WO1994013829A1 - Test de separation differentielle par focalisation isoelectrique - Google Patents

Test de separation differentielle par focalisation isoelectrique Download PDF

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Publication number
WO1994013829A1
WO1994013829A1 PCT/US1993/004154 US9304154W WO9413829A1 WO 1994013829 A1 WO1994013829 A1 WO 1994013829A1 US 9304154 W US9304154 W US 9304154W WO 9413829 A1 WO9413829 A1 WO 9413829A1
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Prior art keywords
labeled
binding agent
complex
analyte
test sample
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PCT/US1993/004154
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English (en)
Inventor
Rajeev Ramanathan
Robert S. Dubrow
Vartan Ghazarossian
Paul G. Hayter
Robert Justice Shartle
Louis J. Dietz
Bala S. Manian
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Biometric Imaging Inc
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Biometric Imaging Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G01N33/561—Immunoelectrophoresis

Definitions

  • This invention is in the field of detecting analytes using agents which specifically bind to the analyte to form complexes, such as antigen/antibody com- plexes.
  • U.S. Pat. No. 5,006,473, Bouma shows the mi ⁇ gration of an alkaline phosphatase labeled antibody in a liposome embedded electrophoresis media. After electro ⁇ phoresis, the liposome is lysed and a staining dye or reactant is released.
  • U.S. Pat. Nos. 4,205,058 and 4,301,139 describe a chromato ' graphy column which separates antigen and anti ⁇ gen-antibody labeled complexes by selectively immobiliz ⁇ ing the complexes.
  • Radio-labeled antigen competes with unlabeled antigen in a sample for binding with the antibody and the concentration of unlabeled antigen is determined by measuring the percentage of radio-labeled . antigen which migrates on the column.
  • T4-I 125 and anti-T4 are reacted and separated on a cross- linked polyvinyl alcohol column.
  • the antibody/T4 complex is retained at the bottom of the column while the T4-I 125 migrates up the column.
  • the patents represent an example of the direct binding of a labeled hapten T4-I 125 and antibody and separation of these complexes. in U.S. Pat. No. 4,811,218, M. Hunkapiller et al.
  • the gel may in ⁇ corporate an antibody which interacts with a migrating antigen.
  • the two lanes may be calibrated so that differ ⁇ ent degrees of retardation, for different concentrations of the migrating macromolecule, are known.
  • microscopic beads treated with ligands can be entrapped in the gel and similarly serve as a retardant. Beads have the advantage of tight packing in the gel if they are of the appropriate size.
  • Activation of the gel in ⁇ volves partial cross-linking so that the gels do not melt on heating.
  • Alternative methods of gel preparation are described, all with the result that a macromolecular re- tardant is immobilized. Electrophoresis proceeds in the usual way.
  • the differential rate of migra ⁇ tion of the labeled binding agent and complex are deter- mined by detecting the label as the substances migrate past a fixed detector.
  • the migration time of the labeled binding agent is used to define a time window in which the complex should be found.
  • the labeled substances are separated on the basis of their charge to mass ratio making it difficult to distinguish between large molecules with small charge differences.
  • the labeled substances are mov- ing through the separation medium during detection which causes the peaks to spread, thereby decreasing resolution of the peaks.
  • use of the fixed detector neces ⁇ sitates waiting for the slowest migrating peak to move completely past the detector which may take 30 minutes or more.
  • An object of the invention is to devise a rapid, sensitive and high resolution method for sepa ⁇ rating large molecules of similar molecular weight with small charge differences.
  • a further object is to in- crease the resolution and sensitivity of differential separation assays.
  • the above objects have been achieved in a method for detecting and quantitating target analytes which specifically bind to a labeled binding reagent to form a complex.
  • the binding agent is labeled with a de ⁇ tectable marker so that binding between the labeled binding agent and analyte provides a reaction mixture which contains labeled binding agent and a complex of analyte and labeled binding agent.
  • the amount of labeled binding agent in the reaction mixture is in excess of what will react with the target analyte.
  • the reaction mixture is placed on a suitable medium and the labeled substances are separated by isoelectric focusing.
  • the term "separation medium" as used herein refers to the materials and material containers within which isoelec- trie focusing takes place.
  • the excess amount of binding agent which has a different focus point than the analyte/binding agent complex, serves as a reference point or marker for the complex. Differences in focus point arise because of differences in isoelec ⁇ tric point (pi) .
  • a second labeled marker of known concentration is included which has a pi different from the labeled binding agent and complex.
  • the in ⁇ vention encompasses a method for detecting and quantitat ⁇ ing a target analyte in a test sample.
  • a labeled binding agent which specifically binds to a target analyte to form a complex having a characteris ⁇ tic isoelectric point different from the characteristic isoelectric point of unbound labeled binding agent.
  • One also provides a separation medium for isoelectric focus- ing on which the labeled binding agent and complex are focused to different locations. Then one contacts the test sample with an amount of the labeled binding agent in excess of what will react with the target analyte to form a complex with analyte in the test sample.
  • Identification of a peak corre ⁇ sponding to the complex indicates the presence of the target analyte in the test sample.
  • the area under the complex peak is then normalized and compared with the normalized peak areas of peaks containing known concen ⁇ trations of the target analyte to determine the concen ⁇ tration of the target analyte in the test sample.
  • the analyte may be an antigen and labeled binding agent a fluorescently labeled antibody or the active fragment of an antibody such as an Fab.
  • This method can be applied to the simultaneous measurement of multiple analytes or to the differential measurement of multiple isoforms of the same analyte.
  • one again provides a specific binding agent which binds to the analyte to be detected.
  • the complex and excess labeled sub ⁇ stance are separated by isoelectric focusing.
  • the recorded relative spatial distributions are compared to the known relative spatial distributions of unbound labeled substance and complex to identify a complex peak the area under the complex peak is normalized so that one can compare the normalized peak area to the normalized complex peak area of a sample which does not contain the analyte.
  • a finding of a reduced amount of complex indicates presence of the analyte in the test sample.
  • a preferred assay utilizes a second labeled marker of known concentration which has a pi different from the labeled binding agent and complex and therefore focuses at a different location.
  • This second labeled marker serves as a quality control check on the system. For example, if the separation media or other reagent in the test kit are not operative, the failure of the second labeled marker to focus as expected can be readily detected and any results from that assay are discarded. Also, if the labeled binding agent or peak corresponding to complex failed to appear in the proper relationship to the second labeled marker, this test would be discarded as faulty.
  • the second labeled marker having a known concentration, can also be used to normalize the peak areas between runs.
  • the present invention takes advantage of the specificity of reaction of specific binding ole- cules, the ability of capillary isoelectric focusing to separate small amounts of materials with high resolution, the sensitivity of detecting labels such as fluorescent labels, and a quality control and signal normalizing agent to provide rapid, sensitive and high resolution quantitative results.
  • Another advantage of the invention is rapid determination of the results of isoelectric focusing without waiting for mobilization of the target substance or production of detectable materials by the separated biochemicals.
  • the invention also discriminates between isoforms of the target analyte and is particular ⁇ ly useful in separating closely related molecules with small charge differences.
  • closely related means not only heavy molecules of close molecular weight, but also molecules whose charge/mass ratio or other char- acteristic is such that both molecules exhibit similar migration rates, e.g., using electrophoresis, so that previous separation efforts have been difficult.
  • analyte refers to a large variety of chemical substances for which there is a specific binding partner. It is contemplated that the present assay may be applied to the detection of any analyte for which there is a specific binding partner.
  • the analyte usually is a peptide, protein, carbohydrate, glycoprotein, ster ⁇ oid, or other organic molecule for which a specific bind- ing partner exists in biological systems or can be syn ⁇ thesized.
  • the analyte, in functional terms, is usually selected from the group consisting of antigens and anti ⁇ bodies thereto; haptens and antibodies thereto; and hor ⁇ mones, vitamins, metabolites and pharmacological agents, and their receptors and binding substances.
  • analytes are immunologically-active polypeptides and pro ⁇ teins of molecular weights between 1,000 and 4,000,000, such as antibodies and antigenic polypeptides and pro ⁇ teins, as well as haptens of molecular weights between 100 and 1,500.
  • antigenic polypep ⁇ tides are angiotensin I and II, C-peptide, oxytocin, vasopressin, neurophysin, gastrin, secretin, and gluca- gon.
  • antigenic proteins are insulin, chorionic gonadotropin (e.g.
  • HCG human growth hormone
  • TSH thyroid stimulating hor ⁇ mone
  • TSG human placental lactogen
  • TSG thyroxine binding globulin
  • enzymes such as alkaline phosphatase and lactic dehydrogenase, and hepatitis, HTLV-III, influenza, herpes, and other viral associated antigens.
  • Representative of antibody ligands are those antibodies of the IgG, IgE, IgM and IgA classes specific for any of the antigens or haptens, or a class thereof, herein described.
  • the class of hapten ligands is exemplified by thyroxine (T 4 ) , triiodothyro- nine (T 3 ) , the estrogens such as estriol, prostaglandins, vitamins such as biotin, vitamin B 12 , folic acid, vitamin E, vitamin A, and ascorbic acid (vitamin C) , and drugs such as carbamazepine, quinidine, digoxin, digitoxin, theophylline, phenobarbital, primidone, diphenylhydan- toin, morphine, nicotine, and so forth.
  • DNA, RNA, and their complementary binding sequences and binding proteins can be determined by the method of this invention.
  • Cytokines such as interleu- kins, interferons, G-CSF, GM-CSF, M-CSF, tumor necrosis factors (TNF) , erythropoietin and the like are represen ⁇ tative of cytokines that may be determined by methods of this invention.
  • labeled binding agent refers to sub ⁇ stances which specifically bind to the analyte and which have a detectable label.
  • the label may be covalently linked or bound to the binding agent indirectly through another specific binding reaction, for example, a labeled goat antihuman antibody could be used to label a human antibody.
  • a labeled goat antihuman antibody could be used to label a human antibody.
  • Those skilled in this art will recognize a wide variety of techniques to label proteinaceous, as well as non-protein specific binding substances. For instance, fluorescent dye labeling of proteins in general and antibodies or antigens in particular is well known.
  • cytokines and monoclonal antibodies to these cytokines are known, for example, interleukin (l , 13, 2, 3, 4, 5, 6, 7, 8, 9, 10) ; inter- feron ( , ⁇ , ⁇ ) ; granulocyte/macrophage colony stimulat ⁇ ing factor (CN-CSF, G-CSF, M-CSF) ; tumor necrosis factor (TNF and ⁇ ) ; and transforming growth factor and their monoclonal antibodies are known.
  • Interleukin l , 13, 2, 3, 4, 5, 6, 7, 8, 9, 10
  • inter- feron , ⁇ , ⁇
  • granulocyte/macrophage colony stimulat ⁇ ing factor CN-CSF, G-CSF, M-CSF
  • TNF and ⁇ tumor necrosis factor
  • transforming growth factor and their monoclonal antibodies are known.
  • Antibodies to creatine kinase (skeletal, cardiac, and brain) are described in U.S. Pat.
  • Lipopolysaccaride an endotoxin
  • LPS Lipopolysaccaride
  • Monoclonal antibodies to LPS are well known, see U.S. Pat. No. 5,092,235 (Williams et al. and references therein) .
  • a specific binding substance may be labeled with any of a variety of dyes, such as fluorescein dyes or rhoda ine dyes by conventional chemical techniques.
  • Representative fluorescent dyes for making the labeling binding agent are fluorescein isothiocyanate (emission at 520 nm) , 4-chloro-7-nitrobenzo- -oxa-l-diazole (emission 550 nm) , tetramethylrhodamine isothiocyanate (emission 580 nm) , Texas Red (emission 610 nm) . These dyes are available from Molecular Probes, Inc.
  • fluorescein 5 or 6 succinimidylcarboxylate fluorescein 5 or 6 iodoacetamide
  • fluorescein 5 or 6 maleimide is available.
  • Similar functional groups are available for tetramethyl-rhodamine dyes.
  • spe- cific binding molecule such as an antibody by specifi ⁇ cally binding a second labeled antibody to the antibody reagent, such that the detected components are labeled antibody-antibody complex and labeled antibody-antibody/ antigen complex.
  • separation media refers to electro ⁇ phoresis, such as slab gel electrophoresis or capillary electrophoresis.
  • Media such as polyacrylamide, cellu ⁇ lose acetate, agar gel, and agarose gel, are suitable for slab gel electrophoresis.
  • the medium in capillary elec ⁇ trophoresis is generally a free solution and separation medium refers to the capillary container for the free solution.
  • separation tech ⁇ niques are well known to those skilled in this art.
  • the invention involves the reaction of a labeled binding agent (LB*) and an ana ⁇ lyte [A] to form a complex A/LB* and separation of these species on a separation media, measuring the differential migration of A/LB* and LB* on the separation media by detecting the label and then using the difference in migration to identify the analyte by comparison with calibration data or other data which establishes expected migration data for the analyte.
  • LB* labeled binding agent
  • A an ana ⁇ lyte
  • the reaction can be determined by measuring the decrease in the size of the LB* peak.
  • multiple analytes can be detected by using different labeled binding agents that specifically bind to each analyte and detecting the LB* and A/LB* complex for each label.
  • two dif ⁇ ferent labeled binding agents can be bound to one analyte to form a sandwich complex and the migration of the sand ⁇ wich complex can be compared with either or both of the labeled binding agents.
  • haptens can be detected by binding the hapten to a labeled carrier [C*] such as a polypeptide to form a conjugate in which the hapten or the carrier is labeled (HC*) .
  • C* labeled carrier
  • the hapten in a test sam ⁇ ple is allowed to compete with HC* for antihapten anti ⁇ body and the mixture is separated on the separation media.
  • the species HC* and antibody/HC* complex are de ⁇ tected after they separate on the media.
  • This reagent is referred to as a "hapten conjugated labeled carrier.”
  • All of these embodiments are preferably prac ⁇ ticed by the inclusion of a second labeled marker which has a pi different from the labeled binding agent and complex and therefore focuses at a different location.
  • the fluorescent dye can be bound to a protein or other substance which will affect its pi so that it will focus at a desired location in a particular medium.
  • the labeled marker having a known concentration, provides for normalization of channel to channel variability in antibody reaction and detection of signal amplitude. It provides an early warning that the particular assay is grossly incorrect.
  • the labeled marker also provides a reference point for discrimination of both the labeled binding agent peak and complex peak, thus further as ⁇ suring the quality of the assay.
  • the second labeled marker also provides a means for quantitation of the analyte by normalizing peak areas.
  • This invention is most advantageously applied in the diagnosis of molecular variants in proteins.
  • creatine-kinase (CK) MB isoforms (MB2 and MB1) have been used in the early diagnosis of cardiac muscle injury following acute myocardial infarction.
  • the determination of the MB2 and MB1 isoforms is also useful in determining the onset of acute cardiac allograft rejection as well as injury following coronary artery bypass grafting.
  • the determination of CKMM isoform is important in monitoring atropic skeletal muscle changes.
  • mitochondrial creatine kinase in patients with cerebrovascular damage and it is critical that there be a rapid assay to assess such damage so that drug therapy can begin.
  • alkaline phosphatase iso ⁇ forms is important in liver, bone, and kidney disease, as well as liver transplant rejection.
  • Those skilled in the medical arts will recognize a large number of macromole- cules with charge-based isoforms or isovariants having clinical diagnostic significance which can be discrimi ⁇ nated by using the method of this invention.
  • Fig. 1 is a plan view of the apparatus of the present invention.
  • Fig. 2 is a top view of a thin film slab gel arrangement for electrophoresis.
  • Fig. 3 is a side view of a capillary arrangement for electrophoresis.
  • Fig. 4 is a plot of detector signals from complex and free fluorescent labeled binding agent.
  • Fig. 5 is a plot of overlapping detector signals of different wavelength from complex and free fluorescent labeled binding agent.
  • Fig. 6 is a top view of a multiple lane arrangement for electrophoresis.
  • Fig. 7 is a plot of detector signals from free fluorescent labeled binding agent and fluorescent labeled binding agent complexed with two different isoforms of a target analyte.
  • Fig. 8 shows the isoelectric focusing on a thin film slab gel of Cy5 labeled human serum albumin (HSA) and Cy5 labeled human serum albumin complexed with an antibody.
  • HSA human serum albumin
  • Fig. 9 shows the isoelectric focusing in a capillary of Cy5 labeled Fab and Cy5 labeled Fab complexed with CKMB2. Best Mode for Carrying Out the Invention
  • a single lane iso ⁇ electric focusing electrophoresis apparatus 10 having a negative electrode 16 and a positive high voltage elec- trode 15 at an opposite end is shown.
  • the electrophore ⁇ sis apparatus consists of a conventional single lane 18 having a substrate 17 and a separation medium 19.
  • the separation medium is a thin film gel
  • the substrate is usually a self-supporting material which may be glass, mylar (trademark) or any well known gel support.
  • the gel itself is usually polyacrylamide or argarose, although other gel materials such as synthetic acrylamide substi ⁇ tutes may also be used. Uniform polymerization and free ⁇ dom from bubbles and irregularities are desirable proper- ties.
  • the gel may be hydrated or rehydratable.
  • the sub ⁇ strate is a grooved insulator such as acrylic.
  • the cap ⁇ illary itself is usually borosilicate glass, although other clear low reflection materials may also be used. Rectangular capillaries are preferred when the label is to be detected by optical scanning.
  • the reaction mixture is applied to the separation medium by placing it on top of the gels or wicking it into the capillaries. High voltage is then applied to the separation medium at elec- trodes 15 and 16 and charged ions migrate to their iso ⁇ electric point.
  • the test sample is a fluid, frequently a frac ⁇ tionated blood sample.
  • Blood may be preprocessed to re ⁇ move constituents which will interfere with the assay. Removal may be by filtering, absorption, centrifuging or precipitating either the desired or undesired components so that a desired target analyte may be obtained for electrophoresis.
  • the desired target analyte must be one for which there is a specific binding agent.
  • Fluorescent tags such as those commercially available are manufac ⁇ tured by Molecular Probes Inc. of Oregon which special ⁇ izes in dyes or dye beads that can be covalently attached to binding agents.
  • Monoclonal antibodies can now be manufactured so that the behavior of this binding agent is uniform and predictable for many assays. Monoclonal antibodies are more expensive than polyclonal antibodies, but the antibodies have greater specificity, are directed toward single epitopes, are easy to produce in large quantities and are generally more useful in precise separation of bound and free material.
  • the labeled binding agent is applied in excess so that the reaction with the -analyte will be driven to completion, or nearly to completion in a reasonable or convenient amount of time.
  • the amount of excess labeled binding agent should not be more than 20 times the maxi ⁇ mum expected amount of analyte, although the number may range between 2 and 50, approximately.
  • the complex of labeled binding agent and analyte should have a different isoelectric point from that of the free labeled binding agent.
  • carrier ampholytes which produce linear pH gradients in a variety of ranges are com er- cially available, such as PHARMALYTE (trademark) carrier ampholytes (copolymers of glycine, glycylglycine, amines and epichlorhydrin available from Pharmacia LKB Biotech ⁇ nology, Piscataway, New Jersey) or ISOGEL (trademark) ampholytes (available from FMC Bioproducts, Rockland, Maine) .
  • the ampholytes are added to the reaction mixture before it is applied to the separation medium.
  • Each solute migrates under the influ ⁇ ence of the electric field to a position in the separa ⁇ tion medium where the pH is equal to the solute's iso- electric point.
  • the separation medium is optically scanned to detect the labeled substance.
  • a strongly emitting light source such as light emitting diode or laser 23 is used to generate a beam 25.
  • the LED 23 has an output power of about 50 milliwatts and a wavelength band which will excite fluorescence in the fluorescent labeling material. Such excitation radiation is known as actinic radiation.
  • the beam is intercepted by a focusing lens 27 which directs the beam through a slit aperture and 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 dichroic mirror 35.
  • Di- chroic mirror 35 is chosen to selectively reflect light at the wavelengths emitted by light source 23 while transmitting light at the wavelengths emitted by the fluorescent labeled binding agents.
  • the reflected beam is directed toward a focusing lens 37.
  • Light passing through the focusing lens carries an image of the slit 29 which is directed onto separation medium 19.
  • the image of slit 29 can be scanned along the longitudinal axis of separation medium 19 by moving separation medium 19 rela ⁇ tive to lens 37.
  • the focusing lens is used by light traveling in each direction.
  • the retrobeam is directed to reflecting surface 33 which is a part of dichroic mirror 35.
  • Light reflected from the separation medium is reflected toward light source 23 while fluores- cent light is passed through.
  • the fluorescent light is then directed by a mirror 41 through a filter 43 which rejects any light other than the desired wavelength from the fluorescent label.
  • Light transmitted through the filter is directed toward a focusing lens 45. From there the beam is directed to a light detector, such as photo- multiplier tube 47 with a slit located at the image plane of the separation medium.
  • the focused locations of the labeled substances are measured relative to one end of the separation medi ⁇ um.
  • Each target substance and the corresponding labeled binding agent are subject to the same procedure in the calibration run.
  • mean locations are determined.
  • the standard deviation is determined for the location of the free binding agent and the loca ⁇ tion of the complex.
  • the relative locations of the complex and free binding agent in calibration runs are used to establish a spatial window for the expected location of the complex.
  • the location of the free binding agent is used to search for the complex in test samples. If the search reveals that a peak is present within a standard deviation or two of the expected location then that peak is identified as the complex.
  • the output of the photomultiplier tube is main ⁇ tained in a buffer memory 49 and a ratio may be formed between the signals representing labeled complex and free labeled binding agent.
  • a data reader 50 is connected to the buffer memory 49 for receiving recorded signals which represent the fluorescent peaks.
  • the data reader is a computer which correlates the various peaks. Each peak is recorded in order to search for complex and free labeled binding agent in the recorded data. Normally the location of the free labeled binding agent is established from prior calibration locations. Once the position of the free labeled binding agent peak is known, a search is conducted for the corresponding complex which should be located a certain distance away, within a spatial window defined by statistical limits. A peak within this window is identified as the complex, i.e. the target analyte.
  • the peak areas of the identified peaks are examined and normalized in computer 50.
  • the method whereby free labeled binding agent is correlated with complex is ex ⁇ plained further below.
  • the computer also stores calibra ⁇ tions of known concentrations of target analyte so that the normalized peak areas may be compared in order to obtain an estimate of the unknown concentration.
  • the top view of a slab gel 11 shows that the image 29' of slit 29 scans between positive high voltage electrode 15 and negative voltage electrode 16.
  • electrodes 15 and 16 are dis- posable conductive polymer films in direct contact with thin film slab gel 11. Elimination of electrode reser ⁇ voirs is possible because optical scanning of the separa ⁇ tion medium obviates mobilization of the focused materi ⁇ als prior to detection.
  • the reaction mixture is placed on top of hydrated gel 11. Small volumes are placed approximately in the middle while larger volumes may uniformly cover the surface.
  • the reac ⁇ tion mixture is used to rehydrate gel 11 giving a uniform initial distribution throughout the gel.
  • the high voltage applied to electrode 15 causes migration of bound and free labeled binding agents, which are posi ⁇ tive or negative charged molecules to a point where they encounter a pH at which their net charges become 0, their isoelectric points, and migration halts.
  • the free labeled binding agent will reach a position corresponding to its pi which is different from the pi of the bound labeled binding agent.
  • Sub ⁇ strate 17 is an insulator platform with elevated ends.
  • Metal electrodes 15 and 16 are located in longitudinal grooves in the top surfaces of opposite ends of platform 17.
  • Capillary 12 is placed horizontally on top of plat- form 17 with the ends of capillary 12 located in the grooves containing electrodes 15 and 16.
  • a gap 13 separates capillary 12 from electrode 15 and a gap 14 separates capillary 12 from electrode 16.
  • capillary 12 is loaded with a reaction mixture containing ampholytes before being placed on platform 17. The end of capillary 12 is dipped in the reaction mixture and capillary action wicks the solution into the capillary.
  • capillary 12 is then placed on platform 17 and a drop of basic catholyte is added to gap 14 bridging capillary 12 with electrode 16 and a drop of acidic anolyte is added to gap 13 bridging capillary 12 with electrode 15.
  • capillary 12 is loaded with reaction mixture after being placed on platform 17.
  • a drop of reaction mixture is placed in gap 13 or 14 and capillary action wicks the solution into the capillary and the other gap making contact with both electrodes.
  • Fig. 4 a plot of the detec ⁇ tor signal is shown where the horizontal axis is distance and the vertical axis is amplitude of the detected sig ⁇ nal. After focusing, the separation medium is scanned to detect the presence of label.
  • a relative- ly large peak 51 is observed, representing free fluores ⁇ cent labeled binding agent of a first color.
  • Another signal 54 discussed below is detected at a second loca ⁇ tion.
  • a weaker signal 53 of the same color is observed.
  • Peak 53 exists in the mid region of a window, Wl, between X2 and X .
  • the existence of window Wl is established by the strong free fluorescent labeled binding agent signal 51.
  • Peak 53 is within win ⁇ dow Wl and is recognized as a fluorescent labeled complex signal.
  • Peak 54 is not within window Wl and is treated as a false positive or artifact, after being checked to determine whether the signal is not mistaken for the free fluorescent labeled binding agent signal 51.
  • a search of all signals is made to determine the most logical posi ⁇ tions for free fluorescent labeled binding agent and com- plex. If no signal is found in spatial window Wl, the absence of target analyte is inferred. Each window W acts as a spatial filter, allowing discrimination of spu ⁇ rious fluorescent signals and noise. Note that all signals are recorded and signal discrimination occurs after recording by analyzing recorded data. Even though separation medium characteristics may vary from lane to lane or run to run, the present invention has immunity to most variations because the complex and free fluorescent labeled binding agent are subject to the same conditions.
  • the ratio of the two signals represented by the areas under peaks 51 and 53 represents an estimate of the ratio of complex to free fluorescent labeled binding agent after normalizing data relative to calibrations, assuming good binding efficiency.
  • another peak 55 is observed.
  • This represents another free fluorescent labeled binding agent.
  • This defines another spatial window W2 at a subsequent location and a lesser peak 57 is measured in the window. This is taken to represent a fluorescent labeled complex.
  • the ratio of bound to free label is computed and once again the target analyte associated with the second label may be estimated in concentration. It is possible for the peaks to overlap each other as shown in Fig. 5.
  • the first free fluores ⁇ cent labeled binding agent peak 61 having a relatively large amplitude, overlaps a second peak 65 of similar amplitude in a test where two different fluorescent la- beled binding agents were used.
  • Second peak 65 is the second free fluorescent labeled binding agent signal.
  • Peak 61 establishes the spatial window W3 where a peak 63, representing a fluorescently labeled complex of a color which is the same as that associated with free labeled binding agent peak 61, occurs totally within second peak 65. Nevertheless, because of 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 molecular amounts of complex and free labeled binding agent are estimated to be in the same ratio.
  • a spatial window W4 is established, but no fluores ⁇ cent signal is found within the window so the absence of the corresponding target analyte is inferred.
  • a multiple lane electrophoresis cartridge is shown.
  • the cartridge 71 is provided with two lanes 73 and 75.
  • Each of the lanes has a respective slit image 87 and 89 which scan the lanes as indicated.
  • the two lanes are constructed similarly with the separation mediums 74 and 76 being thin film slab gels or free solution capillaries.
  • Lane 73 is used to run a calibrated amount of target analyte and a known amount of free fluorescently labeled binding agent.
  • lane 75 an unknown amount of target analyte is run with free fluorescently tagged binding agent.
  • the two lanes may then be compared to determine the amount of unknown analyte in lane 75.
  • normalization is carried out by running a constant known amount of a second labeled marker in each lane during every run. For greater accuracy, multiple runs may be made in lane 73 of various amounts of target analytes so that many normalized peak areas may be stored in the memory.
  • the normalized peak area from a run containing an unknown amount of target analyte may then be looked up and compared with the normalized peak areas of known con ⁇ centrations, with the best match indicating the amount of target analyte.
  • a single labeled binding agent may be used to determine the ratio of different isoforms of a target analyte as shown in Fig. 7.
  • the free fluorescent labeled binding agent peak 91 is used to establish two windows, W5 and W6, for two isoforms of the target ana ⁇ lyte.
  • a peak 93 is located within window W5 and is rec ⁇ ognized as labeled binding agent bound to one isoform of the target analyte.
  • a second peak 95 is located within window W6 and is recognized as labeled binding agent bound to a second isoform of the target analyte.
  • the ratio of the peak area for peaks 93 and 95 may be used to estimate the relative amounts of the two isoforms of the target analyte in the sample. Both isoforms of the target analyte must contain the epitope recognized by the specific binding agent, but differ in isoelectric point.
  • One of the advantages of the present invention is that analysis of the peaks representing complex and free labeled binding agent can be computed without re ⁇ leasing the applied voltage.
  • Another advantage is that the present system uses only a single lane of an electro- phoresis apparatus so that run to run non-uniformities are nulled.
  • a second lane in an electrophoresis device as a reference or calibration, but such calibrations may be done beforehand and the results stored in a memory. It is also possible to use a second or third or fourth lane for additional analytes of inter ⁇ est creating panels of relevant related analytes.
  • analysis of target analytes usually requires post-focusing mobilization or in situ analysis by a plu ⁇ rality of stains, colored or fluorescent substrates, etc.
  • the analysis may be run in real time as soon as focusing is complete and before the applied voltage is released. This leads to an increase in both resolution and sensitivity.
  • the applied voltage may be maintained during analysis so that the peaks will remain focused. Focusing increases the concentration of the complex peak leading to greater sensitivity. Spatial separation of the peaks may be adjusted without increas ⁇ ing peak width by varying the pH gradient. Amplitude thresholds may be used as further discrimination against noise and artificial signals.
  • dif ⁇ ferent fluorescent wavelengths may be used, so long as filter 43 in Fig. 1 can adequately resolve the different wavelengths. Multiple tests can be run simultaneously, each test associated with a particular wavelength. Exa ple 1 Detection of Antibody to Human Serum Albumen (HSA)
  • HSA fraction V
  • Monoclonal anti-HSA was obtained from Biospacific Inc. (Emeryville, California)
  • Cy5-labeled HSA was synthesized by the cou- pling of Cy5 fluorescent dye (Biological Detection Sys ⁇ tems, Pittsburgh, Pennsylvania) to HSA and removal of free dye using gel filtration • (Molecular Probes, Eugene, Oregon) . This fluorescent substance is the labeled bind ⁇ ing agent.
  • DSA Differential separation assay
  • pHAST IEF gels Puracia, Piscataway, New Jersey
  • the positions of the fluorescent proteins were determined using a post-electrophoretic scan of the gel with a He-Ne laser optic system by moving the gel with a stepper motor.
  • the reflected fluorescence was collected using a R928 PMT and the data was collected using data acquisition software on an IBM (trademark) personal computer. Separation on this type of gel is based on the isoelectric points of the proteins. With reference to Fig. 8, the result is shown as a plot of fluorescence versus distance on the gel.
  • the pH gradient on the gel is also plotted.
  • the Cy5-HSA control peak 97 is focused at 34 mm (pH 4.7) .
  • the immune complex consisting of Cy5- HSA/anti-HSA, on the other hand, has a peak 98 which is focused at 23 mm (pH 5.3) . As shown on the figure, excellent separation (0.6 pH units) is obtained between the immune complex and the excess binding agent (Cy5- HSA) . The peak 99 is residual uncomplexed labeled Cy5-HSA.
  • This example demonstrates that the relevant spatial window for this pair of labeled binding agent (Cy5-HSA) and analyte (anti-HSA) is 11 mm. Peak 97 at 34 mm defines the reference position for the data acquisi ⁇ tion window in which the immune complex Cy5-HSA/anti-HSA peak should be found.
  • Creatine kinase is an enzyme present in various mammalian tissue. It occurs in three different forms known as isoenzymes: CK-MM (skeletal) , CK-MB (cardiac) and CK-BB (brain) . After release from tissue and on cir ⁇ culation in blood the MM and MB forms themselves break ⁇ down to smaller fragments known as isoforms or subforms. In the event of myocardial infarction, the MB isoenzyme, present in cardiac muscle, is released in the plasma. Hence, it serves as a specific diagnostic molecular marker for cardiac ischemia or necrosis. The early and rapid detection of this isoenzyme and its isoforms are very crucial for the diagnosis of myocardial infarction for initiating thrombotic therapy.
  • Cy5 labeled CK-MB antibody Separation of Cy5 labeled CK-MB antibody from its immune complex was performed using a capillary isoelectric focusing system.
  • CK-MB2 human heart
  • monoclonal anti CK-MB were obtained from Biospacific (Emeryville, California) .
  • Fab fragments were prepared by digesting the monoclonal anti CK-MB with the enzyme papain.
  • Cy5 labeled Fab was synthesized by the coupling of Cy5 fluorescent dye (Biological Detection Systems, Pittsburgh, Pennsylvania) to Fab and purified by conventional gel permeation and ion exchange methods. This fluorescent substance is the labeled binding agent.
  • DSA Differential separation assay
  • reaction mixture was then diluted 30-fold with a 2% solution of ampholytes with a 3 to 10 pH range (Biorad Inc., Hercules, Califor ⁇ nia) in deionized water.
  • Capillary action was used to fill a 50 x 0.3 x 0.03 mm borosilicate glass rectangular capillary (R&S Medical, Mountain Lakes, New Jersey) , coated to suppress electroendosmosis (Capillary Electro- phoresis. Academic Press, Inc., San Diego, California
  • the positions of the fluorescent proteins were determined at this point by scanning the capillary with a He-Ne laser optic system by moving the capillary with a stepper motor with the field on.
  • the reflected fluores ⁇ cence was collected using a R928 PMT and the data was collected using data acquisition software on an IBM (trademark) personal computer.

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Abstract

L'invention concerne un procédé pour détecter et quantifier des analytes parmi des substances très proches l'une de l'autre, en faisant réagir l'analyte contenu dans un échantillon de test avec un excédent d'agent de liaison marqué qui se lie spécifiquement à l'analyte pour former un complexe. Le complexe et l'excédent d'agent de liaison marqué, qui ont des pI différents, sont séparés par focalisation isoélectrique dans laquelle l'emplacement (x1) de l'excédent d'agent de liaison marqué définit un emplacement attendu (w1) dans lequel le complexe devrait se trouver. Les substances focalisées sont détectées par balayage optique du milieu de séparation (10) avant ou après l'élimination du champ électrique appliqué. Un second marqueur marqué avec une concentration connue fournit un moyen pour normaliser les zones de crête qui sont comparées avec les données d'étalonnage normalisées stockées pour déterminer la quantité d'analyte cible contenue dans l'échantillon de test.
PCT/US1993/004154 1992-12-04 1993-05-03 Test de separation differentielle par focalisation isoelectrique Ceased WO1994013829A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1101106A4 (fr) * 1998-07-28 2006-05-03 Bd Biosciences Systems And Rea Dispositif et methode d'analyse de motilite cellulaire
US7846676B2 (en) 2004-07-19 2010-12-07 Cell Biosciences, Inc. Methods and devices for analyte detection
US7935308B2 (en) 2004-07-19 2011-05-03 Cell Biosciences, Inc. Methods and devices for analyte detection
US7935479B2 (en) 2004-07-19 2011-05-03 Cell Biosciences, Inc. Methods and devices for analyte detection
US8021611B2 (en) 2005-04-09 2011-09-20 ProteinSimple Automated micro-volume assay system
US8945361B2 (en) 2005-09-20 2015-02-03 ProteinSimple Electrophoresis standards, methods and kits
CN112805416A (zh) * 2018-08-23 2021-05-14 普诺森公司 用于检测和分析自由硫醇的系统和方法

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US5137609A (en) * 1992-01-31 1992-08-11 Biometric Imaging Inc. Differential separation assay

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Title
HOKHAIDO OGAKU ZASSHI, Volume 67, issued January 1992, SAGAWA, "Analysis of Anti-DNA Antibody in Sera from Patients with Systemic Lupus Erythematosus by Isoelectric Focusing with Respect to Autoantibody Isotype and Immune Complex", pages 67-80. *
JOURNAL OF CHROMATOGRAPHY, Volume 539, issued February 1991, R.G. NEILSEN et al., "Separation of Antibody-Antigen Complexes by Capillary Zone Electrophoresis, Isoelectric Focusing and High-Performance Size-Exclusion Chromatography", pages 177-185. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1101106A4 (fr) * 1998-07-28 2006-05-03 Bd Biosciences Systems And Rea Dispositif et methode d'analyse de motilite cellulaire
US7846676B2 (en) 2004-07-19 2010-12-07 Cell Biosciences, Inc. Methods and devices for analyte detection
US7935308B2 (en) 2004-07-19 2011-05-03 Cell Biosciences, Inc. Methods and devices for analyte detection
US7935489B2 (en) 2004-07-19 2011-05-03 Cell Biosciences, Inc. Methods and devices for analyte detection
US7935479B2 (en) 2004-07-19 2011-05-03 Cell Biosciences, Inc. Methods and devices for analyte detection
US9304133B2 (en) 2004-07-19 2016-04-05 ProteinSimple Methods and devices for analyte detection
US9400277B2 (en) 2004-07-19 2016-07-26 ProteinSimple Methods and devices for analyte detection
US8021611B2 (en) 2005-04-09 2011-09-20 ProteinSimple Automated micro-volume assay system
US8945361B2 (en) 2005-09-20 2015-02-03 ProteinSimple Electrophoresis standards, methods and kits
CN112805416A (zh) * 2018-08-23 2021-05-14 普诺森公司 用于检测和分析自由硫醇的系统和方法
CN112805416B (zh) * 2018-08-23 2024-03-01 普诺森公司 用于检测和分析自由硫醇的系统和方法
US12265031B2 (en) 2018-08-23 2025-04-01 ProteinSimple Systems and methods for the detection and analysis of free thiol

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