CA2056076A1

CA2056076A1 - Apparatus for automatically processing magnetic solid phase reagents

Info

Publication number: CA2056076A1
Application number: CA002056076A
Authority: CA
Inventors: Chi-Chin Wang; Robert T. Mc Keever; Marshall L. Salyers
Original assignee: Individual
Current assignee: EIDP Inc
Priority date: 1990-12-14
Filing date: 1991-11-22
Publication date: 1992-06-15
Also published as: NO914917D0; ES2104652T3; ATE154136T1; DE69126410D1; NO914917L; EP0490358B1; DE69126410T2; EP0490358A3; JP3381934B2; IE913944A1; JPH04341356A; US5128103A; EP0490358A2

Abstract

Title Apparatus for Automatically Processing Magnetic Solid Phase Reagents ABSTRACT
An automatic chemistry system having a wheel for mounting reaction vessels is provided with a programmable permanent magnet which is selectively positioned adjacent a reaction vessel to separate magnetizable particles in the vessel from the vessels liquid contents. This facilitates performing heterogeneous immunoassays.

Description

Title Apparatus for Automatically Processing Magnetic Solid Phase Reagents Field of the Invention The present invention relates to an automated apparatus for the separation and concentration of materials in small amounts of complex liquid mixtures.

Backqround of the Invention Separation, isolation and concentration are process steps common to a chemical analysis. Often these steps are taken to remove interfering substances so that a subsequent chemical analysis can be performed. This "separation" stage can be performed several ways including solvent extraction, solvent evaporation and resin exchange. Magnetic separation, another technique for removing interferring substances, is a process of separation, isolation and concentration where the sought-for substance is attached or bound to magnetic particles. The magnetic particles offer advantages of handling including speedr convenience and low energy input. It i5 particularly suited to handling small samples. Advanced Magnetics Inc. of Cambridge, MA has been very active in this field in the application of their super paramagnetic particles to separation techniques. Their usage and properties is described in a product bulletin entitled Magnetic Affinity Chromatography Starter Kit M4001 and Magnetic ~ffinity Chromatography Support BiomagTM
M4100 dated July 1984.

,. : . ,, ., . , . ,: .
, . ~ :.,. , . ,:

~. :
' ~

Magnetic particles are particularly useful in heterogeneous immunoassays as a solid support. To be useful as a solid support, the particles must be derivatized to permit the attachment of bioactive protein. Hersh et al. in their U.S. Patent 3,9S3,997 describe the use of magnetically responsive particles for this purpose and use functionalized silanes as the intermediate between the particles and the bioactive protein.
There are essentially two types of heterogeneous immunoassays. These are competitive immunoassays and sandwich immunoassays. In a competitive assay, an antibody to an antigen contained in a first reagent is attached to the derivatized magnetic particles to make up a solid phase. The second reagent, consisting of antigen attached to a tag ~a measurable entity, including radioactive molecules, fluorescent molecules, or enzymes), and patient sample are mixed with the solid phase in a test tube. In the absence of patient antigen, some 50% of the antigen-tag is bound to the antibody of the magnetic solid phase. In the presence of patient antigen, some of the anti~odies are filled up with patient antigen and are unavailable to the tag antigen. As a result increasing amounts of patient antigen leads to decreasing amount of tag antigen. Thus one can form a calibration chart relating the amount of patient antigen to the amount o~ ta~. The separation stage results from the need to measure the free tag or the bound tag, not the total tag added. The magnetic particle facilitates this separa~ion by forming the particles with the bound tag into a pellet on the side of the tube. The free tag can then be removed as by aspiration. Following the separation and removal of free tag, another reagent i5 added so that the amount of bound tag can be measured. In a typical case, enzyme is , . ~, .

' " .

r~ ~

used as the tag so that the reagent added is a "substrate" for the enzyme permitting the measurement of -the amount of tag that was bound to antibody.
In a typical sandwich immunoassay, an antibody to ~-an antigen is attached to the magnetic particle. This is in high concentration relative to the amount of patlent antigen in a sample. Patient antigen is captured by the antlbody on the magnetic particles and then the particles (and captured patient antigen) separated from lnterfering substances in the sample. To this, a second reagent, containing a second antibody with an attached tag, is added. This second antibody attaches to the patient antigen, captured by the first antibody on the magnetic particle, and results in the formation of a sandwich so ~hat the second antibody tag is held firmly by the antigen to the first antibody on the magnetic particle. At this point, a magnetic separation similar to that described, permits the determination of bound tag which is in proportion to the patient antigen, the excess tag of the second reagent having been removed hy aspiration.
Magnetic particles are particularly useful as the solid support in heterogeneous immunoassays because they can readily separate the free from ~he bound tag. Such immunoassays using magnetic particles as a solid support are described for example in U.S. Patent 4,661,408 (Lau et al.), U.S. Patent 4,628~037 issued to Cha~non et al., U.S. Patent 4,672,040 ~ssued to Jssephson, and U.S.
Patent 4,698,302 issued to Whitehead et al. The methods disclosed in all of these patents relate to manual processes which utilize manual magnetic separation units such as those that are available from Corning Medical, Corning Glass Works, Medfleld, ~A. Such manual techniques are relative slow, requir~ relatively strong magnets which are expensive, require cons~derable manual , ., , , ~ '' ,' , . . ~ ~ ~' ' .

2 ~ 3 dexterity, and require an excPssive amount of time to effect the separation with the purity required, particularly for sandwich type heterogeneous lmmunoassay.
TechIIicon Corporation has offered an automated heterogeneous magnetic immunoassay system ~or some years. In this system the reagents are combined in a continuous flow process. Having reacted the reagents together, the process then brings the stream through a magnetic field where the magnetic particles are captured and, bound tag measured. The pro~lem with this process is that of continuous flow sys~ems in general.
Carryover from Qne sample to the next tends to produce erroneous results, which error is reduced by reducing the number of samples analyzed per hour.
The Du Pont patented ACMIA technology for digoxin (DGN Method) has been used on the aca~ Discrete ~nalyzer using resin based column header as solid separation media. This assay has been adapted to run on the Dimension~ Clinical Chemistry System using chromium dioxide magnetic particles as the solid support.
Unfortunately for both analyzers, manual treatment of the samples with antibody con~ugate reagent (ABC) is required. The Dimension~ assay also involves treatment ~5 with chromium dioxide particle reagent (CPR), maynetic separation and transferring of the superna~ant to the instrument for photometric measurement. This manual step required is not only time consuming but, because of the manual feature, is subject So error.

Many of these problems of the prior art as~ay and other analysis systems particulaxly those prior art systems using magnetic particles, i.e., particles that are responsi~e to a magnetic field, are reduced using - ':

r~

the apparatus of this invention. The invention is particularly useful for those systems using robot~c arms operating in conjunction with an assay wheel as a transport means for the reaction vessels or cuvettes.
This invention permits magnetic particles to be used as a separable solid support in various analytical techniques and in automated fashion. This is particularly true and useful for heterogeneous assays which require that the solid support be removed and/or washed during the assay process.
According to this invention an automatic apparatus is provided for separating particles from a~ueous particle dispersions disposed in a plurality of reaction vessels, ~he particle being responsive to a magnetic field, oomprising: a transport means for moving the vessels in sequence past at least one processing position, a robotic arm for selectively processing the vessels as they move sequentially, and means for selectively subjecting the vessels to a magnetic field as they move sequentially, thereby to separate the particles from the aqueous dispersion.
In the preferred embodiment of the invention, the transport means indexes the vessels stepwise past the processing position and the reaction vessels each have walls and a longitudinal axis that is generally vertically diQposed while the subjecting means field is generally transverse to the reaction vessel's longitudinal axis, whereby the particles are drawn toward a wall of the vessel. The sub~ecting means comprises a permanent ma~netic wh~ch is positioned relative to the cuvette to have the ~lux axis of the magnet intersect the bottom center region of the cuvette. Preferably, the sub~ectiDg means is positioned on the robotic arm i~sel such that wherever the arm is positioned, proper cuvettes are automatically addressed.

, ., .. . . ..

.;

. , .
:
:
: . ' . .

rl ~S

In accordance with a further preferred embodiment of the invention, the sub~ecting means subjects a reaction vessel located ei~her ahead or behind ~indexing wise) the position of the robotic arm relative to a reaction vessel to a magnetic field. In another embodiment, the subjecting means simultaneously subjects three sequentially positioned reaction vessel to the magnetic field, such that a vessel containing a magnetizable dispersion can index through and be subjected to the magnetic field up to three times.
The invention also includes an automatic apparatus for use in assays for separating magnetic particles from a liquid phase, the apparatus comprising: a plurali~y of reaction vessels adapted to hold magnetic particles dispersed in a liquid phase, a reaction vessel mounting wheel adapted to move the reaction vessels in sequence past at least one processiny position, probe means for dispensing liquid into and withdrawing liquid from the vessels, a reaction monitoring arm capable of relative msvement with respect to the wheel and being positioned with its periphery ad~acent any of the reaction vessels, a detector positioned on the arm radially outside the reaction vessels, the monitoring arm being operable to direct a beam of interrogating radiation ~hrough each vessel to the detector, and coupling means coupled to the arm for positioning a first magnet adjacent the location of the beam ~f interrogating radia~ion, but spaced therefrom by the distance separating adjacent reaction vessels on the wh~el.
The pparatus of this invention elimina~es the need for pretreatment of assays involving a magnetic solid support. It permits all necessary reagents to be delivered directly to a reaction vessel to perfoxm the nece~sary incubations, separate the magnetic particles ~CPR) from the solution, withdrawing the reacted ~ . .

material from the reaction vessel, and transferring it to a second reaction vessel for photometric measurement.
This invention greatly facilitates the automation of the assay since the magnet module is controlled by the software used in controlling the automatic clinical chemistry system itself. It facilitates the performance of many heterogeneous assays on automatic clinical chemistry systems such as the ~ffinity Chromatography Media Immunoassay (ACMIA) using magnetic chromium dioxide particles as a solid support. Although this assay does not require washing, the apparatus of this invention also permits the clinical chemistry system to wash the solid support where necessary.

~
The invention will be better understood with reference to the several drawings, in which like reference numerals are used to indicate like components, in which: -Fig. l is a pictorial plan view of an automatic clinical chemistry system in which this invention may find use;
Fig. 2 is a pictorial representation of the sample wheel and arm assemblies, the reagent arm and probe, and transport means of the elinical analyzer system of Fig.
l;
Fig. 3 is a fragmentary plan view of the reaction monitoring arm of Fig. 2 incorporating a magnet of ~his invention to effect removal of the CPR from reaction vessels;
Fig. 4 is a pictorial representation of the magnet holder of Fig. 3;
Fig. 5 is a front elevation view of the magnet holder of Fig. 4;

, ~ ~c ~ rj ~

Fig. 6 is a front elevation view of the cover of the photodetector to which the magnet of Figs. 4 and 5 is attached; and Fig. 7 is a pictorial view of an alternative embodiment of this invention for attaching three magnets to the reaction monitoring arm.

Des~i~tiQn Qf the Preferred Em~diment There is illustrated in Fig. 1 an automated clinical chemistry system with which this invention finds use. This particular clinical chemistry system is an analyzer known as the Dimension~ clinical chemistry system sold by E. I. du Pont de Nemours and Company, Wilmington, Delaware. The system includes a compu~er 10 with appropxiate display and keyboard. It also includes a sample carousel or wheel 12 together with a sample arm 14 and probe (not shown) for transferring samples to a cuvette or reaction vessel 17 ~Fig. 2) on a cuvette wheel or transport means 16.
The Dimension~ clinical ohemistry system uses a FlexTM reagent cartridge 18 (Fig. 2). The cartridge contains a bar code which is read by a bar code reader 20 (Fig. 1) as the cartridges are introduced onto the transport means 16 via a reagent shuttle 22. A reagent arm 24 and probe 26 draws reagent from the appropriate reagent cartridge well in one of the FlexTM units 18 and then dispenses it into an assigned reaction vessel 17.
The FlexTM cartridges 18 ~ach hav~ a number of wells which contains the various reagen~s needed in either liquid or tablet form. The reagent arm 24 positions the reagent probe 26 to hydrate, mix and transfer reagents used ln photometrlc testsO Stepping motors (not shown) rotate ~he arm And pO it~on the probe for aspiration and dispensing of reagents. The reagent probe 26 is an u7trasonic mechanism used for hydrating, aspirating, . ~ .

:

dispensing and mixing reagents. It can access reagent cartridges in one position and move the reaction vessels 17 at any of the active positions around the transport means 16. The hydration and mixing is all part of an existing commercial system and need not be described further.
As is known in the Dimension~ clinical chemistry system, cuvettes are formed by pulling two different composition ribbons of clear film from the cuvette film c~rtridge 30 onto the periphery of the transport means 16. The transport means, in the form of a cuvette wheel, has lO0 separate cuvette cavities. The inner wall o~ each cavity has an inner wall to allow transmission of light. There is a cuvette forming station 32 in which an ionomer film ribbon is heat softened, molded onto the inner wall of the cuvette or reaction vessel cavity and its optical window. The tranSpGrt means is then rotated to stretch the outer ionomer-nylon film ribbon across the molded inner film and the two are heat-bonded to each other. A small opening remains at the top of the cuvette to ~llow the addition of reagent and sample. A drive capstan 40 pulls the cuvette film moving the cuvettes clockwise about the cuvette wheel, i.e., the transport means 16.
After the cuvette ls formed, the sample arm 14 draws a sample fxom a sample cup ln the sample wheel 12 and adds it to the reaction vessel or cuvette 17.
Sample mlxing is performed ultrasonioally by the eample probe. The reagent arm/probe 24/26 hydrates reagents automatically as they are needed. ~he reagent probe then adds hydrated reagent to the cuvette and ultrasonioally mixes the sample and reagen~s together.
A photometer 42 located beneath the reagent arm 24 and under the transport means 16 measures light absorbance through the cuvette at various wavelengths. A s~urce . ~ ~

. .

lamp 44 emits a light beam which pa-qses through various lens housed in a rotatable detector arm 45 to a photodetectox 46 which, being mounted on the outer-end of the detector arm 45 ad~acent the outer periphery of S the cuvettes 17, rotates about the transport means 16.
The photodetector relays absorbance readings through the computer where the readings are converted into concentration units.
In aocordance with this invention a magnet 56, positioned in a mounting block 50 (Fig. 4), is secured to the photodetector 46 whose inner face, facing the cuvettes, is best seen in Fig. 6. The inner face, or cover, of the photodetec or of Fig. 6 has screw holes which conform to the serew hole locations in the mounting block S0. The access window for the photodetector 46 is illustrated at 98 (Fig. 6). The mounting block 50 is formed with an opening 54 to permit the passage of light through the holder. The block 50 positions a permanent magnet 56 to be spaced from the opening 54 a distance to eorrespond to the spacing between two adjacent cuvette positions. The magnet 56 is positioned such that the flux axis of the magnet cylinder will pass through approximately the bottom center of each cuvette such that the magnetic particles are withdrawn from the cuvette toward the bottom and sidewall thereof.
The magnet 56 is mounted in a position such that it has a flat circular surface 58 parallel to and approximately 2/32 inches removed from the wall of the cuvettes 17 which it is facing. The center of the magnet i~ at the same horizontal plane as mentioned at the bottom of the cuvette.
The permanen~ magnet S6 is cylindrical shaped, approximately O.S inch in diameter and 0.25 inches in height with a magnetic strength of approximately 2500 to :
. .

y~ ~

3000 gauss measured on the face of the magnet in a preferred embodiment. Other sizes, shapes, and strengths may be used.
In operation, the magnet is positioned, by its being mounted on the detector arm 45, one cuvette to the right of the optical axis of photometer 45 (cuvette +1), when the photometer arm 45 is instructed by program command to move to a selected cuvette position designated cuvPtte zero. Thus, with the solid phase magnetic particles being in "cuvette +1" position relation to the photometer arm, the magnet 56 pulls the particles out of suspension and against the side-bottom wall of the cuvette. This permits the reagent probe to "sip" the supernatant from the cuvette + 1 position and transfer it to the cuvette zero position in a preferred embod.iment, where it may be read by the photometer. The receptive cuvette may be any position on transport means 16, to where the reagent probe 26 and arm 24 are accessible. This is accomplished through the software of the computer 10.
Although the computer of the chemistry system may be programmed in any desired manner, one method by way of illustration for performing a digoxin assay may be described by the following pseudocode designations when used with the Dimension~ system:
photometr~c method: "DIG"
~ey=Oxe4 first cuvette: {* pretreatment cuvette *?
-108.2 sec: QC cuvette tag G "rlcl"
30 -94.0 sec: add 80 ~l of R1 {* first xeagent ~con~ugate] *}
followed by 20 ~l of water ultra power 8 0 0.0 sec: add 80 ~1 of sample followed by 19 ~1 of water .

~ ' , ' ' ~ ' , ~ ~ 3 60 7 D

ultra power = 2 ultra ontime = 0.5 sec ultra duty cycle = 30 %
ultra cycle time = 105 msec ultra erxor rate ~ 40 %

240.0 sec: add 80 ~l of R2 {* second reagent [chrome] *}
followed by 40 ~l of water remix R2 at power level 7 for 5.0 sec ultra power - ~
ultra ontime = 0.5 sec ultra duty cycle 50 %
ultra cycle time = 105 msec ultra error rate c 40 %
425.0 sec: apply magnetic field for 20.0 sec 1***********~******************************************
note: cuvette 2 time 0.0 lags that of cuvette 1 by 20.0 sec; 430.0 sec -= 450.0 sec of cuvette 1 *************************~****************************}

second cuvette: {* sample rate *}
-125.0 sec: QC cuvette tag - "rl"
25 0.0 sec: form cuvette ~* in absence of sample delivery, we must explicitly ask for cuvette *]
320.0 sec: add 275 ~l of R4 followed by 20 ~l of water ultra power ~ 0 420 . 0 5eC: add 60 ~l of first cuvette (height = 5.0 mm) followed by 30 ~l of watcr ultra power G 8 ultra ontlme = 0.5 sec ultra duty cycle - S0 %

~,;
- ~ . ., .. . .. . . . .

ultra cycle time = 105 msec ultra error rAte ~ 40 %
430.0 sec: hardread cuvette ~ag = "rA"
550.0 sec: hardread cuvette tag = "rB"

calculation:

float mauA ~ rAt~05] - rA~510];
float mauB ~ rB[405~ - rB~510];
legend[0~ = "RDIG"; polished[0] - mauB - mauA;
endcalc flex specification:

l-----R1---~ 2~ --R4--l / 1 / 2 / 3 / g / 5 / 6 / 7 / 8 ll _____________________________________________ ___ I---coniugate---chrome--l I ~~~---onpg---- I I

_________ _______________________________________ Interpretation To simplify the interpretation, the times at which activities occur have been referenced with respect to the time of the first reagent delivery to the ~irst cuvette. Activities which are grouped by cuvette in the specification are here placed ~n time order.
Those activities not relevant to the performance of the assay are ~ot described.
The times appearing in square brackets can ke used to back-reference to the assay specification.
0.0 sec [cu~ette 1, -94.0 sec]

; ,, ~, Using the reagent probe, deliver 80 ~L of conjugate reagent followed with 20 ~L of water to the first cuvette.

94.0 sec: lcuvette 1, 0.0 sec]
Using the sample probe, deliver 80 ~L of sample followed with 19 ~L of water to the ~irst cuvette. Mix the first cuvette using the ultrasonic mixing ca~ability of the sample probe.

334.0 sec: ~cuvette 1, 240.0 sec~
Using the reagent probe, resuspend the particle reagent in the reagent container using the ultrasonic mixing capability of the reagent probe. Deliver 80 ~L of this particle reagent followed by 40 ~L of water to the first cuvette. Mix the first cuvette using ~he ultrasonic mixing capability of the reagent probe.

434.0 sec- [cuvette 2, 320.0 sec]
Using the reagent probe, deliver 275 ~L of ONDG reagent ~ollowed wi~h 20 ~L of water to the second cuvette.

519.0 sec: [cuvette 1, 425.0 sec~
Usin~ the photometer, seguester all particle ~n th~ first cuvette by placing the magnet affixed to the photomet~r arm next to the first cuvette. Remain at this position ~or 20.0 seconds.

7.v~

~' . ` ' ' .

534.0 sec: [cuvette 2l 920.0 sec]
~sing the reagent probe, aspirate 60 ~L of supernatant from the first cuvette.
Deliver ~his volume to the second cuvette, following immediately with 30 ~L of water.
Mix the second cuvette using the ultrasonic mixing capability of the reagent probe.

544.0 sec: [cuvette 2, 430.0 sec]
Using the photometer, take an initial reading of the second cuvette.

664.0 sec: [cuvette 2, 550.0 sec]
Using ~he photometer, ~ake a final reading of the second cuvette.

An alternative embodiment of this invention is illustrated in Fig. 7 which depicts a bracket 70 that may be attached to the photometer arm 45 so as to extend along the outer periphery of the reaction vessels, i.e., cu~ettes 17. Bracket 70 is in the form of an L and at one end of the bracket 70 is a magnet holder 72 which may be formed of a suitable engineering plastic or any other non-ferrous material. Three magnets 7~, which may be the same as those used in connection with the embodiment described in connection with Figs. 3 to 6, are positioned such that the inner surface 78 is flat and circular so as to be parallel to and 2/32 of an inch removed from the wall o~ the cuvettes 17 which it is facing. The magnets are positione~ vertically such ~hat ~he magnet is ~n the ~ame hor~zontal plane as the bottom of the cu~ette as previously described in connection with the embodiment of Figs. 3 t~ 6.

" .
" , : . - , . , ~

, , ' ~ ~ 3 ~ he program commands are similar to those described in connection with the first embodiment of this invention and with this design the magnet is automatically positioned so that when the photometer arm 45 is instructed by a program command ~o move to any cuvette posltion, designed cuvette 0, the magnet is automatically positioned by the b.racket 70 four ~and sequentially thereafter when multiple magnets are used) cuvettes to the right of the photometer arm 45. Hence the magnets are positioned ad~acent to cuvettes 4, 5 and 6. A magnetic test in positions 4, 5 or 6 could be interfered by any photometric measurements performed on cuvettes at positions 0, ~1 or ~2 at a time other than when magnetic separation is desired, because of the magnets in bracket 70. Hence cuvette positions 0, + 1 and ~ 2 are made unavailable ~or those non-magnetic test such that a premature magnetic separation on the magnetic test would never occur. With this embodiment using the bracket 70, a cuvette at a particular position may be subjected to the influence of a magnetic field for separation of the CPR through three cycles typically of 20 seconds each such that a full total of 1 minute would be used to separate the desired CPR. Other than that the operation is the same as that previously described.
~ hichever embodiment is used, various heterogeneous assays may be performed without ~he use of pretreatmen~.
Using these procedures, for example, on ~he Dimension~
clinical analyzer system: (a) Dispense ~C reagent (consists of anti-digoxln antibody to ~-galactosidase conjugate) into cuvette A. (b) Dispense sample into cuvette A. Incubate for 1 to 5 minutes. (c) Dispense CPR (Oubain-BSA-coated CrO2 parkicles). Incubate for 1 to 5 minutes. ~d) Magnetic separation for 15-30 seconds. (e) Transfer an aliquot o~ supernatant to , , , ~ .~ - . . -, . .

3'1'j ' cuvette B, which c~ntains o-nitrophenyl galactoside ~ONPG) substrate solution. (f) Photometric measurement on cuvette B.
In short by providing a "smar~" magnet module of this inventlon, which is software controllable, an automatic system is provided with a minimal of manual operations. In a preferred embodiment, the magnet is located one cuvette position (position + 1) after the cuvette directly in line with the photometric detector (position 0) of the photometer arm 45. Recall, in the embodiment, and where the magnet is mounted on the cover of the photometric detector 46. This provides a system which is capable of performing heterogeneous immunoassays~ The sys~em is capable of operating with or without washing of the particles as part of the assay procedure. In addition to the conventional ACMIA
assays, it i~ also able to perform enzyme immunoassays (EIA~ employing either a sandwich or a competitive mode using magnetizable particles as a solid support. The EIA assays require washing of the solid support and sensitivities of 10-12 mole/liter are obtained.

Example 1 Enzymometri~ Immunoassay for Diqoxin Reagents:
The reagents described below are available commercially under the tradename DIG FlexTM reagent cartridge (Part number 717035.901) which is intended for the detection of digoxin in human specimens using the Du Pont Dimension~ clinical chemistry system.
1. ~NTIBODY-~-GALACTOSIDAS~ CONJUGATE REAGENT, hereafter designated Coniugate, is a eovalently cross-linked aggregate of anti-digoxin antibody and B-galactosidase, formulated in a sodium biphosphate/sodium monophosphate buffer, pH 7.4. The Conjugate solution is . ~.................... . .

`

. . .

~8 contained in wells No. 1 and 2 of the FlexTM reagent cartridge 18 (Fig. 2).
2. CHROMIUM DIOXIDE PARTICLE REAGENT, hereafter designated CPR, is a suspenslon of magnetizable chromium dioxide particles, on which a covalent coating of oubain-bovine serum albumin (Oubain-BS~) molecules has previously been introduced. Appropriate amount of CPR
is formulated in a sodium b.iphosphate/sodium monophosphate buffer pH 7.4, and is contained in wells 3, and 4 of the FlexTM reagent cartridge 18.
3. o-NITROPHENYL GALACTOSIDE REAGENT, hereafter designated ONPG, is a solution of o-nitrophenyl galactoside in a buffer consisting of N-2-hydroxyethyl-piperazine-N'-2-ethanesul~onic acid ~HEPES buffer), pH
7.8, and is contained in wells 7 and 8 of the FlexTM
reagent cartridge 18. ONPG is used as a colorimetric substrate for ~-galactosidase. It is therefore feasible to substitute ONPG with another substrate for the enzyme and achieve similar results for the assay.
The following procedure can be used to perform a digoxin assay using the apparatus of this invention in the Dimension~ clinical chemistry system with ~he embodiment of Fig. 2.
Procedures:
1. Prepare ~he Dimension~ clinical chemistry system per Operator's Guide (Part No. 715813.901 prov~ded with the system.
2. Prior to testing specimens containing unknown concentration of digoxin, five calibrator samples are normally tested under the "Calibrationl' mode of the Dimension~ clinical chemistry system. The "assigned values" of each calibrator ls manually entered into the computer before the tests. Load appropriate calibrators and a DIG FlexTM reagent cartridge on ~he system. After lB

--4_; , '" """ ' ,.

' ~ ~

~ 3 the tests are completed, the on-b~ard computer automatically performs a mathematical regression using the signals and assigned-values of all five samples.
The regression employs an algorithm commonly known as "~OGIT function" and computes a series of "linearization coefficients". The coeEficients are stored in the computer memory.
3. Schedule a digoxin test via the on board computer. Load the specimen and a DIG FLEXTM reagent cartridge containing the same lots of reagents as used in the Calibration step. The magnet-equipped Dimension~
clinical chemistry system performs the entire test automatically and uses the stored linearization coe~ficients to compute the concentration of digoxin by applying the LOGIT function in a reverse manner. The sequence of events by which the system performs the digoxin test follow.

4. Upon receiving commands to perform a digoxin test, the Dimenslon~ clinical chemistry system forms two cuvettes (A and B~. The cuvettes, in a continuous chain-like formation, are situated around the perimeter of the Cuvette Wheel 16 ~Fig. 2). The Cuvette Wheel is contained in a chamber which is maintained at a constant temperature of 37C.

5. ~n aliquot of Conjugate (80 ~L~ is automatically withdrawn from the reagent cartridge and dispensed lnto Cuvette A by the system's Reagent ~robe 26.

6. After 90 seconds, a sample of the specimen (80 ~Lj is withdrawn from the Sample Cup situated on Sample Wheel 12 and dispensed into Cuvette A by the systemSs Sample Probe 14. The Sample Probe ~s equipped with an ultrasonic device. ~ter dispensing the specimen, the Probe is vibrated ultrasonically for 2 seconds while immersed in the solution. Thls provides agitation to , , , ~

, allow thorough mixing of the specimen wi~h the Conjugate solution.

7. After an incubation period of 180 to 360 seconds, an aliquot of CPR suspension (80 ~L) is withdrawn from the reagent cartridge and dispensed into Cuvette A by the Reagent Probe. The Reagent Probe is also equipped with an ultrasonic device. Prior ~o withdrawing the CPR suspension from the reagent cartridge, the probe is vibrated ultrasonically for 5 seconds while immersed in the liquid to ensure consistent re-suspension of the CPR particles. After dispensing the suspension into Cuvet~e A, the probe is again ~ibrated for 2 seconds while immersed in the solution. This provides agitation to achieve consistent suspension of the CPR in tbe reaction mixture.

8. The mixture is allowed to incubate for approximately 2 minutes. During which the Conjugate moleeules stoichiometrically bound to the digoxin molecules provided by the speclmen remain in solution, while excess conjugate ts bound by the Oubain-BSA
coating on CPR particles.

9. While Cuvette A is incubating, an aliquot of ONPG (275 ~L) is wit~drawn from the reagent cartridge and dispensed into Cuvette B by the Reagent Probe.

10. Approximately 2 minutes aftex the dispensing of CPR, a command instructs the Photometer Arm 45 to move ~o a position, such that ~he permanent magnet is directly facing Cuvette A.

11. The Photometer Arm is held stationary ~or 20 s~conds. This magnetizes the CPR and, in effect0 holds the particles to the bottom and one side of the cuvette.

12. The Reagent Probe is oommanded to withdraw a 60 ~L aliquot of liquid, now free of CPR particles, from Cuvette A and dispen~e it ~nto Cuvette B. While the Probe is immersed in Cuvette B, it is vibrated .
: ~

2~ 'ifj ~J~
ultrasonically for 2 seconds. This step isolates the digoxin-bound Con~ugate molecules, which is -stoichiometrical to the unknown digoxin concentration, for enzymatic measurement ln Cuvette B. Any and all exc~ss Conjugate molecules are bound to the CPR and retained in Cuvette A.

13. After 10 seconds, the Photometer Arm is moved to allow the opening 54 of detector 46 (Fig. 3) to face Cuvette B. Absorbance at ten wavelengths are measured.
The difference of absorbance between 405 nm and 510 nm are computed by the computer and recorded as rA (initial reading).

14. One hundred twenty (120) seconds after the initial reading, the Photometer is instructed to measure the absorbance of Cuvette B again. The difference of absorbance between 405 nm and 510 nm is computed and recorded as rB (second readlng).

15. The difference between rB and rA is computed and recorded as ~he photometric signal of the test. The signal is used to compute the concentration of digoxin in the specimen as described in Step 3.
Results:

Table 1: Digox~n Assay Calibration Results Assigned Signals (mA) Mean Values SamPle 1 ~m~le 2 Si~nal ~P# CV*
0.00 ng/mL 62.9 66.1 6405 2.26 3.5%
0.70 ng/mL 82.8 81.4 82.1 0.99 1.2%
1.20 ng/mL 101.8100.9 101 4 0.64 0.6%
302.40 ng/mL 114.8116.5 115.7 1.20 1.0%
5.00 ng/mL 169.2169.5 16g.4 0.21 0.1%
~ SD: Standard Deviation.
* CV: Coefficient of Variance. It is calculated by dividing SD with ~ean.

~ ~ 3 Using this procedure, tests were run on digoxin samples with the following results:
Table 1 exhibits a set of results obtained from five calibrators. The "Assigned Values" (ln ngtmL) and the mean signal (in mili-absorbance unit, mA) are used by the on-board computer to perform a regression analysis ~y the LOGIT function.
Table 2 shows testing results of ten serum specimens. The results, ln ng/mI of digoxin c~ncentration, are calculated by the ~omputer automatically. The data indicates that the test results are in close agreement with those obtained by the Stratus~M system.

Table 2: Dlgoxin Test Results in Comparison to Results by A Commercial Tes~ (Stratus) Dimension~ Stratus*
~erum No Syskem System 1 0.93 ng/mL 0.90 ng/mL
2 1.21 1.20 3 1.92 2.00 ~ 1.17 1.10 0.89 ~.90 6 1.23 1.20 7 0.77 ~.60 8 0.95 0.80 9 l.lS 1.40 1.39 1.20 * Stratus~ Immunoassay System, manufactured ~y Baxter Healthcare Co., Dade Division, Miami~ FL 33152 ~ L~l~b~g~ fQI

~ :~

~ ~-3 ~ '3 En~ymQm~Iic Assav ~I Vitamin ~1 Reagents I. Releasing reagents:
These reagents are provided in glass vials. Their function is to treat the serum specimen in such a way that the vitamin B12 molecules contained therein are released and available for the enzymvmetric detection.
1. DENATURING REAGENTS: Two reagents, a 0.1 N
sodium hydroxide ~NaOH) solution and a 20 mM
dithiothriotol (DTE) solution, are provided in separate vials.
2. NEUTRALIZING REAGENT: A sodium phosphate/N-2-hydroxyethylpiperazlne-N'-2-ethanesulfonic acid (Phosphate/HEPES) buffer, pH 5Ø

II. Reagents provided by FlexTM reagent cartridge:
The reagents described below are presented to the Dimension~ clinical chemistry system in a B12 Flex~M
reagent cartridge. Either embodiment of the invention can be used.
l. INTRINSIC FACTOR TO B-GALACTOSIDASE CONJUGATE
REAGENT, hereafter designed IFC, is a covalently cro s-linked aggregate of ~-galactosidase and calf intestinal intrinsic ~actor. Intrinsic factor is a protein which possesses high affinity to vitamin B12 and whose natural function is to transport vitamin Bl2 molecules through the biological systems. A highly purified form o this protein is used in this assay to detect vitamin Bl2 in human serum with high ~ensitlvity and speeificity. The IFC is formulated in a sodium biphosphate/sodium monophosphate buffer, pH 7.8 and is contained in wells 1 and 2 of the B12 FlexTM reagent cartridge 18 (Fig. 2)o . -:

J
2~
2. VITAMIN B12 - COATED CHROMIUM DIOXIDE PARTICLE
REAGENT, hereafter designated B12-CPR, is a suspension of chromium dioxide particles which have been covalently coated with a layer of a vitamin B12-avidin con~ugate.
The reagent is formulated in a sodium biphosphate/sodium monophosphate buffer, p~ 7.4. Appropriate amount of B12-CPR suspension is contalned in Wells 3 and 9 of the B12 Flex~M reagent cartridge 18.
3. CHLOROPHENOL RED-~-D-GA~ACTOPYRANOSIDE
REAGENT, hereafter designated CPRG, is a solution of chlorophenol red-~-D-galactopyranoside in a ~uffer consisting of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES buffer) pH 7.8, and is contained in wells 7 and 8 of the FlexTM reagent cartridge 18. CPRG is used as a colorimetric substrate for ~-galactosidase.

Procedure:
1. Prepare the Dimension~ clinical chemistry system according to procedures specified in the Operator's Guide.
2. In a plast~c or glass test tube, sequentially dispense 200 ~L of serum specimen or calibrator, ~0 ~L
of NaOH solution and 20 ~L of DTE solution. Allow the mixture ~o stand a~ ambient temperature for 5 minutes.
This step brings the pH of ~he specimen to higher than 12, which denatures most or all the serum proteins, including endogenous tntrinsic factor, such that any and all vitamin B12 molecules are released into th~
solution. The DTE reagent helps to block the sulfohydryl groups on the denatured proteins, therefore pre~enting any re~capturing of the vitamin B12 molecules.
3. Dispense 60 ~L of the neutralizing reagent into the test tube. Vortex to mix the solutions. The 2~

~, .

" ~ . ` . . . - , -:
.:

2 ~ 3~i~;3 pH ls reduced to 7.4 to 7.8 at this stage. This step readies the specimen for the subsequent enzymometric test.
9. Prior to testing specimens containing unknown concentration of vitamin B12, five calibrator samples are tested under the "Calibration" mode of the Dime~sion~ clinical chemistry system. The "assigned values" of each calibrator is manually entered into thè
computer before the tests. Load appropriate calibrators and a Bl2 FlexTM reagent cartridge on the system. After the tests are completed, the computer calculates and stores a set of linearization coefficients much like described in Example 1.
5. Schedule a vitamin B12 test via the on-board computer. Load the specimen processed in steps 2 to 3 and a B12 FlexTM reagent cartridge containing the same lots of reagents as used in the Calibration step. The magnet-equipped Dimension~ clinical chemistry system performs the entire test automatically and uses the stored linearization coefficients to compute the concentration of vitamin B12 by applying the LOGIT
function in a reverse manner. The sequence or events by which the system performs the vitamin B12 test follow.
6. The Dimension~ clinical chemistry system forms two cuvettes ~A and B). The entire cuvette wheel is contained in a chamber which is maintained at approximately 37C.
7. An aliquot of IFC (100 ~L) ~s automatically withdrawn from the reagent cartridge and dispensed into Cuvette A by the system's Reagent Pro~e.
8. After 90 seeonds~ a sample of the specimen (50 ~L) is wi~hdrawn from the Sample Cup and dispensed in~o Cuvette A by the system's Sample Probe. The Probe is vibrated ultrasonically for 2 seconds.

~ " .

-- .

~3~b~rS;1~, 9. After an incubation period of 180 to 360 seconds? an aliquot of B12-CPR suspension (80 ~L) is withdrawn from the reagent cartridge and dispensed into ~uvette A by the Reagent Probe. The Probe is S ultrasonically vibrated for S seconds while immersed in the B12-CPR suspension in reagent cartridge 18 and vibrated for 2 seconds after dispensing the suspension into cuvette 17.
`10. The m~xture is allowed to incubate for approximately 2 minutes.
11. While Cuvette A is incubated, an aliquot of CPRG (275 ~L) is withdrawn from the reagent cartridge and dispensed into Cuvette B by the Reagent Probe.
12. Approximately 2 minutes after the dispensing of B12-CPR, the Photometer Arm is moved to allow the permanent magnet to act on Cuvette A directly.
13. The Photometer Arm is held stationary for 20 seconds. The ma~netized B12-CPR particles are held to the bottom and one side of the cuvette.
14. The Reagent ~robe withdraws a 50 ~L aliquot of the supernatant, now free of B12-CPR particles, from Cuvette A and dispensçs ît into Cuvette B. After dispensing the liquid~ the probe i5 vibrated ultrasonically for 2 seconds.
15. Absorbance measurement on Cuvette B is taken both at 10-second and 100-second aft~r the completion of step 14. The difference of absorbance be~ween 577 nm and 700 nm of both measurements is compu~ed. The results for each measurements are recorded as rA and rB, respectively.

16. ~he difference between r and rA is computed and recorded as the photometric signal. The signal is used c compute the concentration of vitamin B12 using -the stored linearization coefficients by applying the LOGIT function in a reverse manner.

2~

,, , ` ~' .

, 27 ~ 3~

Results:

Table 3: Vitamin B12 Assay Calibration Results Assigned Signals ~m~) Mean Vslues 5~mEl9~l 8gmplQ_~ $i~nal ~ y~
O. pg/mL 697 772 73453.0 7.2%
25 pg/mL 823 842 83313.0 1.6%
10~00 pg/mL 851 84g 8501.3 0.2%
400 pg/mL 890 889 8~00.4 0.05%
1,000 pg/mL 9371007 972~9.0 5.1%
# SD: Standard Deviation.
* CV: Coefficient of Variance. It is calculated by dividing SD with Mean.

Table 3 exhibits the results of vitamin B12 calibrators tested by the procedures described above.
The 400 pg/mL calibrator was diluted with equal volume of the 0 pg/mL calibrator and tested by the system. The sample has a theoretical vitamin B12 concentration of 200 pg/mL. The Dimension~ system produced a result of 220 pg/mL. This result is sufficiently close to the theoretical value to be clinically useful.

E~am~le 3 En~YmeimmunQ~%aY~
It is conceivable that the Dimension(r) system equipped with ~he magnet module o the invention can be used to perform another type of heterogeneous immunoassay, commonly known as Enzyme Immunoassay ~EI~).
The modified system can perform at least two types of EI~, sandwich immunoassay and competit~ve immunoassay, by employing appropriate program commands. The sequence of events required for these two types of ~ests is described as follow.

;
.

--:

r~

SANDWICH IMMUNOA~SAYS: In this type of tests, magnetizable chromium dioxide particles ~CrO2) aré used as solid support, on which an antibody (Capturing Ab) to an analyte of interest is covalently coated. An aliquot of the suspension of CrO2 and a specimen, in which the analyte of interest resides, are dispensed into a cuvette (Cuvette A) of the Dimension~ system in close succession. Either immediately afterward or followinq an incubation period, a solut$on containing an Detector Antibody-enzyme conjugate is dispensed into the same cuvette. F~llowing a second incubation period, the magne~ is moved next to Cuvette A, achieving magnetic separation of CrO2 from the liquid. The Reagent Probe is commanded to withdraw the entire liquid content from the cu~ette.
Thereafter, a cleanin~ solution, consisting of either water or an appropriate buffer, is dispensed into Cuvette A. The Probe is ultrasonically vibrated for a pre-programmed period to enhance mixing of the particles in the cleaning solution. The magnet is held next to the cuvette, such that as soon as the ultrasonic vibration subsides, the particles are attracted to one side and away from the liquid. ~he Particle Washing routine, which consists of removal of contaminated liquid, dispensinq of fresh cleaning solution, ultrasonic mix and CrO2 separation is performed twice thereafter.
These steps, conceivably performed automatically by the Dimension~ system, results in a CrO2-hound "sandwich~' which consists of, in sequence, the Capturing Ab, the analyte of interest and the Detector Antibody-enzyme con~ugate. The solid-phase bound sandwich conglomerate is substantially free of contaminatinq serum components and excessi~e Detector An~ibody-enzyme conjugate molecules due to the trice washinq procedure.

2~

,, ~ -;

:` , , ~ u ~ u J ~J

Upon completing the Particle ~ashing routine, the magnet is moved away from Cuvette A. A solution containing an appropriate substrate of the enzyme is dispensed onto the CrO2 particles, now substantially free of liquid. ~he Reagent Probe is vibrated ultrasonically to achieve mixing of the particles. The enzyme molecules on the solid-phase bound sandwich is allowed to react with the substrate for a pre-programmed period of time.
During the reaction period, a quench solution, which is capable of stopping the enzyme reaction, is dispensed into a second cuvette (cuvette B), which commonly follows Cuvette A. Upon completion of the enzyme-to~substrate reaction, the magnet is moved next to Cuvette A which again separates the CrO2 from the liquid. The Reagent Probe withdraws a portion of the liquid from Cuvette A and dispenses it into Cuvette B.
Upon mixing with the quench solution, any residual enzymatic reaction is stopped. Two photometric measurements are taken on Cuvette B, one before and one after the liquid transfer. The difference between the two measurements, which is stoichiometric to the concentration of the analyte, is computed and recorded as signal.
Similar to the Calibration procedure described in Example 1, a eet of calibrators with known concentration of the analyte is tested before any unknown specimen.
The signals resulted from the testing of the calibrators are computed by a reyression functlon to derive a set of regression coefficient-~. These coefficients are stored ~n the computer memory and used for comput~ng the concentration of the analyte of an unknown specimen tested subsequently.
COMPETITIVE IMMUNOASSAYS: This type of test deviates from t~e Sandwich Immunoa~say in that a . ~
- , . . .

. .

-:

purified form of the analyte or its chemical derivative is covalently coated on the solid support, the chromium dioxide particles ~Ag-cro2)~
An aliquot of Ag-CrO2 suspension, a specimen containing the analyte of interest and a Detector Antibody-enzyme con~ugate solution are dispensed into Cuvette A in a similar fashion as those descrlbed in the Sandwich Immunoassays. Similar incubation periods are allowed.
The particle was~ing routine, as described in the Sandwich Immunoassays, is employed to clean the particles three times. In this type of test, the CrO2-bound analyte molecules compete against the analyte molecules provided by the specimen for the binding sites on the Detector-Antib~dy molecules. The competitive reaction results in a linkage of CrO2--bound analyte to the Detector Antibody-enzyme conjugate. The concentration of such linkage is inversely related to the concentration of the analyte in the specimen.
The substrate solution dispensing, ultrasonic mixing, incubation in Cuvette A are all performed in a manner similar to the Sandwich Immunoassays. Subsequent dispensing of quench solution into Cuvette B, magnetic separation in Cuvette A, liquid transfer, photometric measurements and compu~ation of the test results are all processed in a fashion similar to the Sandwich immunoassays.

~ ~., ~ .

, ~
., , , " ~

Claims

1. An automatic apparatus is provided for separating particles from aqueous particle mixtures disposed in a plurality of reaction vessels, the particles being responsive to a magnetic field, comprising:
a transport means for moving the vessels in sequence past at least one processing position, a robotic arm for selectively processing the vessels as they move sequentially, and means for selectively subjecting the vessels to a magnetic field as they move sequentially, thereby to separate the particles from the aqueous dispersion.

2. The automatic apparatus set forth in claim 1 wherein the transport means indexes the vessels stepwise past the processing position.

3. The automatic apparatus of claim 2 wherein the reaction vessels each have walls and a longitudinal axis that is generally vertically disposed, and the subjecting means field is generally transverse to the longitudinal axis, whereby the particles are drawn toward a wall of the vessel.

4. The automatic apparatus of claim 2 wherein the subjecting means is positioned on the robotic arm.

5. The automatic apparatus of claim 3 wherein the subjecting means is positioned on the robotic arm.

6. The automatic apparatus of claim 4 wherein the subjecting means subjects a reaction vessel located ahead or behind in the sequence of the position of the robotic arm relative to a vessel.

7. The automatic apparatus of claim 6 wherein the subjecting means simultaneously subjects three sequentially positioned vessels to the magnetic field.

8. An automatic apparatus for use in assays for separating magnetic particles from a liquid phase, the apparatus comprising:
a plurality of reaction vessels adapted to hold magnetic particles dispersed in a liquid phase, a reaction vessel mounting wheel adopted to move the reaction vessels in sequence past at least one processing position, probe means for dispensing liquid into and withdrawing liquid from the vessels, a reaction monitoring arm mounted for relative movement with respect to the wheel and to be positioned with its periphery adjacent any of the reaction vessels, a detector positioned on the arm radially outside the reaction vessels, the monitoring arm being operable to direct a beam of interrogating radiation through each vessel to the detector, and coupling means coupled to the arm for positioning a first magnet adjacent the location of the beam of interrogating radiation, but spaced therefrom by the distance separating adjacent reacting vessels on the wheel.

9. The automatic apparatus of claim 8 wherein the magnet is mounted on the detector radially outside of the reaction vessels.

10. The automatic apparatus of claim 8 which includes two additional magnets positioned by the coupling means to be contiguous the first magnet and also the next two sequestral vessel locations on the wheel.

11. The automatic apparatus of claim 10 wherein the coupling means is a bracket secured to the periphery of the arm.

12. The automatic apparatus of claim 10 wherein the probe means is adapted to transfer liquid in a vessel subjected to the magnet to another vessel free of the magnetic particles.