CA2128317A1 - Differential separation assay - Google Patents
Differential separation assayInfo
- Publication number
- CA2128317A1 CA2128317A1 CA002128317A CA2128317A CA2128317A1 CA 2128317 A1 CA2128317 A1 CA 2128317A1 CA 002128317 A CA002128317 A CA 002128317A CA 2128317 A CA2128317 A CA 2128317A CA 2128317 A1 CA2128317 A1 CA 2128317A1
- Authority
- CA
- Canada
- Prior art keywords
- bound
- binding agent
- fluorescent
- analyte
- free
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
Abstract
An electrophoresis-based assay system for detection of one or more target substances, i.e. an analyte tagged with fluorescent binding agents. The analyte is reacted with an excess amount of fluorescently tagged binding agent. The reaction mixture is subjected to electrophoresis and the migration of bound and free fluorescent substances are timed at a location where there is a spatial and optical differentiation of the two substances. An optical detector supplies signals corresponding to fluorescent amplitudes of the two substances. The free fluorescent substance arrives at a time expected from calibration runs. The optical signal is a marker for a second time, either earlier or later, when bound substrate should have arrived. Recorded data is searched to establish the relation between free and bound dye among the recorded optical signals. An absence of a bound dye signal infers the absence of target analyte in a sample. The amounts of bound and unbound amounts of the same fluorescent substance may be related by ratio of amplitudes of the optical signals so that the amount of target analyte may be estimated.
Description
WO93/1539~ PCT/US92/05537 ,,..~-.
2~28~1~
Dîfferential Separation Assay Technical Field - .
The invention relates to an assay for molecular or microbiological agents and in particular to a fluores-cence marker-~ased electrophoretic system for detecting such agents.
~Background Art In U.S. Pat. No. 4,811,218 M. Hunkapiller et .
;~ al. teach a DNA sequencing system using a multiple lane electr~phoresis apparatus. Fluorescent dyes are attached to molecules moving through the lanes. A moving Illumi-: nation;and detection:syst~m scans the multiple lanes.
Pour~color data points a:re re orded for each of several lanes at a particular time at a fixed dista~cs down the gel:. Through a complex:analytic procedure, the four col~
ors;are;related~to the concentrations of four dye-Iabeled DNA::components. :The object is to identify concentrations o~ A,~C,; G, or T or G, G+A j C~T or C which are DN~ piece endings~where A - adenosine, C = cytosine, G = guanine 2~5 ~: and~T = thymine.~ Peak concentrations of a particular dye label~are matched~:with particular bases in DNA sequen~es~
In U.S.~ Pat. No.~4,890,247 Sarrine e al~ de-scribe an appara~us~which robotically handl~s a plurality f liquid:samples in~test tubes, applies the sample~ to 30 electrophoresis matrices and then carries out electropho-resis~ :The electrophoretically separated molecul~s arè
illumina~ed wi~h fluorescent light. An an~log signal is p~oduced, repre entlng the scanned ~ield of view. A com-: puter stores intensi~y levels o~ the analog ~ignal: and performs densitometric analysis to read the electropho-retic data. Densitometry is a conventional prior art technique for reading such data.
~2~3~ 2-In an article entitled "Affinity Electrophore- -~
sis~l by Vaclav Horejsi, report~d~in "Enzyme Purification and Related Techniques", W. ~akob~ ~d., Academic Press, 1984, p. 275 a novel type of ele~rophoresis is de-scribed. One lane of the g~l ~ èdium is impregnated with immobilized liyands capable of reacting with a migrating macromolecule, while another lane, a control gel, is un-treated. Thus, a comparison can be made, using ~lectro-phoresis, ~etween a macromolecule sample retarded by the a~finity gel lane and a similar sample in the control gel lane. In a variation of this technique, the gel may in-corporate an antibody which interacts with a migra~ing antigen. The two lanes may be calibrated so that dif~er-ent degre~s of rstardation, for different concentrations of the migrating macromolecu~e, are known. Moreover, microscopic beads treated with ligands can be entrapped in the gel and similarly servP as a retardant. B~ads have the advantage of tight packing in the gel if they are of appropriate size. Activation of the gel involves partial cross-linking so that the gels do not melt on heating. Alternative methods of gel preparation are de-scribed, all with the result that a macromolecular re-tardant is immobiIized. Electrophoresis proceeds in the : usual way.
:: :: : : ~
: 25 ~ :: Whi}e the analytical systems of the prior art a~e~very use~ul for DNA analysis and the l?ke, they are not:suited for routine clinical laboratory applicatîons -~ ~ where the target substance is a large m~lecule or patho-gen,:such as a single macromslecule or a bact~rium.
, Clinical labs have a need for rapidly analyzing body fluids for an increasingly larger number of target ~:~ biochemical substances present at low concentrations that are indicators of various diseases ~uch as aardioYa~cular eases, immune disorders, cancer, microbial infection, etc. Mor over, the current increase ~nd sev~rity of : sexually transmitted diseases place~ an additional burden on laboratories as more tests are needed. An object of the i~ventisn is to devise a rapid, sensitive and precise WO93/15392 PCTJU~92/05537 21~4317 assay system for bioche~ical substances and pathogens especially suited to, but not limited to, clinical laboratories.
.~ :
Summary of the Invent`ion The above objact has been achieved in an assay system for substances of human origin or derived from pathogens which are typically the subject~ of clinical lab assays, hereafter called target analyte~. In a procedure, a binding agent with fluorescent properties becomes a fluorophore, which is combined with a known amount of target analyte; with the fluorophor~ present in -an excess amount of known concentration. The binding agent can be an antibody, antigen, lectin, receptor, v~
enzyme substrate or inhibitor or protein ligand or other chemical or biochemical with specific affinity for a corresponding molecule. The binding agent i~ specific for a particular substance of human or animal origin or substance derived from a pathogen. Th se substances :~
~could be of diagnostic utility for human or animal disea es or infectious diseases or biotherapeutlc u~ility. Besides bindiny with the target analyte, the excess amount of ~inding agent which has a relatively ~ different mobility than th target analyte and the : ~ 25 : analyte-binding agent pair and serves as a reference ~: ~ pointer or marker:for the target analyte~ Differences in :`
mobility arise because of differences in charge-to-mass :~
ratios.
The sample containing both the bound and free fluorescent binding agents are sub~ected to electrophore-sis in ~ gel. "Bolmd" binding agent is that which has reacted or complexed with another substance. "Free"
binding agent is that which has not reacted. A slit or :: pinhole is used to limit illumination of the gel o a
Dîfferential Separation Assay Technical Field - .
The invention relates to an assay for molecular or microbiological agents and in particular to a fluores-cence marker-~ased electrophoretic system for detecting such agents.
~Background Art In U.S. Pat. No. 4,811,218 M. Hunkapiller et .
;~ al. teach a DNA sequencing system using a multiple lane electr~phoresis apparatus. Fluorescent dyes are attached to molecules moving through the lanes. A moving Illumi-: nation;and detection:syst~m scans the multiple lanes.
Pour~color data points a:re re orded for each of several lanes at a particular time at a fixed dista~cs down the gel:. Through a complex:analytic procedure, the four col~
ors;are;related~to the concentrations of four dye-Iabeled DNA::components. :The object is to identify concentrations o~ A,~C,; G, or T or G, G+A j C~T or C which are DN~ piece endings~where A - adenosine, C = cytosine, G = guanine 2~5 ~: and~T = thymine.~ Peak concentrations of a particular dye label~are matched~:with particular bases in DNA sequen~es~
In U.S.~ Pat. No.~4,890,247 Sarrine e al~ de-scribe an appara~us~which robotically handl~s a plurality f liquid:samples in~test tubes, applies the sample~ to 30 electrophoresis matrices and then carries out electropho-resis~ :The electrophoretically separated molecul~s arè
illumina~ed wi~h fluorescent light. An an~log signal is p~oduced, repre entlng the scanned ~ield of view. A com-: puter stores intensi~y levels o~ the analog ~ignal: and performs densitometric analysis to read the electropho-retic data. Densitometry is a conventional prior art technique for reading such data.
~2~3~ 2-In an article entitled "Affinity Electrophore- -~
sis~l by Vaclav Horejsi, report~d~in "Enzyme Purification and Related Techniques", W. ~akob~ ~d., Academic Press, 1984, p. 275 a novel type of ele~rophoresis is de-scribed. One lane of the g~l ~ èdium is impregnated with immobilized liyands capable of reacting with a migrating macromolecule, while another lane, a control gel, is un-treated. Thus, a comparison can be made, using ~lectro-phoresis, ~etween a macromolecule sample retarded by the a~finity gel lane and a similar sample in the control gel lane. In a variation of this technique, the gel may in-corporate an antibody which interacts with a migra~ing antigen. The two lanes may be calibrated so that dif~er-ent degre~s of rstardation, for different concentrations of the migrating macromolecu~e, are known. Moreover, microscopic beads treated with ligands can be entrapped in the gel and similarly servP as a retardant. B~ads have the advantage of tight packing in the gel if they are of appropriate size. Activation of the gel involves partial cross-linking so that the gels do not melt on heating. Alternative methods of gel preparation are de-scribed, all with the result that a macromolecular re-tardant is immobiIized. Electrophoresis proceeds in the : usual way.
:: :: : : ~
: 25 ~ :: Whi}e the analytical systems of the prior art a~e~very use~ul for DNA analysis and the l?ke, they are not:suited for routine clinical laboratory applicatîons -~ ~ where the target substance is a large m~lecule or patho-gen,:such as a single macromslecule or a bact~rium.
, Clinical labs have a need for rapidly analyzing body fluids for an increasingly larger number of target ~:~ biochemical substances present at low concentrations that are indicators of various diseases ~uch as aardioYa~cular eases, immune disorders, cancer, microbial infection, etc. Mor over, the current increase ~nd sev~rity of : sexually transmitted diseases place~ an additional burden on laboratories as more tests are needed. An object of the i~ventisn is to devise a rapid, sensitive and precise WO93/15392 PCTJU~92/05537 21~4317 assay system for bioche~ical substances and pathogens especially suited to, but not limited to, clinical laboratories.
.~ :
Summary of the Invent`ion The above objact has been achieved in an assay system for substances of human origin or derived from pathogens which are typically the subject~ of clinical lab assays, hereafter called target analyte~. In a procedure, a binding agent with fluorescent properties becomes a fluorophore, which is combined with a known amount of target analyte; with the fluorophor~ present in -an excess amount of known concentration. The binding agent can be an antibody, antigen, lectin, receptor, v~
enzyme substrate or inhibitor or protein ligand or other chemical or biochemical with specific affinity for a corresponding molecule. The binding agent i~ specific for a particular substance of human or animal origin or substance derived from a pathogen. Th se substances :~
~could be of diagnostic utility for human or animal disea es or infectious diseases or biotherapeutlc u~ility. Besides bindiny with the target analyte, the excess amount of ~inding agent which has a relatively ~ different mobility than th target analyte and the : ~ 25 : analyte-binding agent pair and serves as a reference ~: ~ pointer or marker:for the target analyte~ Differences in :`
mobility arise because of differences in charge-to-mass :~
ratios.
The sample containing both the bound and free fluorescent binding agents are sub~ected to electrophore-sis in ~ gel. "Bolmd" binding agent is that which has reacted or complexed with another substance. "Free"
binding agent is that which has not reacted. A slit or :: pinhole is used to limit illumination of the gel o a
3 5 narrow track or spot. The free fluorescent binding : agent, having a known and di~feren~ mobility than the bound material will move past the stationary ~iewing track. This serves as an internal marker indicating that W093/~539~ PCT/US92/05537 ~ 128 3 i~ 4 the assay system is functioning properly~ The bound fluorescent substance may move past the viewing track prior to or subsequent to the unbound material. By means of prior calibration, a time "window~' is associated for motion of one substance past the sli~;~r pinhole relative to the other. Motion of one substa~e past the slit creates an expectation of the ar ~al of the other within predetermined limits. With the peak detected ~ignal corresponding to the free substance b~ing used as a reference, in the actual run if the bound material does not arrive within the expected window, any other peak obtained outside the window is considered an artifact~
- The expected arrival times of free fluore~cent substance peaks are determined by calibration runs, as well as by peak levels. The intensities of bound and free fluorescent ~ubstances at the track are recorded and the :~ two substances are asso~iated by analysis of the time ~ separation between signal peaks, :~ As mentioned abo~e, a calibration procedure ~ establishes expected times when free and bound binding : agent will pass the track where migration times are meas-ured and recorded. These expected times are used to search for the presence of a target substance where the ; : presence is uncertain. For example, if the calibration ~: 25 run establishes that the unbound binding agent will pass - ~ ~ the track at a first time and the bound binding agent will pass the track at a second time~ the time differ-ence, averaged o~er a number of runs, creates xpected times of arrival at the tr~ck in particular gels. When an unknown substance is mixed with a binding agent spe-ific to the target analyte, in an amount in exc~ss of what will react with the target ~ubstance, there will be electrophoretic migration of free and bound binding ayent. The binding agent and the target substance, if : 35 present, will have the same fluorescent wavelength.
electxophoresis of the mixture is conducted, the times when substances having the fluorescent characteristic reach the slit are measured and recorded r After recQrd-W093/15392 ` PC~/US92/05537 ~I~8317 ing, the data is searched for each peak ~xhibiting the ~:;
fluorescent wavelength. The time differential between the arrival of bound and free binding agent at a slit is applied to each peak to see if a second peak lies at the differential time, within certain statistical limits called a "window". If 50~ the second peak is paired with the first peak t~ establish a bound and free dye relationship which reflects bound and free binding agent.
The presence of bound binding agPnt in turn indicates the 1~ presence of target substance and allows quantitation of taryet substances as described below.
Since the fluorescently tagged binding agents -~
~re ~pecific to targ~t analytes, several different tags of different fluor~scent wavelengths may be used in the sam~ sample and gel lane. A filter wheel is used to ob~erve one wavelength at a time at the slit or pinhole.
For each color, a time domain association is formed of the amplitude of the free fluorescent substance, and the ampli~ude of the bound substance as they move past a slit. As a further step, amplitude ratios may bP
compared to calibration measurements to determine the presence and exact concentration of target analytes.
S~ch amplitude rativs preferably use thP area under each peak, rather than the peak height, f~r the ratio compu-25 ~tatio~. When the term "amplitude" is used in connectionwith such ratios,:the area under the peak is intended.
Electrophoretic separation is based on differing ~: charge/mass ratio,~charge alone or charge/mass/shape c :ontributions and can be optimized for a given analyte-3 0 binding ~gent combination .
When multiple fluorescent substances are u ed, the wavelength separation is pref~rably at least 10 nm.
: : By observing the passage o~ free and bound dye-macromolecule at a single location within a track, signal interferences due to any non-uniformities in the ~el, : such as polymerization irregularities, bubble~, etc~ are obviated.
WO93/1~392 PCT/US92/~5537 ~83~ -6-The invention rapidly predicts, in real time, the results of electrophoresis, without waiting for com-pletion of migration of the target substance or without waiting ~or production of colored or fluorescent materi-als by the separated biochemicals.
,~
Brief Description of the Drawings. ~
~ ig. l is a plan view of the apparatus of the present invention.
Fig. 2 is a top ~iew of a single gPl lan.
illustrated in Fig. l.
Fig. 3 is a plot of detector signals from -unbound and bound fluorescent material.
Fig. 4 is a plot of overlapping detector signals of different wavelength from unbou~d and bound fluorescPnt material.
Fig. 5 i~ a top ~iew of a multiple lane gel : arrangement for electrophoresis.
Fi~. 6 iS a plot of signal amplitude in volts :20 ~ersus time for detection of antibody to human serum alb~min~
~:~ Best Mode for Carrying Vut the Invention With~reference to Fig. l a single lane gel 2~5 :~;electrophoresis apparatus ll having a well 13 at one end with~a~negative voltage terminal 16 and a posi~ive high vol~age electrode terminal 15 at an opposite endO The eletrophore~is apparatus consists of a conventional sin-: gl~ lane 18~having a substrate 17, a gel layer 19 and a :30 protective glass cover 2l. The substrate is usually a elf-supporting material which may be glass, Mylar (trademark) or any well known gel support. ~he gel it-self is usually polyacrylamide or agarose~ although sther ~: : gel materi~ls such as synthetic acrylamide substitutes ; 35 may also be used. Unifo~m polymeriæation and fr~edom from bubbles and irregularities are desirabl~ prop~rties.
The glass cover is preferably.nonreflective glass which : : merely serves as a protective cover for the g81 . The W~ g3/15392 P~r/U~g2/0~537 -7--~l 283I 7 well 13 is normally positioned vertically 50 that it will receive a sample without spillage~ The well funnels a prepared sample toward the gel. The well may combine a stacking and separating gel and creates a spot of sample material on the gel. High voltage is then applied to the gel at terminals 15, 1~ and charged ions migrate toward the positively charged voltage electrode. The end of the gel near well 13 is maintained at negative sr ground potential so that there is a substantial potential dif- -ference from one end of the gel to the distant end.
The sample which is placed in well 13 is a fluid, frequently a frartionated blood sample. Blood may ~e pre-processed to remove constituents which will inter-fere with the assay. Removal may be by ~iltering, ab-sorption, centri~uging or precipitating either the de-~ired or undesired components so that a desired taxget an~lyte may be ob.tained for electrophoresis. The desired target analyte must be one for which there is ~ specific : binding agent. Fluorescent tags such as those commer-cially available are manufactured by Molecular Probes : Inc. of Oregon which sp cializes in dyes or dyed beads that can be covalently attached to binding agents9 Where : target analytes are fo~nd in larger structures, such as pathogenic agents, then such a dye-binding agent ¢onju-gate would be appropriate for tracking that pathogenic gent~ ~onoclonal antibodi~s can now be manufactured so that the behavior of this binding agent is unifo~m and : : predickable for m~ny assays. Monoclonal an~ibodie~ are more expensive than polyclonal antibodies, but the anti-bodies hav~ greater specificity, are directed toward I
single epitopes, are easy to produce in larg~ quantities ~; : and are generally more useful and cau5e precise separa-tion o f bound and free mat~rial.
m e tagged binding agent is supplied in excess ~ 35 so that th~ r~acti~n with the analyte will be driven to : completion, or nearly to completion in a reasonable or convenient amou~t of time. The amount of excess tag should not be more ~han twenty times th~ amount of W0~3/15392 PCT/US9~ 537 3~
expected maximum level bound tag, although the number may range between 2 and 50, approximately. The tagging sub- :
stance should alter the mass to charge ratio when com-bined with the analyte and subjected to an electro-phoretic field.
A strongly emitting light source, such as light emitting diode or laser ~liS used to generate a beam 25.
The LED 23 has an outpu~power of about 50 mW in a wave-length band which will-excite fluorescence in the fluo rescent tagging material. Such excitation radiation is known as actinic radiation. The beam i5 intercepted by a focusing lens 27 which directs the beam hrough a slit ~pertu~e in barrier 29. Light emerging from the slit is divergent and is intercepted by the collimating lens 31.
The beam is then directed onto a reflecting surface 33 which is part of a prism 35. The reflective surfac~ 33 is at a 45 degree angle to the beam so that the reflected beam makes a 90 degree angle with the inc:ident beam. The r~flected beam is directed toward fs:)cusing lens 37 where ~:
20 ~he~ beam passes through one half of the focusing lens, while the other half is reserved for li~ht tratreling in the opposite direc~ion, reflected from gel layer 19.
Light passing through the focusing lens carries an image : ~ of the slit 29 which is directed onto the gel layer 19.
~ ~ Fluorescent light emitted from tagged cQmplex and some reflected light from the gel layer travel~ in a retro-beam 39 to the left half of focu~ing lens 370 Note that one half Qf the focusing lens is used by light trav~
el:ing in each direction. The right half is used by the : 30 incoming beam, while the left half is used by the retroD
beam. From there, the retro-beam is directed to reflect-ing ~ur~ace 41 which is part of prism 35. The retro-beam passed through a filter 43 which rejects any light other than the desired wavelength from th~ fluoresce~t target. Light transmitted through the ~ilter is directed toward focusing lens 45. From there the beam is directed to a light detector, such as photomultiplier tube 47 with a slit located ~t the image plane of the gel.
W093/~392 ~ PCT/US92/0~537 The time of arrival of the fluorescent s~bstances is measured r~lative to the starting time, i.e. the application of high voltage which initiates electrophoretic migration. Since the arrival ~ime is not S precise, but rather is a Gaussian curv~, the peak time is recordedL Each target substance and the corresponding fluorescent binding agent are subject to the same procedure in the calibration run. In calibration runs a mean migration time to he measurement slit or pinhol~ is 10 det~rmined. Then, the standard deviation is determined ~-for the time of arrival of the free binding agent, as well as for the bound target substance. In the present ~invention, it is necessary to know the mean migration time, i.e., the expected arrival times of bound and free binding ag~nt for specific target substances because the times will be used to search for target an~lyte in a sample where the target substance is possibly present, but not necessarily present. The difference in arriYal :: times between the bound and free binding agent may be 20 used to establish a time window so that the arrival of :;
one member may be paired with the other member in a search for the other member. If the search reveals ~hat ~: the other member is present within a standard deviation : : or two, that other material is identified as a member of ~5 th~ pair. If nothing is found within the time window, the first member of the pair is regarded to be an ` artifact and is discarded.
The ou~put of the photomultiplier tube is :maintained in a buffer memory 4~ and a ratio may be formed between the signals representing bound and free dye labeled binding agent. A data reader 50 is connected to the bu~er mem~ry 49 for receiving recorded 5ignals which represent the ~luorescent peaks. The data reader : is a computer which correlates the ~arious peaksO Each ~ peak is recorded in order to search for bound and free fluorescQ.nt subs~ance in the recorded data. Normally, the time of appearance of the free fluorescent substance could be established from prior calibration times~ Once ~a3~l -10-th~ position of the free fluorescent substance peak is known, a search is conducted for the corresponding bound fluorescent substance which should be located a certain time interval away, within a time window defined by statistical limits. A peak within this window is identified as the bound fluor~s~cen$ substance, i.e. the target analyte. Next the am~,lltudes of the identified peaks are examined and a r~io is computed in the data ~:
reader 50. The m~thod whereby free fluorescent substance is correlated with bound fluorescent substance is explained further below. The computer also stores cali-brations of known concentrations of target substance so -that ratios may be compared in order to obtain an esti-mat of the unknown co~centration.
15In Fig. 2, the top view of gel 11 shows that the image 29' of slit 29 falls between a positive high voltage terminal 15 and a spot from well 13, coinciding with negative voltage terminal 16. In operation the high ~;~ voltage appli d to terminal 15 causes migration of bound and free tagged binding agents, which are positively or negatl~ely chargQd molecules which respond to the elect:ric field from the high voltags supply. The free tagged binding agen~ will reach the image 29' of slit 29 which is fixed in position at a time different than the :~ :25:: bound tagged binding agent. The unbound tagged ~inding agent serves as~ one marker for a time window which has the bound tagged~binding agent as a corresponding marker, the~two:markers forming a pair of markers which are ::separate~ in time within the statistical limit whirh i5 defined.
I With reference to Fig. 3, a plot of the detec-:~ tor signal is~shown ~here the horizontal axis is time and : the vertical axis;is amplitude of the detected signal.
s an example, electrophoresis begins at a first time, t = 0, and the detector is made op~rati~e. At a second~ime, t2, a relatively large peak 51 i5 ob erved~
representing free fluorescent materia} of a first color.
Ansther signal 54, discussed below, is detected after WO93/15392 PCT/US9?/0~537 -11- 2l283l 7 peak 51. A time later, t3, a weaker signal 53 of the same color is observed. The peak 53 exists in the midregion of a window, Wl, between Xl and X2. The existence of window Wl i5 established by the strong free S fluorescent material signal 51. Peak 53 is within window Wl and is recognized as a bound fluores~ent matsrial signal. Peak 54 is no~ within window ~l and is treated as a false positive or artifact, after being checked to determine whether the-signal is not mistaken for the free fluorescent material signal 51. A search of all signals ~ i5 made to determine the most logical positions for free :~ and bound fluorescent substances. If no signal is found .
-in time window W1, the absence of target analyte is inferred. Each:window W acts as a time domain ~ilter, allowing discrimination of spurious fluorescent signals and noise. No~e that all signals are recorded and signal discrimination occurs after recordin~ by ~nalyzing reoorded data. Even:khough gel to gel characteristics may vary, the present invention has immuni~y to most ~variations because the bound and free fluorescent substances tra~erse the same path.
The ratio of the tWQ signals represented by the area under the peaks 51 and 53 represents an estimate of the ratio o~ a~bound to free fluorescent substances, 2~ after normalizing data relative to calibrations, assuming ood~bindin~ efficiency. A further time latert another : large peak 55: is obserYed. This represents another free luorescent binding agent. This defines another time window W2 at a~subseq~ent time and:a lesser peak 57 is measured in the window. This is taken to represent a bound ~luorescent material. Again, the ratio of bound : to free dye is~computed and once again the target analyte associated with the second dye may be stimated in ~ , : concentration.:
It is possible for the peaks to overlap each other as shown~in Fig. 4. Here, the first free fluorescent substance peak 61, having a rela~ively large ~: amplitude, overlaps ~he second peak 65 of similar :: :
W093/l~39~ PCT/US92/05537 ~ 12-amp~itude in a test where two different fluorescent substances were used~ The second peak 65 is the ~econd free fluorescent ~ubstance signal. However, hecause dif-ferent colors are used, as separated by the filter 43 in Fig. 1, the two peaks may be separately observed. Peak 61 establishes the time window W3r~w~ere a peak 63, representing a bound fluorescently tagged binding agent :~
of a color which is the same--as that associated with the unbound peak 61, occurs totally within the second peak 65. Nevertheless, because of the filter 43, peak 63 may be spatially and optically differentiated from peak 65.
The ratio of bound to unbound signal amplitudes appears to be about 2:1. The corresponding mol~cular amounts of bound and unbound tagging material are estimated to be in ~5 the ~ame rati~. For the peak 65, a time window W4 is :~
established, but no fluorescent signal is found within the window and so the absence of tar~et analyte i~
inferred.
With reference to Fig. 5, a multiple lane elec- .-trophoresis sheet gel is shown. The sheet 71 is prQvided with two lanes 73 and 75. Each of thc lanes has a re- :
spective well 83 and 85 and a rPspective slit image 87 ~ and 89. The two lanes are constructed similarly, with : the slit image locations in the same position. Lane 73 is used to run a calibrated amount of target analyte and a k~wn amount of free fluore~cently labeled binding agent. In lane 75 an unknown amount of target analyte is run with free fIuorescently tagged binding age~t. The ~; two lanes may be compared to determine the amount o~
~nknown analyte in lane 75. For greater accuracy, multiple runs may be made in lan. 73 of various amounts : of target analytes so that many ratios may be stored in a :: memory. A r~tio ~rom a run of an unknown amount o~
target analyte may then be looked up and compar~d with ~nown ratios, with the be5t match indicating the ~mount o~ targe~ analyte.
One of the advantages of the pre~ent i~v~ntion i5 that analysis of peaks representing bound and free dye W~3~15392 P~T/US9~ 537 ~128~17 can be computed before electrophoresis is complete, i.e.
before the migrating substances reach the distant high voltage alectrode. Another advantage is that the present system uses only a single lane of an electrophoresis apparatus so that ~e.l to gel non-uniformities are nulled.
It is possible to use a ~econd lane in an electrophoresis device as a reference or calibration, but such calibra-tions may be done beforehand and results stored in a memory. It is also po~sible to use a second or third or ~ fourth lane for additional analytes of interest cr2ating panels of rele~ant analytes. In the prior art, analysis o~ target analytes usually requires completion of the ~lectrophoresis and subsequent analysis by a plurality of stains t colored or fluorescent substrates, etc. Using the present in~ention, the analysis may be done in real time as soon as sufficient separation exists betwéen the `~: bound and free ~luorescent material. Such a separation ~: can be at a point which is only twenty five percent or thirty three percent of the length of a lane. Once a point is found where adequate separation exists, the image of the slit or pinho~e is p~siti~ned at that ~: location and then all measurements are made from there.
: It is als:o to be noted that this is an open-ended electrophoresis system, i.e. there is no need to stop the electrophoresis at a defined point to get all materials "on scale". Materials that migrate ~lowly can be detected just as well as fast moving targ t analytes.
: Amplitude threshoIds may be used as furth~r dis~rimina-tion against noise and artificial signals.
To discriminate between two or more fluo-rescentiy tagged target substances in the same gel lane, different fluorescent w~velengths can be used, so long a~
filter 43 in FigO 1 can adeguately resolve the different : : wavelengths~ Multiple tests can be run simultaneously, each te~t associated with a partiGular wavelength.
Example l. Detection of proteins present in human blood.
Creatine kinase is an enzyme present in various mammalian WO93J1~392 PCT/US~2/05537 ~2~3~ -14-tissue. It occurs in three different forms known as iso-enzymes: CK-MM (skeletal), CK-MB (cardiac) and CK-BB
(brain). After release from tissue and on circulation in blood the MM and MB forms themselves break down to small-er fragments known as isofo~ms or subforms. In the eventof myocardial infarction, the MB isoenzyme, present in cardiac muscle, is releàsed into plasma. Hence, it serves as a specific diagnostic molecular marker for car-diac ischemia or necrosis. The early and rapid detection of this isoenzyme and iks isoforms are ~ery crucial for the diagnosis of myocardial infarction and for initiating thrombolytic therapy.
To perform the test, a blood sample is separat-ed into pIasma and red blood cells. The plasma is mixed with Qxcess antibody tagged with a fluorescent dye which is directed against CX-M~ . The attachment of f luorescent : a~tibodies for a CK assay is known and is described in : U.S. Pat. No. 4,353,982 to M. Gomez et al. If CK-~B is present in plasma, an immune complex consisting of CK-MB
and fluorescently tagged antibody will be formed. On application of an electric field, the reactio~ mixture : : consisting of the fluorescent immune comp~ex and the un-~reacted fluorescent antihody, will migrate on the gel.
Because of charge and:mass differences, the labeled in tact immune complex will migra~e differently than the labelled antibody. The fluorescence associated with bound and fre2 markers will be detected and arrival times : measured and recorded~ Free marker is id2ntified by a large peak. Any substance within the expected time of the free substance is regarded o be target analyte.
Anything else is an artifact.
~; Example 2. Detection Qf the presence of sexually transmitted diseases. Many sexually transmitted patho-35 gens such as chlamydia, herpes, etc. ~orm lesions in the :~ uro-genital area. For detection of these pathogens, sam-ples are taken With a swab directly from the lesion and a number of different types of tests are performed on this W093/15392 PCT~US92/~5537 extract. These tests include culture and/or immunochemi-cal tests.
After a lesion is sampled with a swab, the swab is treated with a solubilization reagent to liberate mi-5 cro-organisms present. This process will also solubilize target analytes originating from the micro-organisms~
This extracted solution will be filtered and reacted with fluorescently tagged antibody so that there is a substan-tial excess of unreacted tagging substanc~. The differ-lO ential assay proceeds as described above.
The methods described above accslerateselectrophoresis by varying amounts, depen~ing on where ~he image 29' of the~slit (~ee Fig. 2) is positioned. If the difference between the charge, mas~ and shape of the l5 bound and free substances is great, early separat,ion may ~; be expected and the image location 29' may be moved close to~well 13~ the~difference between the two 9uantities i5 not great,~the image location 29' needs to be further away from well 13 to~allow longer separation time o~ the 0~bound~and unbound fluorescent substance. In either ca~a, the results of elec~rop~,oresis are predicted at an earli-er~time than a complete electrophoresis run.
Example 3. ~ Detection of antibody to human serum 25~ album~in (HSA). ~ In~the following example an antibody a~ainst HSA is the;target substance which is detected by tagging w:ith ~luoresc2nt HSA. HSA, Fraction V, was obtained~ from~S~igma :Chemical Company (st. I,ouis, M0~ .
Monoclonal anti~-HSA ~was ob~ained from Biospacific Inc., California. Cy5-labelled HSA was synthesized by the coupling of Cy5 fluorescent dye (Biological Research) to NSA and was obtained from ~ole~ular;Probes (Eugene, Oregon). This fluorescent substan e is ~he binding agent. ~
Di~erential separation assay (DSA) was done as follows: C:y5-labelled HSA (binding agent) was incubated ::
with msnocl~nal anti-HSA (target) at a f inal concentra-tion of 400 ng/ml Cy-HSA and 200 ug/ml anti-HSA in 0. 09 M
: ~ :
W093~5392 PCT/~S92~05~37 ~ 3~ -16-Tris, 0.08 M borate, 0.26 mM EDTA, pH 8.3. A control sample consisted of Cy5-HSA alone at 400 ng/ml without added antibody. Reactions were performed in 1.5 ml Eppendorf tubes in a total reac~ion volume of 20 ul.
5 After incubating the samples at room temperature (20~C3 for 30 minutes, 10 ul aliquots~were loaded onto 6%
nondenaturing (8 cm x 10 cm xji.'75 mm) polyacrylamide gels (Jule labs) containin~;~.9 M Tris, 0.8 M Borate, 2~6 mM EDTA, pH 8.3. Electrophoresis was performed at 100 V
for 40 minutes using a Hoe~er Mighty Small SE200 system.
The real time detection of fluorescent proteins : during electrophoresis was performed using a He-Ne laser beam focussed at a point 1.3 cm below the wells of the gel. The reflected fluorescence was collected u~ing a 15 photomultiplier (PMT) tube. Data was collected using a Labwindows (Trademark3 data acquisition board on an IB~-;~ PC and imported into a Microsoft Windows Excel file for analysis and graphics.
Samples containing excess Cy5-HSA were reacted 20 wi~h excess monocIonal anti-HSA and then were loaded onto m : 6% acrylamide gels:. Figure 6 shows signal amplitude as a ~: ~ signal in volts which is proportional to the reflected f luorescencQ . Separation on this gel system is based on charge/mass characteristics of the proteins and more 25 rapidly migrating species migrate past the laser beam earlier than more slowly migrating protein ~peciesO
The Cy5-HSA peak 91 migrates past the laser beam at approximately 8 minutes. This is a calibration run to establish a:time for free Cy5-HSA~ The immune 30 complex consisting of ~Cy5-HSA~ - Anti-HSA~, on the ~other hand, has a peak 93 which migrates past the laser ~pot at 25.5 minutes~ This example demonstrakes that the rel~ant time window for ~his pair of binding agent ~ANTI-HSA) and fluore~cent tag (Cy5-HSA) is 17~5 minutes.
35 The 8 minute peak 91 defines tha reference posi ion in the data acquisition window for finding the peak g3 of the immune complex ~Cy5-HSA) - Anti-HSA3.
- The expected arrival times of free fluore~cent substance peaks are determined by calibration runs, as well as by peak levels. The intensities of bound and free fluorescent ~ubstances at the track are recorded and the :~ two substances are asso~iated by analysis of the time ~ separation between signal peaks, :~ As mentioned abo~e, a calibration procedure ~ establishes expected times when free and bound binding : agent will pass the track where migration times are meas-ured and recorded. These expected times are used to search for the presence of a target substance where the ; : presence is uncertain. For example, if the calibration ~: 25 run establishes that the unbound binding agent will pass - ~ ~ the track at a first time and the bound binding agent will pass the track at a second time~ the time differ-ence, averaged o~er a number of runs, creates xpected times of arrival at the tr~ck in particular gels. When an unknown substance is mixed with a binding agent spe-ific to the target analyte, in an amount in exc~ss of what will react with the target ~ubstance, there will be electrophoretic migration of free and bound binding ayent. The binding agent and the target substance, if : 35 present, will have the same fluorescent wavelength.
electxophoresis of the mixture is conducted, the times when substances having the fluorescent characteristic reach the slit are measured and recorded r After recQrd-W093/15392 ` PC~/US92/05537 ~I~8317 ing, the data is searched for each peak ~xhibiting the ~:;
fluorescent wavelength. The time differential between the arrival of bound and free binding agent at a slit is applied to each peak to see if a second peak lies at the differential time, within certain statistical limits called a "window". If 50~ the second peak is paired with the first peak t~ establish a bound and free dye relationship which reflects bound and free binding agent.
The presence of bound binding agPnt in turn indicates the 1~ presence of target substance and allows quantitation of taryet substances as described below.
Since the fluorescently tagged binding agents -~
~re ~pecific to targ~t analytes, several different tags of different fluor~scent wavelengths may be used in the sam~ sample and gel lane. A filter wheel is used to ob~erve one wavelength at a time at the slit or pinhole.
For each color, a time domain association is formed of the amplitude of the free fluorescent substance, and the ampli~ude of the bound substance as they move past a slit. As a further step, amplitude ratios may bP
compared to calibration measurements to determine the presence and exact concentration of target analytes.
S~ch amplitude rativs preferably use thP area under each peak, rather than the peak height, f~r the ratio compu-25 ~tatio~. When the term "amplitude" is used in connectionwith such ratios,:the area under the peak is intended.
Electrophoretic separation is based on differing ~: charge/mass ratio,~charge alone or charge/mass/shape c :ontributions and can be optimized for a given analyte-3 0 binding ~gent combination .
When multiple fluorescent substances are u ed, the wavelength separation is pref~rably at least 10 nm.
: : By observing the passage o~ free and bound dye-macromolecule at a single location within a track, signal interferences due to any non-uniformities in the ~el, : such as polymerization irregularities, bubble~, etc~ are obviated.
WO93/1~392 PCT/US92/~5537 ~83~ -6-The invention rapidly predicts, in real time, the results of electrophoresis, without waiting for com-pletion of migration of the target substance or without waiting ~or production of colored or fluorescent materi-als by the separated biochemicals.
,~
Brief Description of the Drawings. ~
~ ig. l is a plan view of the apparatus of the present invention.
Fig. 2 is a top ~iew of a single gPl lan.
illustrated in Fig. l.
Fig. 3 is a plot of detector signals from -unbound and bound fluorescent material.
Fig. 4 is a plot of overlapping detector signals of different wavelength from unbou~d and bound fluorescPnt material.
Fig. 5 i~ a top ~iew of a multiple lane gel : arrangement for electrophoresis.
Fi~. 6 iS a plot of signal amplitude in volts :20 ~ersus time for detection of antibody to human serum alb~min~
~:~ Best Mode for Carrying Vut the Invention With~reference to Fig. l a single lane gel 2~5 :~;electrophoresis apparatus ll having a well 13 at one end with~a~negative voltage terminal 16 and a posi~ive high vol~age electrode terminal 15 at an opposite endO The eletrophore~is apparatus consists of a conventional sin-: gl~ lane 18~having a substrate 17, a gel layer 19 and a :30 protective glass cover 2l. The substrate is usually a elf-supporting material which may be glass, Mylar (trademark) or any well known gel support. ~he gel it-self is usually polyacrylamide or agarose~ although sther ~: : gel materi~ls such as synthetic acrylamide substitutes ; 35 may also be used. Unifo~m polymeriæation and fr~edom from bubbles and irregularities are desirabl~ prop~rties.
The glass cover is preferably.nonreflective glass which : : merely serves as a protective cover for the g81 . The W~ g3/15392 P~r/U~g2/0~537 -7--~l 283I 7 well 13 is normally positioned vertically 50 that it will receive a sample without spillage~ The well funnels a prepared sample toward the gel. The well may combine a stacking and separating gel and creates a spot of sample material on the gel. High voltage is then applied to the gel at terminals 15, 1~ and charged ions migrate toward the positively charged voltage electrode. The end of the gel near well 13 is maintained at negative sr ground potential so that there is a substantial potential dif- -ference from one end of the gel to the distant end.
The sample which is placed in well 13 is a fluid, frequently a frartionated blood sample. Blood may ~e pre-processed to remove constituents which will inter-fere with the assay. Removal may be by ~iltering, ab-sorption, centri~uging or precipitating either the de-~ired or undesired components so that a desired taxget an~lyte may be ob.tained for electrophoresis. The desired target analyte must be one for which there is ~ specific : binding agent. Fluorescent tags such as those commer-cially available are manufactured by Molecular Probes : Inc. of Oregon which sp cializes in dyes or dyed beads that can be covalently attached to binding agents9 Where : target analytes are fo~nd in larger structures, such as pathogenic agents, then such a dye-binding agent ¢onju-gate would be appropriate for tracking that pathogenic gent~ ~onoclonal antibodi~s can now be manufactured so that the behavior of this binding agent is unifo~m and : : predickable for m~ny assays. Monoclonal an~ibodie~ are more expensive than polyclonal antibodies, but the anti-bodies hav~ greater specificity, are directed toward I
single epitopes, are easy to produce in larg~ quantities ~; : and are generally more useful and cau5e precise separa-tion o f bound and free mat~rial.
m e tagged binding agent is supplied in excess ~ 35 so that th~ r~acti~n with the analyte will be driven to : completion, or nearly to completion in a reasonable or convenient amou~t of time. The amount of excess tag should not be more ~han twenty times th~ amount of W0~3/15392 PCT/US9~ 537 3~
expected maximum level bound tag, although the number may range between 2 and 50, approximately. The tagging sub- :
stance should alter the mass to charge ratio when com-bined with the analyte and subjected to an electro-phoretic field.
A strongly emitting light source, such as light emitting diode or laser ~liS used to generate a beam 25.
The LED 23 has an outpu~power of about 50 mW in a wave-length band which will-excite fluorescence in the fluo rescent tagging material. Such excitation radiation is known as actinic radiation. The beam i5 intercepted by a focusing lens 27 which directs the beam hrough a slit ~pertu~e in barrier 29. Light emerging from the slit is divergent and is intercepted by the collimating lens 31.
The beam is then directed onto a reflecting surface 33 which is part of a prism 35. The reflective surfac~ 33 is at a 45 degree angle to the beam so that the reflected beam makes a 90 degree angle with the inc:ident beam. The r~flected beam is directed toward fs:)cusing lens 37 where ~:
20 ~he~ beam passes through one half of the focusing lens, while the other half is reserved for li~ht tratreling in the opposite direc~ion, reflected from gel layer 19.
Light passing through the focusing lens carries an image : ~ of the slit 29 which is directed onto the gel layer 19.
~ ~ Fluorescent light emitted from tagged cQmplex and some reflected light from the gel layer travel~ in a retro-beam 39 to the left half of focu~ing lens 370 Note that one half Qf the focusing lens is used by light trav~
el:ing in each direction. The right half is used by the : 30 incoming beam, while the left half is used by the retroD
beam. From there, the retro-beam is directed to reflect-ing ~ur~ace 41 which is part of prism 35. The retro-beam passed through a filter 43 which rejects any light other than the desired wavelength from th~ fluoresce~t target. Light transmitted through the ~ilter is directed toward focusing lens 45. From there the beam is directed to a light detector, such as photomultiplier tube 47 with a slit located ~t the image plane of the gel.
W093/~392 ~ PCT/US92/0~537 The time of arrival of the fluorescent s~bstances is measured r~lative to the starting time, i.e. the application of high voltage which initiates electrophoretic migration. Since the arrival ~ime is not S precise, but rather is a Gaussian curv~, the peak time is recordedL Each target substance and the corresponding fluorescent binding agent are subject to the same procedure in the calibration run. In calibration runs a mean migration time to he measurement slit or pinhol~ is 10 det~rmined. Then, the standard deviation is determined ~-for the time of arrival of the free binding agent, as well as for the bound target substance. In the present ~invention, it is necessary to know the mean migration time, i.e., the expected arrival times of bound and free binding ag~nt for specific target substances because the times will be used to search for target an~lyte in a sample where the target substance is possibly present, but not necessarily present. The difference in arriYal :: times between the bound and free binding agent may be 20 used to establish a time window so that the arrival of :;
one member may be paired with the other member in a search for the other member. If the search reveals ~hat ~: the other member is present within a standard deviation : : or two, that other material is identified as a member of ~5 th~ pair. If nothing is found within the time window, the first member of the pair is regarded to be an ` artifact and is discarded.
The ou~put of the photomultiplier tube is :maintained in a buffer memory 4~ and a ratio may be formed between the signals representing bound and free dye labeled binding agent. A data reader 50 is connected to the bu~er mem~ry 49 for receiving recorded 5ignals which represent the ~luorescent peaks. The data reader : is a computer which correlates the ~arious peaksO Each ~ peak is recorded in order to search for bound and free fluorescQ.nt subs~ance in the recorded data. Normally, the time of appearance of the free fluorescent substance could be established from prior calibration times~ Once ~a3~l -10-th~ position of the free fluorescent substance peak is known, a search is conducted for the corresponding bound fluorescent substance which should be located a certain time interval away, within a time window defined by statistical limits. A peak within this window is identified as the bound fluor~s~cen$ substance, i.e. the target analyte. Next the am~,lltudes of the identified peaks are examined and a r~io is computed in the data ~:
reader 50. The m~thod whereby free fluorescent substance is correlated with bound fluorescent substance is explained further below. The computer also stores cali-brations of known concentrations of target substance so -that ratios may be compared in order to obtain an esti-mat of the unknown co~centration.
15In Fig. 2, the top view of gel 11 shows that the image 29' of slit 29 falls between a positive high voltage terminal 15 and a spot from well 13, coinciding with negative voltage terminal 16. In operation the high ~;~ voltage appli d to terminal 15 causes migration of bound and free tagged binding agents, which are positively or negatl~ely chargQd molecules which respond to the elect:ric field from the high voltags supply. The free tagged binding agen~ will reach the image 29' of slit 29 which is fixed in position at a time different than the :~ :25:: bound tagged binding agent. The unbound tagged ~inding agent serves as~ one marker for a time window which has the bound tagged~binding agent as a corresponding marker, the~two:markers forming a pair of markers which are ::separate~ in time within the statistical limit whirh i5 defined.
I With reference to Fig. 3, a plot of the detec-:~ tor signal is~shown ~here the horizontal axis is time and : the vertical axis;is amplitude of the detected signal.
s an example, electrophoresis begins at a first time, t = 0, and the detector is made op~rati~e. At a second~ime, t2, a relatively large peak 51 i5 ob erved~
representing free fluorescent materia} of a first color.
Ansther signal 54, discussed below, is detected after WO93/15392 PCT/US9?/0~537 -11- 2l283l 7 peak 51. A time later, t3, a weaker signal 53 of the same color is observed. The peak 53 exists in the midregion of a window, Wl, between Xl and X2. The existence of window Wl i5 established by the strong free S fluorescent material signal 51. Peak 53 is within window Wl and is recognized as a bound fluores~ent matsrial signal. Peak 54 is no~ within window ~l and is treated as a false positive or artifact, after being checked to determine whether the-signal is not mistaken for the free fluorescent material signal 51. A search of all signals ~ i5 made to determine the most logical positions for free :~ and bound fluorescent substances. If no signal is found .
-in time window W1, the absence of target analyte is inferred. Each:window W acts as a time domain ~ilter, allowing discrimination of spurious fluorescent signals and noise. No~e that all signals are recorded and signal discrimination occurs after recordin~ by ~nalyzing reoorded data. Even:khough gel to gel characteristics may vary, the present invention has immuni~y to most ~variations because the bound and free fluorescent substances tra~erse the same path.
The ratio of the tWQ signals represented by the area under the peaks 51 and 53 represents an estimate of the ratio o~ a~bound to free fluorescent substances, 2~ after normalizing data relative to calibrations, assuming ood~bindin~ efficiency. A further time latert another : large peak 55: is obserYed. This represents another free luorescent binding agent. This defines another time window W2 at a~subseq~ent time and:a lesser peak 57 is measured in the window. This is taken to represent a bound ~luorescent material. Again, the ratio of bound : to free dye is~computed and once again the target analyte associated with the second dye may be stimated in ~ , : concentration.:
It is possible for the peaks to overlap each other as shown~in Fig. 4. Here, the first free fluorescent substance peak 61, having a rela~ively large ~: amplitude, overlaps ~he second peak 65 of similar :: :
W093/l~39~ PCT/US92/05537 ~ 12-amp~itude in a test where two different fluorescent substances were used~ The second peak 65 is the ~econd free fluorescent ~ubstance signal. However, hecause dif-ferent colors are used, as separated by the filter 43 in Fig. 1, the two peaks may be separately observed. Peak 61 establishes the time window W3r~w~ere a peak 63, representing a bound fluorescently tagged binding agent :~
of a color which is the same--as that associated with the unbound peak 61, occurs totally within the second peak 65. Nevertheless, because of the filter 43, peak 63 may be spatially and optically differentiated from peak 65.
The ratio of bound to unbound signal amplitudes appears to be about 2:1. The corresponding mol~cular amounts of bound and unbound tagging material are estimated to be in ~5 the ~ame rati~. For the peak 65, a time window W4 is :~
established, but no fluorescent signal is found within the window and so the absence of tar~et analyte i~
inferred.
With reference to Fig. 5, a multiple lane elec- .-trophoresis sheet gel is shown. The sheet 71 is prQvided with two lanes 73 and 75. Each of thc lanes has a re- :
spective well 83 and 85 and a rPspective slit image 87 ~ and 89. The two lanes are constructed similarly, with : the slit image locations in the same position. Lane 73 is used to run a calibrated amount of target analyte and a k~wn amount of free fluore~cently labeled binding agent. In lane 75 an unknown amount of target analyte is run with free fIuorescently tagged binding age~t. The ~; two lanes may be compared to determine the amount o~
~nknown analyte in lane 75. For greater accuracy, multiple runs may be made in lan. 73 of various amounts : of target analytes so that many ratios may be stored in a :: memory. A r~tio ~rom a run of an unknown amount o~
target analyte may then be looked up and compar~d with ~nown ratios, with the be5t match indicating the ~mount o~ targe~ analyte.
One of the advantages of the pre~ent i~v~ntion i5 that analysis of peaks representing bound and free dye W~3~15392 P~T/US9~ 537 ~128~17 can be computed before electrophoresis is complete, i.e.
before the migrating substances reach the distant high voltage alectrode. Another advantage is that the present system uses only a single lane of an electrophoresis apparatus so that ~e.l to gel non-uniformities are nulled.
It is possible to use a ~econd lane in an electrophoresis device as a reference or calibration, but such calibra-tions may be done beforehand and results stored in a memory. It is also po~sible to use a second or third or ~ fourth lane for additional analytes of interest cr2ating panels of rele~ant analytes. In the prior art, analysis o~ target analytes usually requires completion of the ~lectrophoresis and subsequent analysis by a plurality of stains t colored or fluorescent substrates, etc. Using the present in~ention, the analysis may be done in real time as soon as sufficient separation exists betwéen the `~: bound and free ~luorescent material. Such a separation ~: can be at a point which is only twenty five percent or thirty three percent of the length of a lane. Once a point is found where adequate separation exists, the image of the slit or pinho~e is p~siti~ned at that ~: location and then all measurements are made from there.
: It is als:o to be noted that this is an open-ended electrophoresis system, i.e. there is no need to stop the electrophoresis at a defined point to get all materials "on scale". Materials that migrate ~lowly can be detected just as well as fast moving targ t analytes.
: Amplitude threshoIds may be used as furth~r dis~rimina-tion against noise and artificial signals.
To discriminate between two or more fluo-rescentiy tagged target substances in the same gel lane, different fluorescent w~velengths can be used, so long a~
filter 43 in FigO 1 can adeguately resolve the different : : wavelengths~ Multiple tests can be run simultaneously, each te~t associated with a partiGular wavelength.
Example l. Detection of proteins present in human blood.
Creatine kinase is an enzyme present in various mammalian WO93J1~392 PCT/US~2/05537 ~2~3~ -14-tissue. It occurs in three different forms known as iso-enzymes: CK-MM (skeletal), CK-MB (cardiac) and CK-BB
(brain). After release from tissue and on circulation in blood the MM and MB forms themselves break down to small-er fragments known as isofo~ms or subforms. In the eventof myocardial infarction, the MB isoenzyme, present in cardiac muscle, is releàsed into plasma. Hence, it serves as a specific diagnostic molecular marker for car-diac ischemia or necrosis. The early and rapid detection of this isoenzyme and iks isoforms are ~ery crucial for the diagnosis of myocardial infarction and for initiating thrombolytic therapy.
To perform the test, a blood sample is separat-ed into pIasma and red blood cells. The plasma is mixed with Qxcess antibody tagged with a fluorescent dye which is directed against CX-M~ . The attachment of f luorescent : a~tibodies for a CK assay is known and is described in : U.S. Pat. No. 4,353,982 to M. Gomez et al. If CK-~B is present in plasma, an immune complex consisting of CK-MB
and fluorescently tagged antibody will be formed. On application of an electric field, the reactio~ mixture : : consisting of the fluorescent immune comp~ex and the un-~reacted fluorescent antihody, will migrate on the gel.
Because of charge and:mass differences, the labeled in tact immune complex will migra~e differently than the labelled antibody. The fluorescence associated with bound and fre2 markers will be detected and arrival times : measured and recorded~ Free marker is id2ntified by a large peak. Any substance within the expected time of the free substance is regarded o be target analyte.
Anything else is an artifact.
~; Example 2. Detection Qf the presence of sexually transmitted diseases. Many sexually transmitted patho-35 gens such as chlamydia, herpes, etc. ~orm lesions in the :~ uro-genital area. For detection of these pathogens, sam-ples are taken With a swab directly from the lesion and a number of different types of tests are performed on this W093/15392 PCT~US92/~5537 extract. These tests include culture and/or immunochemi-cal tests.
After a lesion is sampled with a swab, the swab is treated with a solubilization reagent to liberate mi-5 cro-organisms present. This process will also solubilize target analytes originating from the micro-organisms~
This extracted solution will be filtered and reacted with fluorescently tagged antibody so that there is a substan-tial excess of unreacted tagging substanc~. The differ-lO ential assay proceeds as described above.
The methods described above accslerateselectrophoresis by varying amounts, depen~ing on where ~he image 29' of the~slit (~ee Fig. 2) is positioned. If the difference between the charge, mas~ and shape of the l5 bound and free substances is great, early separat,ion may ~; be expected and the image location 29' may be moved close to~well 13~ the~difference between the two 9uantities i5 not great,~the image location 29' needs to be further away from well 13 to~allow longer separation time o~ the 0~bound~and unbound fluorescent substance. In either ca~a, the results of elec~rop~,oresis are predicted at an earli-er~time than a complete electrophoresis run.
Example 3. ~ Detection of antibody to human serum 25~ album~in (HSA). ~ In~the following example an antibody a~ainst HSA is the;target substance which is detected by tagging w:ith ~luoresc2nt HSA. HSA, Fraction V, was obtained~ from~S~igma :Chemical Company (st. I,ouis, M0~ .
Monoclonal anti~-HSA ~was ob~ained from Biospacific Inc., California. Cy5-labelled HSA was synthesized by the coupling of Cy5 fluorescent dye (Biological Research) to NSA and was obtained from ~ole~ular;Probes (Eugene, Oregon). This fluorescent substan e is ~he binding agent. ~
Di~erential separation assay (DSA) was done as follows: C:y5-labelled HSA (binding agent) was incubated ::
with msnocl~nal anti-HSA (target) at a f inal concentra-tion of 400 ng/ml Cy-HSA and 200 ug/ml anti-HSA in 0. 09 M
: ~ :
W093~5392 PCT/~S92~05~37 ~ 3~ -16-Tris, 0.08 M borate, 0.26 mM EDTA, pH 8.3. A control sample consisted of Cy5-HSA alone at 400 ng/ml without added antibody. Reactions were performed in 1.5 ml Eppendorf tubes in a total reac~ion volume of 20 ul.
5 After incubating the samples at room temperature (20~C3 for 30 minutes, 10 ul aliquots~were loaded onto 6%
nondenaturing (8 cm x 10 cm xji.'75 mm) polyacrylamide gels (Jule labs) containin~;~.9 M Tris, 0.8 M Borate, 2~6 mM EDTA, pH 8.3. Electrophoresis was performed at 100 V
for 40 minutes using a Hoe~er Mighty Small SE200 system.
The real time detection of fluorescent proteins : during electrophoresis was performed using a He-Ne laser beam focussed at a point 1.3 cm below the wells of the gel. The reflected fluorescence was collected u~ing a 15 photomultiplier (PMT) tube. Data was collected using a Labwindows (Trademark3 data acquisition board on an IB~-;~ PC and imported into a Microsoft Windows Excel file for analysis and graphics.
Samples containing excess Cy5-HSA were reacted 20 wi~h excess monocIonal anti-HSA and then were loaded onto m : 6% acrylamide gels:. Figure 6 shows signal amplitude as a ~: ~ signal in volts which is proportional to the reflected f luorescencQ . Separation on this gel system is based on charge/mass characteristics of the proteins and more 25 rapidly migrating species migrate past the laser beam earlier than more slowly migrating protein ~peciesO
The Cy5-HSA peak 91 migrates past the laser beam at approximately 8 minutes. This is a calibration run to establish a:time for free Cy5-HSA~ The immune 30 complex consisting of ~Cy5-HSA~ - Anti-HSA~, on the ~other hand, has a peak 93 which migrates past the laser ~pot at 25.5 minutes~ This example demonstrakes that the rel~ant time window for ~his pair of binding agent ~ANTI-HSA) and fluore~cent tag (Cy5-HSA) is 17~5 minutes.
35 The 8 minute peak 91 defines tha reference posi ion in the data acquisition window for finding the peak g3 of the immune complex ~Cy5-HSA) - Anti-HSA3.
Claims (26)
1. A method for detecting target substances during elec-trophoresis comprising, mixing a specific binding agent with a sample which may contain a target substance, the binding agent being specific to the target substance and having a fluo-rescent characteristic, the amount of binding agent in excess of what will react with the target substance so that there is free and bound binding agent in the sample, both the bound and free binding agent having expected electrophoretic migration times from a starting location to a measuring place, conducting electrophoresis of said mixture in a path defined by electrodes, measuring and recording the times wherein sub-stances having said fluorescent characteristic reach the measuring place, searching said recorded times for bound binding agent in relation to free binding agent using said ex-pected electrophoretic migration times in comparison to said measured times wherein finding of said bound binding agent indicates presence of said target substance.
2. The method of claim 1 wherein said binding agent is selected from the group consisting of a fluorescent antibody, antigen, receptor, enzyme substrate, enzyme inhibitor and lectin.
3. The method of claim 1 wherein said binding agent is conjugated with a fluorescent dye.
4. The method of claim 1 wherein said measuring place is a slit along an electrophoretic trajectory of said bound and unbound binding agent.
5. A method for detecting target substances during elec-trophoresis comprising, (a) reacting one or more known target analytes each with an excess amount of a characteristic fluores-cent substance, creating a mixture of bound and free fluorescent substance, (b) causing electrophoretic migration in a gel of the bound and free fluorescent substance starting from a known starting position, the fluorescent substance mi-grating toward an electrode along a path, (c) directing actinic radiation to said fluo-rescent substance, thereby causing fluorescence, (d) detecting the characteristic fluorescence of the unbound fluorescent substance at a fixed position on said path, (e) detecting the characteristic fluorescence of the fluorescent substance bound to the analyte at a time different from the time of the free substance meas-urement at said fixed position, thereby creating a time difference, (f) recording the time difference to form a time window linking the travel times of the bound and free substances past said fixed position, (g) repeating steps (a)-(e) for patient am-ples in which the presence of target analyte is not known, (h) using the time window to associate bound fluorescent substance with free fluorescent substance in order to establish the presence of target analyte in the patient samples.
6. The method of claim 5 wherein said reacting of target analyte is by means of a fluorescent dye or dye embedded in beads or microspheres attached to binding agent.
7. The method of claim 5 wherein said binding agent is selected from the group consisting of a fluorescent anti-body, antigen, receptor, enzyme substrate, enzyme inhibi-tor and lectin.
8. The method of claim 5 wherein said electrophoretic migration is within a single lane.
9. The method of claim 5 wherein said electrophoretic migration is within a plurality of lanes, one or more of which is used for a known amount of target analyte.
10. The method of claim 9 where the lanes are gel filled capillaries or cuvettes.
11. The method of claim 5 wherein the area under each peak of each detected characteristic fluorescence is measured.
12. The method of claim 6 wherein said detection of the amplitude of characteristic fluorescence is measured through a slit or pinhole as analytes migrate past said slit or pinhole.
13. The method of claim 5 wherein said time window is formed with approximately one statistical variance.
14. Apparatus for detecting target substances during electrophoresis comprising, an electrophoresis apparatus having a voltage supply terminal, a gel lane and a sample supply source, the voltage supply terminal establishing a path of migration for mobile substances, first optical means including a beam, a slit or pinhole, and first optical elements projecting an image of the slit or pinhole on the gel lane in said path between the voltage supply terminal and the sample supply source, a light detector isolated from light of the beam, second optical means including a reflected beam, a filter and second optical elements projecting a reflected image of the slit onto said detector, said first and second optical elements sharing a focusing lens, a sample of free fluorescent substance and analyte material tagged with fluorescent. substance, both being mobile substances in said gel lane, means for measuring and recording times of signals representing detection of fluorescent substances, and a data reader means for correlating the presence of free fluorescent substance with bound fluorescent substance.
15. The apparatus of claim 14 further defined by a prism having first and second reflective surfaces, the first reflective surface inclined at an angle to receive said beam and directing said beam to said shared focusing lens and the second reflective surface inclined at an angle to receive the reflected image of the slit and directing said image to the detector.
16. The apparatus of claim 14 wherein a plurality of gel lanes are arranged in parallel relation, one gel lane being a test lane and one or more gel lane being calibra-tion lanes, said slit or pinhole image being in the same relative location in said lanes.
17. A diagnostic system for determining presence of an analyte comprising, an excess amount of fluorescence labeled binding agent forming a complex with a specific target analyte to be determined, the combination having free fluorescence labeled binding agent and bound analyte, an elongated migration path for the bound analyte and the labeled binding agent, the bound analyte and the labeled binding agent having different migration rates, said path having a migration start zone and a downstream migration analysis zone, optical means in communication with the analy-sis zone for timing the migration of fluorescence labeled binding agent and bound analyte from the start zone to the analysis zone, and means for comparing the migration times or rates of fluorescence labeled binding agent and bound analyte, whereby the presence of analyte is determined.
18. The system of claim 17 wherein said migration path is an electrophoretic path.
19. The system of claim 17 wherein said binding agent is selected from the group consisting of a fluorescent antibody, antigen, receptor, enzyme substrate, enzyme inhibitor and lectin.
20. The system of claim 17 wherein said complexed target analyte comprises a monoclonal antibody.
21. The system of claim 17 wherein said fluorescent labeled binding agent and bound analyte have substantial-ly different charge-to-mass characteristics.
22. The system of claim 17 wherein said migration path is established in a sheet gel.
23. The system of claim 17 wherein said migration path is established in a cuvette.
24. The system of claim 17 wherein said migration path is established in a capillary,
25. The system of claim 22 wherein said sheet gel con-tains a plurality of paths, one path having a calibrated amount of bound analyte and a known amount of free fluo-rescence labeled binding agent.
26. The system of claim 25 wherein said plurality of paths contain a plurality of analytes.
Applications Claiming Priority (2)
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|---|---|---|---|
| US828,407 | 1986-02-11 | ||
| US07/828,407 US5137609A (en) | 1992-01-31 | 1992-01-31 | Differential separation assay |
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| CA2128317A1 true CA2128317A1 (en) | 1993-08-05 |
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|---|---|---|---|
| CA002128317A Abandoned CA2128317A1 (en) | 1992-01-31 | 1992-06-29 | Differential separation assay |
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| US (1) | US5137609A (en) |
| EP (1) | EP0624246B1 (en) |
| JP (1) | JP2556659B2 (en) |
| CA (1) | CA2128317A1 (en) |
| DE (1) | DE69218002T2 (en) |
| WO (1) | WO1993015392A1 (en) |
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- 1992-01-31 US US07/828,407 patent/US5137609A/en not_active Expired - Fee Related
- 1992-06-29 WO PCT/US1992/005537 patent/WO1993015392A1/en active IP Right Grant
- 1992-06-29 CA CA002128317A patent/CA2128317A1/en not_active Abandoned
- 1992-06-29 EP EP92915251A patent/EP0624246B1/en not_active Expired - Lifetime
- 1992-06-29 DE DE69218002T patent/DE69218002T2/en not_active Expired - Fee Related
- 1992-06-29 JP JP5513168A patent/JP2556659B2/en not_active Expired - Lifetime
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| Publication number | Publication date |
|---|---|
| US5137609A (en) | 1992-08-11 |
| JPH07503072A (en) | 1995-03-30 |
| DE69218002T2 (en) | 1997-06-12 |
| JP2556659B2 (en) | 1996-11-20 |
| WO1993015392A1 (en) | 1993-08-05 |
| EP0624246B1 (en) | 1997-03-05 |
| DE69218002D1 (en) | 1997-04-10 |
| EP0624246A4 (en) | 1995-08-09 |
| EP0624246A1 (en) | 1994-11-17 |
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