion of the compound 303 to be tested coming from nozzle 307 of ~ r i- ing valve 311 and one portion of the cell suspension containing a cell type to be tested coming from the intake tubing 348 which is "normally opened" to the output port 346 of diverting valve 347. After a delay provided by Step 721, which is needed for the mixing proress to stabilize, detector 355 registers a signal, SNj, produced by the cells in the presence of the given compound, Nj ISteP 722). After the SNj signal is rL~ d, its value is saved as a reference value IStep 723). The value of the SNj signal is then compared with the value of the reference basal signal, BS ~Step 724). If the SNj signal is greater than the BS signal, the computer 123 ~Fig. 1) will "flag" the r_ 1~, iing compound as "positive" ~Step 725), which means that the ~ pr ' stimulates the cell signal. If SNj is greater than BS, the computer 123 controls a set of antagonists and calculates ca ,~v ,~a d v coordinates for positioning the nozzle 323 of autosampler 315 over the antagonist: i E ~e~ Iocated in rack 317. If SNj is less than BS, computer 12.~ will "flag" the , .

CA 0226l740 l999-0l-28 W O ~3~'13~3~ PCT~US97/13602 c ~ ding compound as "negative" IStep 726). In this case, the computer 123 controls a set of agonists and calculates corresponding coordinates for positioning the nozzle 323 of autosampler 315 over the agonist containin~q located in rack 319.
Each time the condition, "SN; > BS", is satisfied, the program flow will go throuph loop K-O of the incremental counter M lSteP 730). The incremental counter, M, increases the count number by one each time it is trig,qered and thus determines the - e~ i- positions of nozzle 323 of autosampler 315 which serves the set of standard ~: D- t located on rack 317. Next, computer 123 checks if the numerical content of the incremental counter IM;) exceeds the maximum number of standards to be tested IMmaX) entered by the operator IStep 732).
If Mj is less than or equal to MmaXl autosampler will position nozzle 323 into the standard antagonist reservoir, located on rack 317 C~r,~r~ldin9tO the numerical content of the incremental counter, Mj ~Step 734).
Nsxt, ~, r~rticring valve 327is switched over to connect its "normally closed" intake port 323 with the outlet port 328 (Step 736). During Step 736, the combined flow through mixin~ zone 343, reaction r!l . '(, v lines 353 and detector 355iS composed of one portion of standard l,1s. : coming from proportioning valve 327, one portion of the compound to be tested, coming from p,.,~crtil ing valve 311 and one portion of the cell suspension containing a cell type to be tested coming from diverting valve 347.
Next, detector 355 registers and saves (Step 738) for further comparison, a siqnal, SNjMj, produced by a given compound, Np in the presence of a given standard antagonist, Mj. Then, both proportioning valves 311 and 327 are turned off (step 740). This closes intake ports 307 and 323 and opens intake ports 309 and 325to the corresponding outlet ports 312 and 328. Autosampler 315 then positions nozzle 323 into "zero" position occupied by wash buffer reservoir 305. The proportioning valve 327iS then turned on again to open its "normally closed"
intake port 323 into the outlet port 328 IStep 744) and the content of nozzle 323is washed out (Step 746). Next, pr.r ; ~9 valve 327is turned off to close intake port 323 and to open "normally opened" intake port 325. During this process, the fluidic lines are washed with buffer coming from nozzle 325 of autosampler 315.
Next, Computer 123 IFig. 1) compares signal, SNj, measured in the presence of a compound Nj alone, with the signal, SNjMj, measured in the presence of both a compound Nj and a standard anta~qonist, Mj (Step 750). If the signal in the presence of a standard a tn~q~ 1, Mj, is lower than the signal " atod by a compound Nj alone, SNjMj < SNp then both the compound Nj and the standard antagonist, Mj, is flagged as "positive" IStep 751). If both signals are equal to each other, then the compound Nj is flagged as "positive" and the standard antagonist Mj is flagged as "negative" IStep 752). After the data is flagged, the data, containing the ID number of the compound Nj with its flag value and ID number of the standard antagonist Mj with its flag value is transferred into a database IStep 753). After the data is saved the program loops back to trigger the incremental counter M ISteP 730), to increase its count by one and Step 754iS repeated until the condition 732is achieved.
- When the condition determined in the decision step 732is met, that is the compound has been tested against all standard antagonists in a given set, the program resets the content of incremental counter M to zero (Step 755i, and both autosampler 301 and autosampler 315 position their ~~ nozzles 307 and 323to "zsro"
position, where the reservoirs with a washing buffer 305 are located (Step 756). Next, both, ~p~ri ring valves CA 0226l740 l999-0l-28 WO gX~U5~9 PCT/US97/13602 ~26-311 and 327 are turned on IStep 758). This opens "normally closed" intake ports 307 and 323 to the c~ ,1, ding outlet ports 312 and 328. During this washing delay period d~: , ' by Step 760, the contents of both nozzle 312 and nozzle 328 are washed out with buffer 305. After Step 760 iS finished, both, r .i' dngvalves 311 and 327 are turned off lSteP 762). After the washing delay period IStep 760~, the program loops back to the 5 incremental counter N content of which is incremented by one IStep 714) by triggering signal Str from the computer 123 IFig. 1). Then, the numerical content Nj of counter N is compared with the maximum number, NmaX~ of the compounds to be tested. If Nj exceeds NmaX~ the content of incremental counter N is zeroed (Step 717) and the screening mode is stopped IStep 719). Otherwise, Step 715 will be repeatPvd with the next compound.
If the condition of the decision process 724, SNj > BS, is not satisfied, the Nj compound is flagged as "negative" IStep 726) and the inc,L,.,vnlvl counter L is triggered IStep 764). The incremental counter L is increased by one IStep 764) each time it is triggered and thus determines the ... positions of nozzle 323 of autosampler 315 relative to the set of standard agonists located on rack 319. Next, the numerical content, Lk, of incremental counter L is compared with the maximum number, LmaX~ of standard agonists to be used Istep 766).
If Lk is less than or equal to the LmaX~ autosampler 315 positions nozzle 323 into the standard agonist reservoir in rack 319, which corresponds to the numerical content, Lk, of incremental counter L. Next, proportioning valve 327 iS turned on to open its "normally closed" intake port 323 to outlet port 328 and proportioning valve 311 is turned off to close its intake port 307 and open intake port 309 to outlet port 312 IStep 770). During step 770, the combined flow coming through mixing zone 343, reaction d~ r: 9 lines 353 and detector 355 iS composed of one portion of standard agonist 319 coming from inlet port 323 of proportioning valve 327, one portion of buffer 305, coming from inlet port 309 of . ~r i' dng valve 311 and one portion of the cell s~spr-- containing a cell type to be tested coming from the diverting valve 347.
After a delay period provided by Step 772, which is needed for the mixing process to stabilize, a signal, SLk, produced by a given standard agonist, Lk is Ivy;vlv,vd and saved IStep 774). Next, proportioning valve 311 is turned on to open its "normally closed" intake port 307 to outlet port 312 IStep 776). A delay period needed for the mixing process to stabilize is provided in Step 778, after which a signal, SLkNj, is stimulated by a given standard agonist, Lk, in the presence of a given compound, Nj is ,~v t.,,.,d and saved in (Step 780).
After the signal SLkNp is registered IStep 780), both proportioning valves 311 and 327 are turned off to close their ", ';.v "normally closed" intake ports 307 and 323 and to open intake ports 309 and 325 to the C 1- r ding outlet ports 312 and 328 ISteP 782). Nexl, autosampler 315 positions the nozzle of intake port 323 to its "zero" position which corresponds to washing buffer reservoir 305 IStep 784). Then, the proportioning valve 327 iS turned on IStep 785). This opens the "normally closed" intake port 323 to the outflow port 328, and after some delay provided by step 786, which is needed for washing out the content of nozzle 323, the, ~r- i - 9 valve 327 is turned off IStep 787), and the two signals, SLkNj and SLk, are compared IStep 788).
If SLkNj is less than SLk, then the r,~r~, n~lding standard agonist, Lk, is flagged as "positive" IStep 790)-If both signals are equal to each other, then the standard agonist, Lk, is flagged as "negative" IStep 792). After the data is flagged, the data, containing the ID number of the compound Nj with its flag value and the ID number W O ~ 5~ PCT~US97/13602 ~27 ot the standard agonist Lk with its flavn value is transferred into a database IStep 794). After the data is saved the program triggers the incremental counter L ISteP 764) to increase its count by one and Step 796 is repeated until condition 766 is achieved.
When the condition determined in Step 766 is met, that is the compound has been tested against all 5 standard agonists in a given set, the content of tal counter L is reset to zero (Step 755) and both autosampler 301 and autosampler 315 position their respective nozzles 307 and 323 to "zero" position where the reservoirs with washing buffer 305 are located ~Step 756). The system then turns on both proportioning valve 311 and p I, i ving valve 327 to open "normally closed" intake ports 307 and 323 to the r ,., ding outlet ports 312 and 328 I- pc ~ (Step 758). During a washingv period provided by Step 760, the contents of both nozzle 307 and nozzle 323 are washed out with the buffer 305. Next, both proportioning valves 311 and 327 are turned off IStep 762) and incremental counter N is incremented by one (Step 714). Next, the new triggered numerical content Nj of the incrementai counter N is compared with the maximum number, NmaX~ of compounds to be tested IStep 716). If Nj, exceeds NmaX~ the counter N is set to zero in step 717 and the screening mode is stopped IStep 719). Otherwise, the whole cycle will be repeated with a subsequent compound.
After the screening mode is stopped (Step 719), the operator is prompted by the computer to choose either potency profilinvn mode or to repeat screeninvn mode with a new set of compounds.
A continuous flow diagram of the presently preferred pQI ~ u~iling mode with the negative pressure fluidics system of Fig. 3 is shown in Fiys. 8a - 8e.
The potency profiling program begins from a start state 80D and enters state 802 where it sets all incremental counters, L, M, and N, to zero and sets all parameters to their initial values. This initial parameters include, but are not limited to, the internal initialization of hardware and software variables and subroutines. Next, the u",~"ldl counter N is started ISteP 804), and its numerical content, Nj, is compared with the maximum number, NmaX~ of ~tanda,ds, entered by the operator before starting the program IStep 806). If Nj exceeds NmaX~
the incremental counter N is zeroed ISteP 808), and the program stops IStep 810). 0therwise, the program will proceed by turning on pump 357 ISteP 816). At this point, in ?e~er-' e with initial parameters setup in Step 802, the nozzles 307 and 323 of autosamplers 301 and 315 are in zero position, proportioning valves 311 and 327, priming valves 313, 329, 341, and 345, and diverting valves 335, 333, and 347 are turned off. During step 816, the combined flow coming through mixing zone 343, reaction developing lines 353 and detector 355 is composed of one portion of buffer 305 coming from inlet port 309 of proportioning valve 311, one portion of buffer 305, coming from inlet port 325 of pl~rS i- C valve 327 and one portion of the cell suspension coming from the diverting valve 347. Next, diverting valve 333 is turned on ~Step 820), opening "normally closed" intake port 340 into outlet port 342. During this process, the calibration max solution 337, which determines maximum response, - flows through tubing 336 which is c tod to outlet tubing 334 of diverting valve 335, through intake port 340 of diverting valve 333, through priming valve 341 and then into mixing zone 343, where it is mixed with cells coming from the cell , through intake tube 348 and outlet tube 346 of diverting valve 347, and then through priming valve 345 into the mixing zone 343. The cells calibration max mixture will then flow to da~c'( p v lines 353, ., WO ~8~ , PCI/US97/13602 ~28-and then to detector 355. Next, the maximum signal is measured and registered lStep 824). Diverting valve 335 is then turned on (Step 832) which opens the "normally closed" intake port 338 to outlet port 334 which allows a calibration min solution 339 to flow through diverting valve 333, priming valve 341 and into mixing zone 343 where it is mixed with cells coming from the cell suspension, through intake tube 348 and outlet tube 346 of diverting valve 347, and then through priming valve 345,into mixing zone 343. The cellslcalibratiDn min. mixture then flows to ~ LF ~, lines 353 and then to detector 335. The minimal response signal is then measured and registered (step 836). Next, both diverting valves 335 and 333 are turned off IStep 844). During step 844, the combined flow coming through mixing zone 343, reaction developing lines 353 and detector 355 is composed of one portion of buffer 305 coming from inlet port 309 of proportioning valve 311, one portion of buffer 305, coming from 10 inlet port 325 of ~r-po i dng valve 327 and one portion of the cell s , - coming from the diverting valve 347.
This is needed to wash out the remains of calibration solution from the fluidics line. Next, the "flag" value of the compound, Nj, is checked (Step 812).
If the 'flagN value of the Nj compound is "positive", then the system will go to step 850, otherwise it will go to step 876. If the "flag" value of the N; compound is "positive", autosampler 301 will position nozzle 307 into 15 a reservoir corresponding to the Nj compound, located on rack 303 (Step 850). Next, proportioning valve 311 is operated in a gradient mode (step 852). In the gradient mode, the, ~r_ i Iing valve, 311, combines flows from two intake ports, 307 and 303, in such a way that the, ~r- i' of a Nj compound flow and a buffer flow in an outlet port, 312, changes in time to create a discontinuous change of the compound concentration. While the n( ~ l~ is being changed in time, the fresh portions of the cells, coming into the mixing zone 313 from the 20 cell suspension, at any given time will react with the different cc It~al - of the Nj compound. The resulting dose response ', ' of the signal is registered in Step 854 with the ' 1r, t calculation of the activation parameters for the Nj compound in Step 856 and then saving the data in a database (Step 858).
Next, an incremental counter of the standard antagonists, M, is started ~Step 860) and the numerical content, Mj, of the counter M is compared with the maximum value, MmaX~ entered by the operator IStep 861). If 25 the numerical value Mj does not exceed MmaX~ then the 'flag' value of the Mj standard is checked ~Step 862). If the Uflag'' value of the Mj standard is not Npositive", the incremental counter, M, is increased by one (Step 860), and the steps following Step 860 will be repeated as described above. If the UflagN value of the Mj standard is Upositive", autosampler 315 positions nozzle 323 into the reservoir cc ,. pc ding to the Mj standard ~ t, located on rack 317 (Step 864). After nozzle 323 is immersed into the standard solution, proportioning valve 311 30 is turned on ~Step 866). When the r ~ epoL i ~o.ing valve 311 is turned on, it opens its "normally closed" intake port 307 which is immersed, in Step 850, into a reservoir CGIl~ pr- " g to the compound Nj, so that the Nj compound flows through the fluidics system and g.!.,o,at~s a signal when it is mixed with the cells, coming through intake tubing 34B and outlet tubing of diverting valve 347, priming valve 345 to mixing zone 343 and further to d .~
line 353 and detector 355. Next, proportioning valve 327 is operated in a concentration gradient mode (Step 868).
35 In the gradient mode" .po i ing valve 327 combines flows coming from the two intake ports, 323 and 325 into outlet port 328 in such a way that the p r, liun of a Mj standard i ~o ~t flow and a buffer flow changes in WO ~J~5~, PCTrUS97/13602 time to create a discontinuous change of the standard antagonist concentration. While the concentration is being changed in time, the fresh portions of the cells flow into mixing zone 343 from the cell suspension and react with the mixture composed of the Nj compound at constant rq ,r ll and the standard antagonist Mj at the sr t~ - different at any ~iven time. The resulting dose response inhibition by the Mj standard lgr t of 5 theJ Nj compound stimulated signal is registered in Step 870, with the subsequent calculation of the inhibition parameters for the Mj standard antagonist in a Step 872 and saving the data in a database ~Step 874).
After the curve is ,.~ : od in Step 870 both proportioning valves 311 and 327 are closed (Step 871) and the program flow returns to Step 860 where incremental counter M is incremented by one and the program returns to Step 861 where Mj is compared to MmaX~
When all of the standard antagonists Mj are counted and those with "positive flags" are measured, the numerical value Mj becomes greater than MmaX~ and the decision process in Step 861 returns to Step 804 where the incremental counter N increases its numeric value by one. The value of Nj is then compared to NmaX~ the maximum number of compounds to be tested (Step 806). If Nj exceeds NmaX~ the incremental counter N is reset to zero (Step 808), and the program stops (Step 810). If multiple cell types are to be evaluated, this process can be repeated for each of the cell types in the series of cell types to be tested.If, in Step 812, the flag value of the Nj compound is determined to be "negative", a incremental counter L which counts the standards, Lk ,in a set of agonists, located in rack 319, is updated (Step 8761. Next, the numerical value Lk is compared with the maximum number, LmaX~ of standard agonists entered by an operator (Step 877).
Until the numerical value Lk ~f the L counter is greater than the LmaX value, the program will go to Step 878, where the 'flagged value of the Lk standard is checked. If the flag value is not positive, the program returns to Step 876. If the flag value of the Lk standard is positive, autosampler 301 positions its nozzle 307 into a c .. pr-ding reservoir containing the Nj compound located in rack 303 (Step 880), autosampler 315 positions its nozzle 323 into a cGr,. por ding reservoir containing the Lk standard agonist, located in rack 319 IStep 881).
Next, the concentration gradient mode of proportioning valve 327 iS activated IStep 882) and a dose response stimulation of a signal with the Lk standard agonist is then registered (step 884). In the gradient mode, proportioning valve 327 combines flows coming from the two intake ports, 323 and 325 , into outlet port 328 in such a way that the P.-rsri of a Lk standard agonist flow and a buffer flow changes in time to create a c~ , gradient of the standard agonist. While the gradient is being created in time, fresh cells flow into mixing zone 343 from the cell . r through "normally opened" intake tubing 348 and outlet port 346 diverting valve 347 , "normally opened" priming valve 345, and discontinuously react with the standard agonist Lk at the discontinuously changing concentration of the standard. Once the dose dc,- ' ee of the signal stimulation by the - Lk standard agonist is ~ t~"d (Step 884), the a~li.ai - parameters are - ' ' t~d ~Step 892) and then saved in a database (Step 894).
- 35 After the gradient mode of ~ r - i Iing valve 327 is finished, the valve stays either at the ~ ~F ~ rate reached by the end of the gradient mode or at p,~d~t~. -' prJr~ i rate which e: '' hes the constant W O 98/05959 PCTrUS97/13602 ~30-concentration of the Lk standard agonist which will be used in further steps. Next, the ~ ~ 'x a~ion gradient mode of proportioning valve 311 is activated (Step 886). A dose response inhibition by the Nj compound of the Lk standard agonist stimulated signal is then registered IStep B88~ with a subsequent calculation of the inhibition parameters in Step 896. this data is then saved in a database (Step 894). When the registration of the - - ~.. 1 ', d ~e is finished both proportioning valves 311 and 327 are turned off (Step 8901 to close their intake ports 307 and 323 and to open "normally opened" intake ports 309 and 325. During this process, the fluidics lines are washed with buffer 305 coming from nozzles 309 and 325 of autosamplers 301 and 315 through outlet ports 312 and 328 of ~I-, D:i- ling valves 311 and 327, priming valves 313 and 329, mixing zone 331, "normally opened" intake port 332 and outlet port 342 of diverting valve 333, "normally opened" priming valve 341, mixing zone 343, developing line 353 and detector 355. Next, the program returns to Step 876, at which point the above cycle is repeated until all the standard agonists are counted, and those with a positive flag are measured, and the numerical value Lk ~f the incremental counter becomes greater than the b~ax value, at which point, the program will return to Step 804.
When all of the compounds Nj are counted (Step 804) and tested, the Nj number becomes greater than the NmaX value and the incremental counter N is reset to zero (Step 808) and the program stops (Step 810).
Cells for use in the apparatus can be selected for the presence of particular known receptors or for their ability to provide pr~d~t~"uined cellular ,l pc to particular stimuli. A large number of such cells are known.
For example, to measure the effect of compounds on calcium mobilization induced by different types of receptors, one may wish to use Jurkat T Cells, Platelets, Umbilical Uein, '-~LI' '' ' Cells, or Chines Hamster Lung ri~
20 for thrombin receptor; Cerebellar Purkinje Cells, Cortical Astrocytes and Cortical Glial cells for AMPA r~cLr; a, Hippocampal Neurons for NMDA Receptor; P-12 cells for Purineroic Rec~ , Oligodendrocytes for Platelet-Derived Growth Factor Receptor, Human Neli,oDla~ cells and Pituitary Cells for Neuropeptide Y nuc~pt s and protein tyrosine kinase and protein-tyrosine phosphatase ,~.,.,.i a, Human Medulloblastoma cells for Endothelin Receptor;
Neutrophils for TNFa Receptor; NG108-15 cells for opioid, bradykainin and ATP; Synovial Fibrobtasts for Plasminogen 25 Receptors and so on. If one wishes to measure an intracellular ion c~ for example, one can preincubate the cells with a dye or other dl I~.,l ' ' material having ;,~..;,iti.;ly to conr,~ r of that particular ion. (An actual working example illustrating preparation of cells for detection of calcium ion is set forth in Example 1.) Alternatively, if one wishes to determine the pattern of natural t.~..l )r of receptors responsible for Ca2+
signaling pathway, then one may use the cells of particular interest and then using a set of agonists known to exert 30 their activity through Ca2+ mobilization, to ch~ LIL.i~ the cells by what type of the receptors are expressed in these particular cells. This set of agonists may consist of ac. Ih~' 'Dlin, adrenaline, noradrenaline, 5-hydroxitriptamine, DOPA, NMDA, AMPA, Ans l~;..;,;.. Il, Bradykinin, Bcm~ Opioid, Endothelin-1 Neuropeptide Y, TNF, PDGF, FGF and so on.
The following examples illustrate specific, non-limiting ~ s, or compound profiling nr dC s, in , -~ with the present invention.

WO ~1'~. 5.~, PCI/US97/13602 ~31 TE 671 cells, human medulloblastoma ~ATCC CRL 88051, naturally express endothelin subtype A receptor ETAR. This receptor belongs to the family of seven transmembrane spannin3 G-protein coupled receptors and is known to activate calcium mobilization in the cell Ll~t~ m upon binding to its specific agonist endoehelin 1, a 21-amino acid peptide. To ChdldCt~ the affinity of the agonist for the receptor,: ~. i ' methods measure the physiological response of the cell in the presence of several concentrations of the agonist or antagonist ~Sakamoto, A. et al., 1994, incorporated herein by II.~L~I 1 0 Fig. 9 illustrates a measurement of the activation of intracellular calcium mobilization ICa2+ measured in nanomolar cr '~dIi~ n) as a function of the ~ r I~dth of endothelin-1 IET-1, measured in picomolar 10 c- I~ , using the apparatus and method of the present invention. The apparatus was a negative pressure computer controlled unit as described above. This run was performed in the presence of several concentrations of ETAR specific - ,: .r, antagonist BQ 123 Ishown in nanomolar c~
TE 671 cells were prepared for use in the cell physiometer of the present invention by growing them in a T75 flask until c '1I ~ ~ The growth media was decanted and the cells were washed twice with Dulbecco s 15 phosphate buffered saline IDPBS~ and supplemented with a fresh DPBS containing 2 ~M FURA-2AM, which easily pr : alL~ into cell cytoplasm and is hydroly2ed there with nonspecific esterases to form FURA 2, a dye which is sensitive to the ionized form of calcium. After 30 min incubation, the cells were supplemented with the equal volume of fresh DPBS and incubated for further 30 min. The cells, now loaded with the dye, were detached from the flask bottom with 0.05% Trypsin-EDTA solution. The cells were then washed twice with DPBS, containing 0.1 % soybean 20 trypsin inhibitor, and r~ in calcium-free, magnesium-free and phenol red-free Dulbecco's modified Eagle s medium IDMEM).
Briefly, with reference to Figure 3, after calibration with minimum and maximum standards 1339, 337), ET-1 solution, an agonistic test compound, was mixed with DPBS buffer in continuously changing r ~r l r by the proportioning valve 311 and was directed through the p~l, Ou.ling valve 311, mixing zone 331, diverting valve 333, 25 and priming valve 341 into the mixing zone 343, where it was mixed with a s ~p - of TE-671 cells to provide a final concentration of cells 4 x 105 cellslml. At the same time, BQ 123, as a standard ~, : solution, was also directed to the mixing zone 331 at a predetermined concentration prepared by mixing BQ-123 with DPBS in the proportioning valve 327, in a ratio of 1:1 with the ET-1 solution. In mixing zone 343, the mixed flow of BQ 123 at constant corc : d60n,and ET-1 at a continuously changing in time c~ UL-' lr is mixed with cells in a ration 30 of 2:1. This gives three fold final dilution for each component of the flow passing through mixing zone 343 and eventually, detector 355. The proportioning valve 327, was programmed for the first, second and third runs to give final BQ-123 concentration of zero, 10 nM and 40nM, IJS~ c The prcpt tiL. ' 9 valve 311 was preprogrammed to give a fina1 ~m "at of ET1 in each run from 0 to 100 pM. Changes in intracellular calcium ion concentration in the TE 671 cells passing through the detector 315, were measured in real time as a function of ET 1 35 cr I.dIil using fluorescent dye FURA 2 as described above WO 98~5~3~ PCT/US97113602 The flow rate and the length of the reaction developiny line were chosen so that the time interval from the point of mixing the cells with the compound to the point of signal detection in the flow through optical cell was 40 seconds. The time required for the complete GGr ~lalion gradient run to generate the curves in Figure 9 was five minutes using the present invention. In contrast, in a ~ .. ' assay using the phosphatidylinositol turnover rate as an indicator of ETAR stimulation, it usually takes several hours to get only a few c~ I~dtion pùints on the response curve, and it would take an inordinate amount of time to generate a complete curve as shown in Figure 9.
Figure 10 is a logarithmic transformation of the data in Fig. 9, which is used in the art to calculate maximal cell response in the presence of ~ as well as EC50 value for agonist and PA2 value for antagonist. The EC50 value calculated from the activation curve without B~ 123 is equal to 10 pM and one can see that the a.,li.,l - curves in Figure 10 are parallel shifted to the right in the presence of Bû-123, which is known in the art as Lha~a~ IiL for competitive type of inhibition. The average PA2 value calculated from the two different Cc ~ lldliuns of BQ-123 is equal to 7.96. This ~ ~- ;pr ~ '~ to inhibition constant, K1, for BQ 123 of 11 nM. The EC50 and PA2 values are in close 19 L~ with the literature data IMasaki Ihara et al., 1992, is ~ rv l.e dted herein by ,~.f."~ ~e) Fig. 11 illustrates the inhibition of ET-1 induced intracellular calcium mobilization with Bn-123 in TE-671 cells. The same cell rh, ~Pter apparatus and cell preparation pr. 1l ~ was used here as in Example 1.
In this experiment, ET-1 was introduced at a constant cor : from the "standard" sipper nozzle of the inventive apparatus and B~-123 was Il~' Gd through the "c~ " nozzle of the apparatus. In this example, as in Example 1, the c~c~ '~àl run was five minutes and the reaction Time If rom mixing to d was forty seconds.
Briefly, with reference to Figure 3, after calibration with minimum and maximum standards 1337, 339), ET 1 solution, an agonist standard solution, was directed at a predetermined CG lldi prepared by proportioning valve 327 by mixing it with DPBS buffer, through priming valve 329, mixing zone 331, diverting valve 333 and priming valve 341 and then into mixing zone 343, where it was mixed with a -~-r ~i . of TE-671 cells to provide a final s ~alion of cells of 4 x 105 cellslml. At the same time, DPB in volumetric ratio of 1:1 to ET-1 solution was directed through intake port 309 and outlet port 312 Of r I ~ Q i'~ ling valve 311, priming valve 313 to mixing zone 331 and further through fluidics system to the detector 355. After the intracellular calcium G-n~ lldi- . in the presence of the standard agonist is stabilized, B0 123, as an antagonistic test compound, was directed from intake port 307 of proporlioning valve 311, instead of DPBS, to the mixing zone 331 at continuously changing ~ ~ Idt' ~ prepared by mixing Bn-123 with DPBS in, .pc i ~, valve 311, through the system in a volumetric ratio of 1:1 with the ET 1 solution. In mixing zone 343, the mixed flow of ET 1 at constant cDre ~,dlion, and BG
123 at a continuously changing in time s--ne ~ was mixed with cells in a ratio of 2:1. This gives three fold final dilution for each component of the flow passing through mixing zone 343 and, .i.a,.l 'Iy, detector 355. The W O 98/05959 rcTrusg7/l3602 ~33 proportioning valvs 327 was preprogrammed to give a final cv ~: aliOn of ET-1 of 40 pM at the detector. The proportioning valve 311 was preprogrammed to give final CDr'E l~dtion of BQ-123 from 0 to 300 nM. Changes in intracellular calcium ion sDnrl ~ ~n in the TE 6712 cells passing through the detector 355, were measured in real time as a function of Bû-123 ~Drl llal ) using fluorescent dye FURA 2 as described above.

Fig. 12 shows the Unull method" experiment design ILazareno & Birdsall, 1993, incorporated herein by reference). This example uses the same instrument and the same reagents as in Examples 1 and 2.
In this example, a first concentration gradient of ET 1 coming from the " l -' d nozzle 323 of the 10 inventive ~, ~ dluS is prepared with the buffer solution coming from the "compoundN nozzle. When the final ET-1 ~ dliun has reached the value of 40 pM, the gradient device 1 keeps the ET 1 -- t~alion constant and gradient device 2 starts raising the cDr l ll 'il of the BQ 123 coming from the Vcompound" nozzle. One can switch the agonist yradient over to an antagonist at any, ~d~: ~ 1C~ CF - ~. of the agonist.

The devices of the present invention may be used to determine the pattern of cell surface receptors expressed in one or more cell types as follows. Each of the different cell types to be tested is placed in one of a plurality of cell s ~pr- - r~S~ , or, if a single cell type is to be examined, an apparatus with a single cell - reservoir may be used to hold the cells. The effects of one or more test agents known to influence the activity of particular receptors are measured by combining the cell suspensions with the test agents to form a test 20 mixture, directing the test mixture through a detection zone, and ~ g the cellular response using the procedures described above. Preferably, the test agents comprise one or more receptor agonists. However, in some embodiments the test agent may be an a 11~ : or a mixture of an agonist and an antagonist IIIYA, PEEASE
CONFIRM THIS SENTENCEI.
The cellular response may be a~li.ai ~1 of cellular activity if the test agent is a receptor agonist.
25 Alternatively, if the test agent is an ~ ' v nk ~ the cellular response may be inhibition of cellular activity. Likewise, if the test agent is a combination of an agonist and an antagonist the cellular response may be a reduction or absence ot the activity normally obtained in response to the agonist due to the presence of the ~ i ~v : If the test agent is a test compound the cellular response would be the response expected to be observed with that agent.
If a cellular response is observed with an agonist, - g - ~:, agonistlantagonist mixture or test agent 30 known to interact with a particular receptor, the cell type being evaluated pos~ ~9 that receptor. By examining the effects of several agonists, 3rt1g~r s, agonistlantagonist mixtures or test compounds on cellular ph~
the spectrum of receptors on the cell type being evaluated may be determined. If multiple cell types are to be tested, this process is repeated for each of the cell types in the series to determine the spectrum of receptors present in each cell type. To facilitate the analysis of multiple cell types, each of the cell types may be placed in 35 one of a plurality of cell s , r~a~O~ ~ in the devices described above.

W O 98/05959 PCT~US97/13602 ~34 The devices of the present invention may be used to confirm that a test agent influences the activity of a particular receptor as follows. The test agent may be an agonist, an antagonist, or a mixture of an agonist an antaganist. The test agent may be an agent which is known to influence the activity of the receptor or an agent whose effect on the receptor is unknown. A cell type lacking the receptor serves as a negative control and is placed in one of the cell s ~pon~;,n reservoirs. A cell of the same cell type as the negative control which has been engineered or induced to express the receptor is placed in another cell suspension reservoir. The effects of one or more test agents is assessed in each of these cell types by contacting the cells with the test agent to form a negative control mixture and a test mixture, directing the negative control mixture and the test mixture through a detection zone, and measuring the cellular responses of the negative control cells and the engineered or induced cells.
10 A difference in the response of the engineered or induced cells relative to the response of the negative control cells indicates that the test agent has an effect on the activity of the receptor.
The negative control cell is commonly referred to as the "host cell" and the engineered cell is commonly referred to as the "transfected cell" by those in the industry. To obtain engineered cells, the gene encoding the receptor may be Il.' d into a cell of the same type as the negative control cell using techniques such as 15 l~ calcium ,'- ,' mediated l~ 'e: n, el~ r . ~. viral infection, tra pr n, or other techniques familiar to those skilled in the art. Expression of the receptor gene may be directed by a variety of vectors familiar to those skilled in the art. AIIL~ ly, the cells may be induced to express the receptor through treatment with chemical agents or bioactive agents such as growth factors, cytokines, cell diff-,.~'i.~: factors, or other agents known to those skilled in the art.

The activity of a particular receptor may also be influenced by the cl,a,~Ll~ .s of the particular cell type in which it is ~AI,.essEd. Thus, the same receptor may exhibit vd(ial 1~ in activity between different cell types.
The present invention may also be used to assess such cell type d, ~ : differences in receptor activity. Each of the cell types to be assessed is placed in a cell - , reservoir and the effects of one or more test agents on the activity of one or more receptors on the cells are determined by contacting the cells with one or more test agents to form test mixtures, directing the test mixtures through a detection zone, and measuring the cellular response of the cells as the test mixtures flow through the detection zone. The test agents may be receptor known receptor agonists, known antagonists, mixtures of a known agonist and a known antagonist, or compounds whose activity is unknown as described above. This process is performed for each of the cell types to be tested to determine the nature and 1nS ~ P of the cellular response in each cell type. To facilitate the analysis of multiple cell types, each of the cell types may be placed in one of a plurality of cell ~ r.~ in the devices described above.
Fig. 13 shows an algorithm that may be used in the invention to perform cell mapping and cell receptor "fingerprinting". First, cells are mixed with standard agonist IsteP 1301) and the mixture is provided to a detector (step 1302). Next, the apparatus determines if this standard agonist, upon contact with the cells, tri~qgers any cell response IsteP 13031. There are two possibilities: either the standard agonist does not produce any response IN0~, WO ~ 3)~ PCT~US97/13602 ~35 or it induces the cell response (YES). Cell response is determined by monitoring the signal from the detector for the particular standard agonist being c. ' ' The control system first calibrates to establish a baseline and a maximal response, and any signal from the detector falling between those values is considered to be a positive response.
If the standard agonist causes no cell response, IN0), as measured by the monitoring part of the apparatus, 5 the system increments an agonist counter (step 1309) which indicates that a next standard agonist in a specified set of agonists will be tested with a particular cell line. The system then determines if all available agonists have been tested Istep 1305). If all standard ayonists have not been tested with a particular cell line, fresh cells from the original cell line are then supplied to the system (step 1306). These new cells are then automatically brought into contact with the next standard ayonist from the predetermined set of agonist solutions Istep 1301). Each 10 agonist solution in the set contains one or more ingredients that are known to initiate cell response through the stimulation of a known cell receptor, ion pump or ion channel molecules. The 9Pt- d~ will keep repeating an admixture of different standard agonist substances with the cells until it detects that the cell response is triygered with a particular standard agonist, or until all agonists available to the machine have been tested.
If in step 1303, it is determined that a particular agonist triggers a cell response, the process is switched 15 over to the potency mode of action IsteP 1306) in which the cellular response at various concentration levels of the standard agonist is recorded. After step 1306 has been completed, the process then records the potency parameters, or profile, lI,~ ..i v the level of cellular response produced in a given cell line at various ro1.e tldi- - levels of the standard ayonist (step 1307). The process then once ayain determines if all available standard ayonists have been tested IsteP 1304). If all of the available agonists have been tested (YES), the system then determines if all 20 the available cell lines have been tested (step 1308). If the answer to this question is yes the process is complete Istep 1312).
If in step 1308 it is determined that not all available cell lines have been tested, a cell counter is incremented lstep 1310) which indicates that a next cell line in a specified set of cell lines will be tested. Next, the standard agonist counter is reset ~step 1310) which indicates that the new cell line will be tested with all 25 available standard ayonists, startiny with a first designated agonist in the specified set of standard ayonists. Cells from the new cell line are then supplied to the system Istep 1311) and s barr1 ntly mixed with the specified agonist ~step 1301). The above process steps 1301 1311 are then repeated. In one embodiment, the apparatus will keep repeatino an admixture of different standard agonist substances with the cells until it detects that all agonists available to the apparatus have been tested with all cell lines available to the apparatus.
Fig. 14 shows an algorithm that may be used in the invention to perform transfected receptor chal~Ll~ ion, particularly, of an orphaned receptor. First, in step 1401, an apparatus supplies cells lla.lsI~.~lod with a gene which produces a known orphan receptor, and mixes them with a standard agonist Istep 1402). The mixture is provided to a detector IsteP 1403). Next, the 1, r ~lu~ determines if this standard agonist, upon contact with the cells, trigyers any cell response Istep 1404). There are two possibilities: either the standard agonist does - 35 not produce any response IN0~, or it induces the cell response IYES). Cell response is dPtermined by monitoring the signal from the detector for the particular standard agonist being evaluated. The control system first calibrates to .... . . . . . ..

W O 98/05959 PCTrUS97/13602 ~36 establish a baseline and a maximal response, and any signal from the detector falling between those values is considered to be a positive response.
If the standard agonist causes no cell response, IN0), as measured by the monitoring part of the apparatus, the system increments a standard agonist counter ~step 1405) which indicates that a next standard agonist in a 5 specified set of standard agonists will be tested with the transfected cells. The system then determines whether all available standard agonists have already been tested with the l~ d cells ~step 1406). If not, the process moves back to step 1401 in which fresh cells from the same transfectdd cell line are automatically brought into contact with the next standard agonist from the predetermined set of agonist solutions (step 1402). Each standard agonist solution in the set contains one or more ingredients that are known to initiate cell response through the 10 stimulation of a known cell receptor, ion pump or ion channel molecule. The apparatus will keep repeating an admixture of different standard agonist b~: e with the l~ d cells until it detects that the cell response is triggered with a particular standard agonist, or until all agonists available to the machine have been tested.
If in step 1404 it is determined that the contact of the l, ~ d cells with the standard agonist does initiate the cell response, (YES), as measured by the . t i v part of the , p, dlUS, the process then supplies host cQlls (step 1407) to be mixed with the same standard agonist (step 1408). This mixture is then supplied to the detector (step 1409) for measurin~ the cellular response produced by the standard agonist on the host cells. Next, the system determines if the standard agonist produces a cellular response in the host cell (step 1410). If a cell response is detected IYES) the system then increments the standard agonist counter Istep 1405) after which the system determines if all available agonists have been tested (step 1410). If all available agonists have been tested, the process is complete (step 1412). If all available standard agonists have not been tested, the process moves back to step 1401 where fresh transfected cells are supplied to be mixed with the next standard agonist in the set of standard agonists (step 1402), and the process steps described above are repeated.
If in step 1410, no cell response is produced in the host cell by the standard agonist the process has detected a difference between the host cell and the 1, ~c.,l~d cell. At this point, the process is complete (step 1412). In one embodiment, the apparatus will keep repeating an admixture of different standard agonist substances with the cells until it detects that a particular agonist stimulates a response in the cells l- ~s~L~l~d with an orphaned receptor, and do not stimulate a response on host cells or until all standard solutions available to the machine have been tested.
A continuous flow diagram of the presently preferred cell mapping and cell receptor "fingerprinting" mode with the negative pressure fluidics system of Fig. 3 is shown on Figs. 15a - 15f. To practice this mode, diverting valve 347 is replaced by a mu': ' - ' diverting valve 347 which allows multiple ,~",~ s, each with a different cell suspension, to be ccr".~ d p- dlLI~ to the common outlet 346 so that once an experiment with a particular cell line is complete, the valve 347 may switch over to a next cell reservoir.
The screening program starts from a state 1500 and enters state 1502 where it sets all parameters to their initial values. These initial parameters include, but are not limited to, zeroing counters, the internal initialization of software variables and subroutines.

WO ~X~'~3,~ PCTrUS97/13602 ~37 Once the initial parameters are set, the software requests an operator to enter values for a maximum numbet of standard agonists, LMAX~ and a maximum number of cell lines, CMAX, to be used in the experiment IStep 1503). The autosamplers 301 and 315 (Fig.3~ position the intake nozzles 307 and 323 at the ~r,., ding "zero"
positions occupied by a wash buffer reservoir 305 (Step 1504), and turns off proportioning valves 311 and 327, diverting valves 333, 335 and 347, as well as priming valves 313, 329, 341 and 345 IStep 1506). In a turned off state, priminy valves 313, 329, 341 and 345, are normally opened, proportioning valves 311 and 327 connect, respectively, their outlets 312 and 328 with cs 1~ pr ding "normally opened" intake ports 309 and 325, diverting valve 335 connects its common outlet 334 with "normally opened" intake port 336, diverting valve 333 connects its common outlet 342 with the "normally opened" intake port 332, and multichannel diverting valve 347 connects its common outlet 346 with the first intake port 348 c~ ~d with the reservoir containing first cell suspension under in.. ~ig-t-After the system has been initialized, pump 357 is started IStep 1508). This will force the wash buffer from the "zero" p eo ,~ of both autosamplers 301 and 315 to run through nozzles 309 and 325 and outlet ports 312 and 328 of proportioning valves 311 and 327, respectively, priming valves 313 and 329, mixing zone 331, intake port 332 and outlet port 342 of diverting valve 333, priming valve 341, mixing zone 343, a reaction ' ~ r V line 353, detector 355, pump 357 and then into a drain container 359. In mixing zone 343 this flow is mixed with the cell suspension 349 coming from intake tubing 348 connected with the first cell line, and an outlet port 346 of the multichannel diverting valve 347 through priming valve 345. Thus, the total flow passing mixing zone 343, is a sum of three flows, a first flow coming from the proportioning valve 311, a second flow coming from the valve 327 and a third flow coming from the multichannel diverting valve 347. During this step, both proportioning valves supply wash buffer 305 from the reservoirs located at "zero" position of each autosampler in such a way that the final flowing mixture consists of one part of cell , 349 and two parts of wash buffer 305.
After a delay determined in step 1509, which is needed for the flow of the mixture of cell suspension 349 and wash buffer 305 to stabilize, detector 355 registers a basal signal produced by the cells alone IStep 1510).
After the basal signal is registered, it is saved as a reference signal, BSj IStep 1512) and computer 123 IFig. 1 increments counter L IStep 1514).
The numerical content of the incremental counter L is increased by one each time it is triggered. The numeric value of the counter determines the position of the nozzle of intake port 323 of autosampler 315 that samples the sets of standard agonists 319 to be tested. Next, in step 1516, the numerical content, Lk, of the incremental counter L is compared with the, r~... '~ entered (Step 1503) value of maximal number of the standard agonists to be tested, LmaX .
If Lk does not exceed LmaX~ autosampler 315 positions nozzle 323 into the standard agonist reservoir located at the Lk position of a rack of standard agonists 319 IStep 15181. Next, proportioning valve 327 is - 35 switched ON to open its "normally closed" intake port 323 to the common output port 328 (Step 1520). During Step 1520, the combined flow in mixing zone 343 and a~lo.~ dj in detector 355, is composed of one portion of W O ~ 5~ PCT~US97/13602 ~38-buffer 305 coming throu~h nozzle 309 of proportioning valve 311, one portion of the standard agonist 319 to be tested, coming from nozzle 323 of proportioning valve 327 and one portion of particular cell suspension 349 coming from one of the multitude of the cell IL~ through the intake tubing 348 to the output port 346 of multichannel diverting valve 347. After a delay provided by Step 1521, which is needed for the mixing process to stabilize, 5 detector 355 registers a standard agonist induced signal, SLk, produced by given cells in the presence of the given standard agonist, Lk IStep 1522). After the SLk signal is re~istered, its value is saved as a value, SLk ISteP 1523) and the apparatus proceeds to step 1530 where the proportioning valve 327 is turned OFF, thus connecting normally opened intake port 325 with buffer 305. In step 1532, autosampler 315 moves its nozzle 323 to a zero position occupied with reservoir filled with washing buffer. Proportioning valve 327 is turned ON Step 1534), connecting the normally closed inlet port 323 with outlet port 328. Delay, determined in step 1536, allows for the flow path composed of intake port 323 and outlet port 328 of the proportioning valve 327, priming valve 329, mixing zone 331, intake port 332 and outlet port 342 of the diverting valve 333, priming valve 341, mixing zone 343, reaction developing lines 353 and detector 355 to be washed clean of the remains of the previous compound. After the delay, the r-~r5 i )r v valve 327 is turned OFF in the step 1538, thus bringing the system into original state..
Next, the value of the standard agonist induced signal, SLk, is compared with the value of the reference basal signal, BSj ISteP 1544). If the SLk signal is greater than the BSj signal, the computer 123 IFig. 1) will "flag"
the ~ ,. po ding standard agonist as "positive" IStep 1540), which means that the particular cells express a receptor which is stimulated by the particular agonist. If SLk is not greater than BSp computer 123 will "flag" the corresponding standard agonist as "negative" IStep 1542), which means that the particular cells do not contain a 20 receptor which can be stimulated by the particular agonist.
Each time the condition, "SLk > BSj", is satisfied, the program flow will go through loop K-O initiating thegradientmodeofaction. Inthegradientmode,step1550movesa~ ,'e 315atapositiono ll pcnding to the Lk value determined in step 1514. After that, the, ~p i ~, valve 327 is turned on to operate in the gradient mode IsteP 1552). In step 1554, the dose response curve is ", ~..Ld for the standard agonist stimulated signal. Step 1556 - ' ' Its a potency parameter that could be determined as some specific r ~, Kr of the agonist causing a specific level of a signal. The data are saved in step 1558.
After the data are saved, the proportioning valve 327 is turned OFF ~step 1560), autosampler 315 is moved to a zero position (step 1562),, ~, ~iuning valve 327 is turned ON (step 1564) and, after a washing delay period determined in step 1566, the valve 327 is turned OFF again (step 1568). After step 1568 has been performed, the process goes back, through line 0, to the incremental counter L IsteP 1514).
If in step 1544, the SLk signal is not larger then BSp then step 1542 flags the standard agonist as Ar ~9, .a-, meaning that the particular cells do not have the particular receptor expressed, and the program flow is returned, through line M, to the step 1514.
If in step 1516 IFig. 15b), Lk exceeds LmaX~ indicating that all agonists have been tested, the process moves through line N to step 1570 were a computer 123 ;.. ~.~ a cell counter C. The numeric value of the counter is compared Istep 1572) with the maximum number of cells entered in step 1503. If the counter value, Cj does not ... ..

W O ~U3,3~ PCTrUS97/13602 ~39 exceed CMAX, then controller L is set to zero in step 1574, multichannel diverting valve 347 is switched to the next position, connecting a new cell reservoir with the output 346, and the process returns to step 1509 (Fig. 15a) through line P. If in step 1572 the Cj value is higher then CMAX~ then the process moves ~o step 1578, where autosampler 315 is moved to its zero position and, proportioning valve 327 is turned ON ISteP 1580). After a washing delay, determined in step 1582, the valve 327 is turned OFF IStep 1584) and pump 357 is turned OFF ISteP
1586), and the whole program is stopped in step 1588.
A continuous flow diagram of the presentlv preferred orphaned receptor detection mode with the negative pressure fluidics system of Fig. 3 is shown on Figs. 16a ~ 16g. To practice this mode, diverting valve 347 is replaced by a multichannel diverting vaive 347 which allows for at least two It~3~ 349, containing host cells, native or transfected with an empty vector, and cells transfected with the orphaned receptor of intersst, to be sr ' ~i~, a~ately to the common outlet 346.
The program begins from a start state 1600 and enters state t602 where it sets all parameters to their initial values. These initial parameters include, but are not limited to, the internal initialization of software variables and subroutines. Then the program requests an operator to enter a maximum number of agonists to be tested (Step 1603).
Once the initial parameters are set up, the autosamplers 301 and 315 IFig. 3) move their intake nozzles 307 and 323 to the ce " pr ding "zero" positions occupied by a wash buffer reservoir 305 IStep 1604).
Proportioning valves 311 and 327, diverting valves 333, 335 and 347, and priming valves 313, 329, 341 and 345 are turned OFF IStep 1606). In that turned off state, priming valves 313, 329, 341 and 345, are normally opened, the outlets 312 and 32B of r ~ ri' ling valves 311 and 327 are cr ~tcd". pc ~ .ly, with corresponding "normally opened" intake ports 309 and 325, outlet 334 of diverting valve 335 is c~ : ~ with "normally opened"
intake port 336, common outlet 342 of diverting valve 333 is c~ - I~d with the "normally opened" intake port 332.
After the system has been initialized, diverting valve 347 is set to connect its common outlet 346 with the reservoir containing host cells, position 1, ISteP 1608). The pump 357 is started to pump liquid IStep 1610~. This will force the wash buffer from the "zero" positioned reservoirs of both autosamplers 301 and 315 to run through nozzles 309 and 325 and outlet ports 312 and 328 of proportioning valves 311 and 327, respectively, priming valves 313 and 329, mixing zone 331, intake port 332 and outlet port 342 of diverting valve 333, priming valve 341, mixing zone 343, a reaction developing line 353, detector 355, pump 357 and into a drain container 359. In mixing zone 343, this flow is mixed with the host cell suspension coming from intake tubing 348 and an outlet port 346 of the multichannel diverting valve 347 through priming valve 345. Thus, the total flow passing through mixing zone 343 is a sum of three flows, a first flow coming from the proportioning valves 311, a second flow coming from r ~~ _ 1 ing valve 327 and a third flow coming from the ' ' ' diverting valve 347. During this step, both proportioning valves supply washing buffer 305 from the reservoirs located at "zero" position of each autosampler in such a way that the final mixture consists of one part of the host cell - i, and two parts of washing - 35 buffer.

WO ~J~5,~ PC~rrUS97/13602 ~40 After a delay determined in step 1612, which is needed for the flow of the mixture of cell s r ~ and wash buffer to stabilize, detector 355 registers a basal signal produced by the host cells ~Step 1614). After the basal signal is registered, it is saved as a reference signal, ~BS)HC (Step 1616). In step 1620, multichannel diverting valve 347 is switched over to position 2 to supply the outlet 346 with the s , of the transfected cells. After a delay determined in step 1622, which is needed for the flow of the mixture of new cell suspension and wash buffer to stabilize, detector 355 registers a second basal signal produced by the ~lan~fect~d cells ~Step 1624). After the basal signal is reg ~d, it is saved as a reference signal, IBS)TC (Step 1626) and the pump is turned OFF ~Step 1628). The computer 123 ~Fig. 1) triggers the incremental counter L (Step 1630~.The numerical content of the incremental counter L is increased by one each time it is triggered, thus 10 determining the position of the nozzle of intake port 323 of autosampler 315 which samples the sets of agonists 319 to be tested. Next, the numerical content, Lk, of the incremental counter L is compared with the maximum number of the standard agonists to be tested, LmaX (Step 1632).
If Lk does not exceed LmaX~ autosampler 315 positions nozzle 323 into the agonist reservoir located in the rack 319 at the position determined by the numerical value Lk ~Step 1634). The pump 357 is turned ON in step 15 1636 and proportioning valve 327 is switched ON to open its "normally closed" intake port 323 to the common outlet port 32B (Step 1638~ thus allowing the particular agonist flows through the priming valve 329, mixing zone 331 intake port 332 and outlet port 342 of the diverting valve 333, priming valve 341, mixing zone 343, where it is mixed with the suspension of l, 'c~ d cells coming from outlet port 346 of multichannel diverting valve 347 and priming valve 345. After being mixed with the transfected cells in mixing zone 343, the liquid flow goes through 20 reaction ~ ' r- J lines 353, into detector 355 and further into waste reservoir 359. After a delay provided by Step 1640, which is needed for the mixing process to stabilize, detector 355 registers an agonist induced signal, (SLk)Tc, produced by the particular orphaned receptor transfected cells in the presence of the given agonist, Lk ISteP
1642). After the signal is ~ .ed, its value, ~SLk)TC, is saved in step 1643 and the a,, dt~.~ proceeds to step 1644 where proportioning valve 327 is turned OFF, thus connecting normally opened intake port 325 with buffer 25 305 to outlet port 328. In step 1646, autosampler 315 moves its nozzle 323 to position zero occupied with reservoir filled with washing buffer. Proportioning valve 327 is turned ON again (Step 1648) connecting inlet 323 with outlet 328. A delay, dLl 1 -~ in step 1650, allows for the flow path, composed of intake port 323 and outlet port 328 of the proportioning valve 327, priming valve 329, mixing zone 331, intake port 332 and outlet port 342 of the diverting valve 333, priming valve 341, mixing zone 343, reaction ;'~..' r- V lines 353, and detector 355 to be washed out of the remains of the agonist. After the delay (Step 1650), the proportioning valve 327 is turned OFF in step 1652 and the pump 357 is turned OFF in step 1654, thus bringing the system into its original state.
Next, the value of the registered signal for lldil~le~l~d cells, (SLk)Tc, is compared with the value of the corresponding reference basal signal, ~BS)TC ~Step 1658). If the agonist induced signal, ~SLk)Tc, is not greater than the basal value, ~3S)TC~ the computer 123 ~Fig. 1) will "flag" the ~ " pc ' agonist, Lk as "negative" ~Step 1660), which means that the cell does not contain a receptor which can be stimulated by the particular agonist, and the flow returns through line M back to the incremental counter L IStep 1630). If the lSLk)TC signal is greater than WO ~ ,3, PCT/US97/13602 ~41 the IBS)TC signal, the computer 123 will "flag" the CD~I_ pr ding agonist as "POSITIVETC" (Step 1656), which means that the Il '~ct.!d cells do express a receptor which can be stimulated by the particular agonist.
The next task is to determine if the host cells also express this receptor to distinguish it from the orphaned receptor which should be ~AI.ltssLd only in I~ 'e l~d cells. To do so, after the standard agonist Lk has been flagged as positive in the step 1656, the program flow enters loop K O, where, in step 1662, the autosampler 315 moves its nozzle 323 to the position CG,I~, ~rding to the value obtained in counter L, Lk. Step 1664 switches diverting valve 347 to position 1, which connects host cell s-~sr ~ reservoir 349 with mixing zone 343 through common output 346,through intake port 348 of the multichannel diverting valve 347, and through priming valve 345.
Steps 1666 and 1668 turn valve 327 and pump 357 ON, I- " ~;.. 'y, allowing the buffer 305 coming from intake 309 of the autosampler 301, and standard agonist Lk, coming from the intake 323 and output port 328 of the proportioning valve 327, to be brought together in mixing zone 331 with the subsequent mixing of this solution with the host cells in the mixing zone 343. After a delay determined by step 1670, which is needed for the stream of the mixture of the cells and agonist to stabilize, detector 355 registers the agonist induced signal in the host cells, ISLk)HC (Step 1672).
After the signal is ~ d, it is compared ISteP 1674) with the r ,., 3nding basal signal, IBS)HC, measured in step 1614. Da~ ' 5 on the results of the comparison, the agonist Lk is marked either as ''POSITIVETclPOSlTlVEHc'' IStep 1676) or as ''POSITIVETclNEGATlVEHc'' lStep 1678). In either case, the data are saved in step 1680 for further c~, ' ; by the r~ed.che .
After the data are saved, the multichannel diverting valve 347 is switched over to position 2 in step 1682, the proportioning valve 327 is turned OFF (step 1684), autosampler 315 is moved to the zero position Istep 1686), proportioning valve 327 is turned ON (step 1688) and, after a washing delay, determined in step 1690, is turned OFF again Istep 1692~ and the pump 357 is turned OFF (Step 1694). After the step 1694 has been performed, the flow goes back to the incremental counter L Istep 1630) through the line 0.
If in step 1632 IFig. 16c), Lk does exceed LmaX~ then the process moves to step 1700 IFig. 169) where the counter L is set to zero. Next, the autosampler 315 is moved to the zero position in step 1702, the proportioning valve 327 is turned ON in step 1704 and after a washing delay determined in step 1706, both proportioning valve 327 and pump 357 are turned OFF in steps 1708 and 1710 ,., e ti.~'), after which the program stops in step 1712.
Although the invention has been described in detail with reference to certain particular embodiments thereof, it will be understood that any ~ariai ne and modifications apparent to those of skill in the art will still fall within the spirit and scope of the invention. Other embodiments not ,~r 'iL 11~ described herein may fall within the spirit and scope of the present invention as provided by the following claims.

Claims (93)

WHAT IS CLAIMED IS:

1.A method for identifying compounds having biological activity, comprising the steps of:
(a) combining in a mixing chamber a homogeneous suspension of living cells with a test compound having an unknown cellular effect to form a test mixture;
(b) directing the test mixture through a conduit from the mixing chamber to a detection zone;
(c) directing the test mixture through the detection zone; and (d) measuring a cellular response of said suspended cells to said test compound as said test mixture is flowing through said detection zone.

2. The method of Claim 1, further comprising the steps of:
(e) combining a homogeneous suspension of said cells with a standard compound having a known effect on said cellular response of said cells to form astandard mixture;
(f) directing the standard mixture through the conduit from the mixing chamber to the detection zone;
(g) directing the standard mixture through the detection zone; and (h) measuring the cellular response of said cells to said standard compound.

3. The method of Claim 2, wherein said standard compound and said test compound are simultaneously mixed with said cells in said combining steps, and said measuring step detects said known effect or an alteration of said known effect.

4. The method of Claim 3, wherein said standard compound is an antagonist of said cellular response.

5. The method of Claim 3, wherein said standard compound is an agonist of said cellular response.

6. The method of Claim 3, in which steps (a) and (e) are performed simultaneously;
steps (c) and (g) are performed simultaneously; and steps (d) and (h) are performed simultaneously using a single suspension of said cells.

7. The method of Claim 3, in which steps (a), (b), and (c) and (d) are performed first, and then steps (e), (f), (g), and (h) are performed, wherein the test compound is added together with the standard compound in step (e).

8. The method of Claim 7, in which said cellular response is detected in step (d) to indicate that said test compound is active to generate said response, and in which said standard compound is an antagonist, whereby a decrease in said cell response from step (d) to step (h) is indicative that said test compound is an agonist of said known effect.

9. The method of Claim 7, in which said cellular response is not detected in step (d), indicating that said test compound is not active to generate said response, and in which said standard compound is an agonist, whereby an alteration of said known effect detected in step (h) is indicative that said test compound is an antagonist of said know effect.

10. The method of Claim 7, performed automatically under the direction of a programmable computer on a plurality of test compounds and a plurality of standard compounds;

in which a successive series of antagonists are automatically added as said standard compound in step (e) if said cellular response is detected in step (d) to indicate that said test compound is active to generate said cellular response, whereby a decrease in said cellular response detected in step (h) is indicative that said test compound is an agonist of said known effect; and in which a series of agonists are automatically added as said standard compound in step (e) when said cellular response is not detected in step (d), whereby an alteration of said known effect detected in step (h) is indicative that said test compound is an antagonist of said known effect.

11. The method of Claim 10, further comprising the steps of:
if step (h) indicates that said compound is an agonist of said known effect, then automatically determining the concentration dependence of agonist activity of said test compound by repeating steps (a), (b), (c), (d), (e), (f), (g), and (h) while varying the concentration of said test compound and said standard compound and recording resultant changes in said cellular response; and if step (h) indicates that said compound is an antagonist, then automatically determining the concentration dependence of inhibition of said cellular response in the presence of said agonistic standard compound by repeating steps (e), (f), (g), and (h) while varying the concentration of said test compound and said standard compound and recording resultant changes in said cellular response.

12. The method of Claim 11, further comprising the step when step (h) indicates that said compound is an antagonist of:
(i) automatically determining the concentration dependence of cell response activation by repeating steps (e), (f), (g), and (h) for a zero concentration of said test compound while varying the concentration of said standard compound and recordingresultant changes in cellular response, and then repeating this step (i) for different concentrations of said test compound.

13. The method of Claim 11, further comprising the step when step (h) indicates that said compound is an agonist, of:
(j) automatically determining the concentration dependence of cell response activation by repeating steps (e), (f), (g), and (h) for a zero concentration of said standard compound while varying the concentration of said test compound and recording resultant changes in cellular response, and then repeating this step (j) for different concentrations of said compound.

14. The method of Claim 11, in which the varying of the concentration of the test compound is done continuously.

15. The method of Claim 11, in which the varying of the concentration of the test compound is done stepwise.

16. The method of Claim 11, in which the varying of the concentration of the standard compound is done continuously.

17. The method of Claim 11, in which the varying of the concentration of the standard compound is done stepwise.

18. The method of Claim 11, further comprising the step of graphically displaying the recorded changes in said cellular response.

19. The method of Claim 1, wherein said cellular response is a change in intracellular ion concentration.

20. The method of Claim 19, wherein said ion is calcium, magnesium, proton, sodium, or potassium.

21. The method of Claim 9, wherein said ion is detected using an intracellular dye.

22. The method of Claim 21, wherein said dye is fluorescent.

23. The method of Claim 1, further comprising the step of repeating steps (a)-(d) on each of the cell types in a series of cell types to be tested until the effect of said test compound has been measured in each cell type of said series of cell types.

24. The method of Claim 23, further comprising the steps of:
combining a homogenous suspension of each of said cell types in said series of cell types with a standard compound having a known effect on said cellular response of said cells to form a series of standard mixtures;
directing said series of standard mixtures through the conduit from the mixing chamber to the detection zone;
directing said series of standard mixtures through the detection zone; and measuring the cellular response of each of said cell types to said standard compound.

25. The method of Claim 23 wherein the standard compound and the test compound are simultaneously mixed with said cells in said combining steps and said measuring step detects said known effect or an alteration of said known effect.

26. The method of Claim 25 wherein said standard compound is an antagonist of said cellular response.

27. The method of Claim 26 wherein said standard compound is an agonist of said cellular response.

28. An apparatus for automatically measuring the effect of a plurality of test compounds on living cells, comprising:
a test compound sampler for sequentially providing samples of multiple test compounds;
a cell suspension input for providing a suspension of living cells;
a mixing zone, coupled to the test compound sampler, for receiving the samples of the test compounds from the test compound sampler, receiving the suspension of living cells from the cell suspension input and mixing each test compound with the suspension of living cells so as to provide a respective test solution for each test compound;
a detector, coupled to the mixing zone, for receiving each respective test solution and measuring a cellular response of the suspended cells for each said test solution; and a conduit establishing a flow channel between the mixing chamber and the detector, such that each said test solution flows from the mixing chamber to thedetector.

29. The apparatus of Claim 28 further comprising a standard compound sampler, coupled to said mixing zone, for providing a sample of a standard compound having a known effect on said cellular response of said suspended cells, wherein said mixing zone receives the sample of the standard compound from the standard compound sampler and mixes the standard compound with the suspended cells and said detector measures the cellular response of the suspended cells to the standard compound.

30. The apparatus of Claim 29 wherein said mixing zone simultaneously mixes saidtest compound and said standard compound with said suspended cells and said detector detects said known effect or an alteration of said known effect.

31. The apparatus of Claim 29 further comprising:
a first gradient device, coupled to said test compound sampler, for automatically adjusting the concentration level of said test compound transferred to said mixing zone from said test compound sampler; and a second gradient device, coupled to said standard compound sampler, for automatically adjusting the concentration level of said standard compound transferred to said mixing zone from said standard compound sampler.

32. The apparatus of Claim 31 further comprising a switching valve, coupled to the first and second gradient devices at an input of the switching valve and coupled to said mixing zone at an output of the switching valve, for selectively switching the flow of a concentration of said test compound or a concentration of said standard compound or both to the mixing zone where the test compound and/or the standard compound is then mixed with said suspension of cells.

33. The apparatus of Claim 32 further comprising a calibration unit, coupled to said switching valve, wherein the switching valve also selectively switches the flow of a calibration solution provided by the calibration unit into said mixing zone where the calibration solution is mixed with said suspension of cells.

34. The apparatus of Claim 33 wherein said conduit comprises reaction developinglines coupled to the output of said mixing zone, for receiving a mixture of said cell suspension mixed with either said test compound, said standard compound or said calibration solution, and providing a flow path for the mixture such that there is adequate time for the suspension cells to react with the test compound, the standard compound or the calibration solution, wherein the reaction developing lines is further coupled to the input of said detector which receives the mixture from the reaction developing lines.

35. The apparatus of Claim 34 wherein said detector detects changes in intracellular ion concentration.

36. The apparatus of Claim 35 wherein said ion is calcium, magnesium, proton, sodium, or potassium.

37. The apparatus of Claim 35 wherein said detector detects changes in intracellular ion concentration using an intracellular dye.

38. The apparatus of Claim 37 wherein said dye is fluorescent.

39. The apparatus of Claim 34 further comprising:
a controller, coupled to said first and second gradient devices, said test compound sampler, said standard compound sampler and said switching valve, for controlling their operation; and a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, wherein the computer is also coupled to said detector in order to send and receive cell response measurement signals to and from the detector.

40. The apparatus of Claim 28 wherein said test compound sampler is an automatedrobotic sampler capable of selecting a specified test compound from a library of test compounds.

41. The apparatus of Claim 40 further comprising:
a controller, coupled to said test compound sampler, for controlling the operation of the test compound sampler; and a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, thereby controlling the selection and retrieval of test compounds by the test compound sampler from said test compound library.

42. The apparatus of Claim 41 further comprising a gradient pump having an inputand an output, coupled to said test compound sampler, for adjusting the concentration level of said test compound transferred to said mixing zone from said test compound sampler, wherein:
said test compound sampler comprises:
a first intake nozzle for receiving said specified test compound;
a second intake nozzle for receiving a buffer solution; and wherein said gradient pump is coupled to the first and second intake nozzles andreceives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound.

43. The apparatus of Claim 42 further comprising a standard compound sampler forproviding a sample of a standard compound to said mixing zone.

44. The apparatus of Claim 43 wherein said standard compound sampler is an automated robotic sampler capable of selecting a specified standard compound from a library of standard compounds.

45. The apparatus of Claim 44 further comprising a second gradient pump having an input and an output, coupled to said standard compound sampler, for adjusting the concentration level of said standard compound provided to said mixing zone from said standard compound sampler, wherein:
said standard compound sampler comprises:
a third intake nozzle for receiving said specified standard compound;
a fourth intake nozzle for receiving a buffer solution; and wherein said second gradient pump is coupled to the third and fourth intake nozzles and receives specified concentrations of the standard compound by adjusting the amount of standard compound and buffer solution received by the third and fourth intake nozzles, respectively, wherein the buffer solution is a diluting agent of the standard compound.

46. The apparatus of Claim 45 further comprising a second mixing zone coupled tothe outputs of the first and second gradient pumps, for receiving and mixing said specified concentrations of said specified test compound and said specified standard compound, such that the output of the second mixing zone is provided to said first mixing zone.

47. The apparatus of Claim 46 further comprising:
a calibration unit for providing a calibration solution; and a switching valve, having a first input coupled to said second mixing zone, a second input coupled to the calibration unit, and an output coupled to said first mixing zone, for switching between the flow of either a compound mixture from the second mixing zone or the calibration solution from the calibration unit and then providing the flow to the first mixing zone where it may be mixed with said cell suspension.

48. The apparatus of Claim 47 wherein said calibration unit comprises:
a calibration maximum solution which provides for maximal cell response when mixed with said cell suspension;
a calibration minimum solution which provides for minimal cell response when mixed with said cell suspension;
a diverting valve having a first input coupled to the calibration maximum solution and a second input coupled to the calibration minimum solution, for switching between the flow of either the calibration maximum solution or calibration minimum solution; and a pump, coupled to the output of the diverting valve and an input of said switching valve, for pumping either the calibration maximum or calibration minimum solution from the diverting valve into the switching valve.

49. The apparatus of Claim 48 further comprising a second pump, coupled to an input of said first mixing zone, for pumping said suspension of cells from said cell suspension input into the first mixing zone.

50. The apparatus of Claim 49 wherein said conduit comprises a reaction developing line, having an input coupled to an output of said first mixing zone and an output coupled to an input of said detector, for providing a flow path and a reaction time delay for a mixture received from the first mixing zone and for providing the mixture to the detector.

51. The apparatus of Claim 50 further comprising:
a controller, coupled to said first and second gradient pumps, said test compound sampler, said standard compound sampler and said switching valve, said first andsecond mixing zones, said first and second pumps and said diverting valve for controlling their operation; and a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, wherein the computer is also coupled to said detector in order to send and receive cell response measurement signals to and from the detector.

52. The apparatus of Claim 51 wherein said detector detects changes in intracellular ion concentration.

53. The apparatus of Claim 52 wherein said ion is calcium, magnesium, proton, sodium, or potassium.

54. The apparatus of Claim 52 wherein said detector detects changes in intracellular ion concentration using an intracellular dye.

55. The apparatus of Claim 54 wherein said dye is fluorescent.

56. The apparatus of Claim 40 further comprising:
a pump, coupled to the output of said detector, for providing negative pressure to the apparatus;

a proportionating valve, coupled to said test compound sampler, for adjusting the concentration level of said test compound transferred to said mixing zone from said test compound sampler, wherein said test compound sampler further comprises:
a first intake nozzle for receiving said specified test compound;
a second intake nozzle for receiving a buffer solution; and the proportionating valve receives specified concentrations of the test compoundby adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound.

57. The apparatus of Claim 56 further comprising:
an automated standard compound sampler capable of selecting a specified standard compound from a library of standard compounds, said standard compound sampler including a third intake nozzle for receiving said specified standard compound and a fourth intake nozzle for receiving a buffer solution; and a second proportionating valve, coupled to the third and fourth intake nozzles, for receiving specified concentrations of the standard compound by adjusting theamount of standard compound and buffer solution received by the third and fourthintake nozzles, respectively, wherein the buffer solution is a diluting agent of the standard compound.

58. The apparatus of Claim 57 further comprising:
a first priming valve, coupled to the output of said first proportionating valve, for receiving said specified concentration of said test compound and providing the test compound to said mixing zone;
a second priming valve, coupled to the output of said second proportionating valve, for receiving said specified concentration of said standard compound and providing the standard compound to the mixing zone.

59. The apparatus of Claim 58 further comprising:
a calibration unit including a calibration maximum solution which provides for maximal cell response when mixed with said cell suspension and a calibration minimum solution which provides for minimal cell response when mixed with the cell suspension;
a first diverting valve, having a first input coupled to the calibration maximumsolution and a second input coupled to the calibration minimum solution, for switching between the flow of either the calibration maximum solution or calibration minimum solution;
a second diverting valve, having a first input coupled to the output of said mixing zone and a second input coupled to the output of said first diverting valve, for switching between the flow of either a calibration solution from the first diverting valve or a mixture from the mixing zone;
a third priming valve, coupled to the output of the second diverting valve, for receiving a mixture from the second diverting valve; and a second mixing zone, coupled to the output of the third priming valve, for mixing a mixture provided by the third priming valve with said cell suspension, wherein said cell suspension input and said detector are coupled to the second mixing zone instead of the first mixing zone.

60. The apparatus of Claim 59 wherein said conduit comprises a reaction developing line, having an input coupled to the output of said second mixing zone and an output coupled to an input of said detector, for providing a flow path and a reaction time delay for a mixture received from the second mixing zone before the mixture reaches said detector.

61. The apparatus of Claim 60 wherein said cell suspension input comprises:
a cell suspension reservoir;
a buffer reservoir;
a third diverting valve, having a first input coupled to the cell suspension reservoir and a second input coupled to the buffer reservoir, for adjusting the concentration of the cell suspension, wherein the buffer is a diluting agent of the cell suspension; and a fourth priming valve, coupled to the output of the third diverting valve, for receiving the cell suspension mixture from the third diverting valve and providing this mixture to said second mixing zone.

62. The apparatus of Claim 61 wherein said detector detects changes in intracellular ion concentration.

63. The apparatus of Claim 62 wherein said ion is calcium, magnesium, proton, sodium, or potassium.

64. The apparatus of Claim 62 wherein said detector detects changes in intracellular ion concentration using an intracellular dye.

65. The apparatus of Claim 64 wherein said dye is fluorescent.

66. The apparatus of Claim 61 further comprising a plurality of cell suspension reservoirs.

67. A method of characterizing the receptors present in a cell comprising the steps of:
(a) combining in a mixing chamber a suspension of cells with a test agent known to influence the activity of a particular receptor to form a test mixture;
(b) directing said test mixture through a conduit from the mixing chamber to a detection zone;
(c) directing said test mixture through the detection zone;
(d) measuring a cellular response of said suspension of living cells to said test agent as said test mixture is flowing through said detection zone wherein a response to a test agent indicates that said cell expresses a receptor known to respond to said test agent; and (e) repeating steps (a)-(d) with a series of test agents until the effects of each test agent has been measured.

68. The method of Claim 67, wherein the test agent comprises an agonist.

69. The method of Claim 67 wherein the test agent comprises an antagonist.

70. The method of Claim 67 wherein the test agent comprises a mixture of an antagonist and an agonist.

71. The method of Claim 67 further comprising repeating steps (a)-(e) on a series of different cell types to determine the receptors expressed by each cell type.

72. A method of confirming that a test compound has an effect on the activity of a receptor comprising the steps of:

combining in a mixture chamber a negative control cell type which lacks said receptor with said test compound to form a negative control mixture;
directing said negative control mixture through a conduit from the mixing chamber to a detection zone;
directing said negative control mixture through said detection zone;
measuring the cellular response of said suspension of living cells to said test compound as said negative control mixture is flowing through said detection zone;
combining in said mixing chamber cells of the same cell type as the negative control which have been engineered or induced to express said receptor with said test compound to form a test mixture;
directing said test mixture through said conduit from said mixing chamber to said detecting zone;
directing said test mixture through said detection zone;
measuring the cellular response of the cells in the test mixture to said test compound, whereby a difference in the measured response of said cells in said test mixture relative to the measured response of said negative control cells indicates that said test compound has an effect on the activity of said receptor.

73. The method of Claim 72, wherein said test agent comprises an agonist.

74. The method of Claim 72, wherein said test agent comprises an antagonist.

75. The method of Claim 72, wherein said test compound comprises a mixture of an agonist and an antagonist.

76. A method of determining the activity of one or more receptors in a series of cell types comprising:
(a) combining in a mixing chamber a suspension of living cells comprising a member of said series of cell types with a test agent known to influence the activity of a particular receptor to form a test mixture;
(b) directing said test mixture through a conduit from the mixing chamber to a detection zone;
(c) directing the test mixture through said detection zone;
(d) measuring the cellular response of said suspension of living cells to said test agent;
(e) repeating steps (a)-(d) on each member of the series of cell types until theeffect of said test agent has been measured in each cell type of said series.

77. The method of Claim 76, wherein said test agent comprises a known receptor agonist.

78. The method of Claim 77, wherein said test agent comprises a known receptor antagonist.

79. The method of Claim 77, wherein the test agent comprises a mixture of a known agonist and a known antagonist.

80. The method of Claim 77, wherein the test agent comprises a compound whose activity is unknown.

81. The method of Claim 77 wherein the test agent comprises a mixture of a compound whose activity is unknown and a compound which is a known agonist or a known antagonist.

82. An apparatus for measuring the effect of a test compound on living cells, comprising:
a test compound reservoir;
a cell suspension reservoir providing a suspension of living cells;
a first channel, coupled to the test compound reservoir, through which the test compound can flow;
a second channel, coupled to the cell suspension reservoir, through which the suspension of living cells can flow;
a mixing zone, coupled to the first and second channels, so that the test compound and cell suspension can flow into the mixing zone to provide a desired mixture of the test compound and the cell suspension in said mixing zone;
a fluid flow path through which said mixture can flow from the mixing zone; and a detector, coupled to the fluid flow path, which measures a cellular response of the suspended cells to the test compound, wherein the desired mixture of the test compound and the cell suspension flows along the fluid flow path from the mixing chamber to the detector.

83. The apparatus of Claim 82 further comprising:
a diluent reservoir for providing a diluting agent of the test compound;
a third channel, coupled to the diluent reservoir, through which the diluting agent can flow; and wherein said mixing zone is coupled to the third channel, to receive the diluting agent so as to provide a desired concentration of said test compound.

84. The apparatus of Claim 83 further comprising:
a standard compound reservoir, providing a standard compound having a known effect on said suspension of cells; and a fourth channel, coupled to the standard compound reservoir, through which the standard compound can flow, wherein said mixing chamber is coupled to the fourth channel, receives the standard compound from the fourth channel, and mixes the standard compound with the suspension of cells; and wherein said detector measures a cellular response of the suspended cells to the standard compound.

85. The apparatus of Claim 84 wherein said mixing chamber simultaneously mixes said test compound and said standard compound with said suspended cells and said detector detects said known effect or an alteration of said known effect.

86. The apparatus of Claim 85 further comprising a controller, coupled to the first, second, third and fourth channels, which automatically controls a flow of test compound, buffer solution, cell suspension and standard compound through the first, second, third and fourth channels, respectively.

87. The apparatus of Claim 82 further comprising a controller, coupled to the first channel, which automatically controls a flow of the test compound through the first channel.

88. An apparatus for measuring the effect of a test compound on living cells, comprising:
a test compound reservoir;
a first gradient device, coupled to the test compound reservoir, for automatically adjusting the concentration level of a test compound transferred to said mixing chamber from said test compound reservoir;
a cell suspension reservoir;
a mixing chamber, coupled to the first gradient device and the cell suspension reservoir, which receives the test compound and a suspension of living cells from the cell suspension reservoir and mixes the test compound with the suspension of living cells; and a detector, coupled to the mixing chamber, for measuring a cellular response of the suspended cells to the test compound.

89. The apparatus of Claim 88 further comprising a standard compound reservoir which provides a standard compound having a known effect on said suspension of cells, wherein said mixing chamber is coupled to the standard compound reservoir, receives the standard compound, and mixes the standard compound with the suspension of cells, and wherein said detector measures a cellular response of the suspended cells to the standard compound.

90. The apparatus of Claim 89 wherein said mixing chamber simultaneously mixes said test compound and said standard compound with said suspended cells and said detector detects said known effect or an alteration of said known effect.

91. The apparatus of Claim 89 further comprising a second gradient device, coupled to said standard compound reservoir, for automatically adjustingthe concentration level of said standard compound transferred to said mixing chamber from said suspension of cells.

92. The apparatus of Claim 91 further comprising a controller, coupled to the first and second gradient devices, for automatically controlling a flow of the test compound and automatically controlling a flow of the standard compound, into said mixing chamber.

93. The apparatus of Claim 88 further comprising a controller, coupled to the first gradient device, for automatically controlling a flow of the test compound into said mixing chamber.