WO1994009185A1 - Quantification par electrophorese de complexes de liaison specifiques - Google Patents

Quantification par electrophorese de complexes de liaison specifiques Download PDF

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Publication number
WO1994009185A1
WO1994009185A1 PCT/US1993/009701 US9309701W WO9409185A1 WO 1994009185 A1 WO1994009185 A1 WO 1994009185A1 US 9309701 W US9309701 W US 9309701W WO 9409185 A1 WO9409185 A1 WO 9409185A1
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WO
WIPO (PCT)
Prior art keywords
sample
analyte
gel
amount
antibody
Prior art date
Application number
PCT/US1993/009701
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English (en)
Other versions
WO1994009185B1 (fr
Inventor
Erich A. Gombocz
David H. Rammler
Original Assignee
Labintelligence, Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Labintelligence, Inc. filed Critical Labintelligence, Inc.
Publication of WO1994009185A1 publication Critical patent/WO1994009185A1/fr
Publication of WO1994009185B1 publication Critical patent/WO1994009185B1/fr

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Classifications

    • 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
    • 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
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules

Definitions

  • the field of this invention is electrophoretic identification of analytes.
  • the apparatus affords real time monitoring of the course of the electrophoresis, so that one may control various parameters to change the parameters during the course of the electrophoresis.
  • the apparatus affords real time monitoring of the course of the electrophoresis, so that one may control various parameters to change the parameters during the course of the electrophoresis.
  • Gel electrophoresis protocols are provided for determining simultaneously one or a plurality of analytes in one or a plurality of samples simultaneously using a single gel plate.
  • the methodology comprises combining the sample with a labeled complementary binding member in a pre-determined amount which allows for the formation of soluble complexes, removing insoluble material, and then applying the liquid medium at one of a plurality of wells present on the gel.
  • the gel may be characterized by having a plurality of lanes, with a plurality of sample wells or sample application sites in each lane, spaced apart a sufficient distance to preclude transport of a first sample past the next successive sample well.
  • the difference between the amount of unbound labeled specific binding member in the gel band and the amount of the labeled specific binding member originally employed with the sample will indicate the amount of analyte present in the sample.
  • DESCRIPTION OF THE SPECIFIC EMBODIMENTS Protocols are provided for performing a plurality of gel electrophoretic determinations for the presence of one or more analytes.
  • the method can be used to determine a multiplicity of samples, from the same or different source, on a single gel plate having a plurality of lanes, where each of the lanes has a plurality of application sites for applying the sample(s) .
  • the application sites are separated a sufficient distance, so as to allow for separation between a labeled specific binding member and a complex between the labeled specific binding member and the analyte.
  • the method comprises combining the sample with a pre ⁇ determined amount of the labeled specific binding member to provide for soluble complexes.
  • any insoluble material may be separated ana the liquid used as the sample.
  • the 5 sample(s) are then applied to the gel at the various application sites.
  • the electrophoresis is then performed, whereby the labeled specific binding pair member and the complex of analyte and the labeled specific binding pair member are separated.
  • the labeled specific binding pair member may be any compound, either ligand or receptor compound, which binds
  • the ligand may be a hapten, antigen, protein, sugar, lipid, nucleic acid, naturally occurring or synthetic small organic compound, combinations thereof, or the like.
  • the receptor may be any compound which is complementary to the ligand,
  • nucleic acids 20 either naturally occurring or synthetic, usually being protein, but may also include nucleic acids.
  • Common receptors include antibodies, surface membrane proteins, enzymes, lectins, etc., and one may also use nucleic acids where the nucleic acids may specifically hybridize to a
  • the analyte will not be restricted by size for the most part, ranging from about 1 kD to 1,000 kD, usually not exceeding about 500 kD.
  • the analyte should not be more than about 10 fold different in molecular weight
  • Various labels may be used, including radioisotopes, fluorescers, enzymes, particles, and the like.
  • radioisotopes including radioisotopes, fluorescers, enzymes, particles, and the like.
  • fluorescers include fluorescers, enzymes, particles, and the like.
  • Fluorescent labels include fluorescein, Texas red, phycoerythirin, allophycocyanin, ethidium bromide, dimeric ethidium bromide, umbelliferone. fluos, resos, cascade blue, luciferin yellow, TOTO, YOYO, bisethiduim, chelated rare earth metals, and the like.
  • the fluorescer will absorb in the range of about 250 to 600 nm and fluoresce in the range of about 350 to 750 nm.
  • the gels can be employed with any convenient thickening agent, including agarose, acrylamide, gelatin, etc.
  • concentrations would generally range between about 0.2-2% T. Descriptions of forming gels for gel electrophoresis may be found in: The Practice of Quantitative Gel Electrophoresis, Chrambach, VCH Publishers.
  • the gel plate may be of any convenient size, generally ranging from about 5 to 40 cm in the direction of migration, and about 0.5 to 2 cm normal to the direction of migration, preferably about 0.5 to 1.5 cm.
  • the gel thickness will generally range from about 0.5 to 1.5 mm.
  • capillary electrophoresis may be carried out, where the capillaries are channeled which allows for application of a plurality of samples along the gel length.
  • a buffer solution is employed, which may be any convenient buffer for preparing the reagents for combination with the sample.
  • the sample may be any convenient physiological or non-physiological fluid, including blood derivatives, particularly plasma or serum, saliva, cerebrospinal fluid, etc. ; fermentation and processing mixtures and streams; samples from natural sources, such as water, soil and air; food; etc.
  • the buffer solution may employ any of the common buffers employed with gel electrophoresis, including Tris, glycine, Hepes, Mops, etc.
  • the pH will be chosen to support the stabilization of complex formation between the specific binding pair members, generally the pH will be in the range of about 6 to 10.
  • the buffer concentration will generally range from about 0.5 to 50 mM.
  • the particular concentration will be determined in accordance with optimizing the procedure emperically t or determining the concentration of the antibody.
  • the antibody will be present in an amount at least about 1.25 fold the highest concentration expected to be encountered in the sample and not more than about 5 fold, more usually not more than about 2 fold the highest concentration anticipated to be encountered in the sample.
  • the ratio of antibody to analyte will be chosen in accordance with the Heidelberger curve (Heidelberger and Kandal (1929) J. Exp. Med. 50, 809; (1935) Ibid 62, 697; Heidelberger et al. (1946) Ibid 83, 303; and Heidelberger and Wolfram (1954) Fed. Proc.
  • the analyte and its complementary binding member are usually combined in a volume ratio of about 0.1-1:1-0.1. The particular ratio will be determined by the relative concentration of the analyte in the sample, the potential for interfering agents in the sample, or the like. If desired, the sample may first be treated to remove interfering components, concentrate the analyte, change the nature of the analyte, for example, separating subunits or providing for subunits coming together, or the like. The sample and its complementary binding member are combined, usually conveniently at ambient temperature, or temperatures of from 4° to 40°C may be employed, and the mixture incubated for sufficient time for reaction to occur.
  • controls will also be employed which allow for comparison of the sample results with the control results.
  • the negative control may be water, buffer, the medium in which the analyte is present, a synthetic medium to mimic the analyte medium, or the like.
  • the positive control will provide the analyte or comparable compound in the same or different medium in which the analyte is obtained.
  • any sedimentation which occurs may be removed. The sedimentation may be removed conveniently by centrifugation, although other techniques may be employed, such as filtration.
  • Aliquots or the entire sample may then be transferred to the gel at appropriate sites.
  • the conditions which are employed for the electrophoresis may be optimized for particular analytes, usually being conventional conditions based on the nature of the receptor and the complex. Temperatures, voltages, and currents may be controlled as appropriate. Desirably, a separation will occur between the complex and the unbound labeled compound of at least about 5 mm, preferably at least about 10 mm, and more usually not more than about 25 mm, where there is a single complex. Where there is a plurality of complexes, the closest complex will generally be spaced at least about 5 mm from the unbound labeled complementary binding member.
  • the bands may then be read and the amount of label in the bands quantitated.
  • the amount of labeled complementary binding member By knowing the amount of labeled complementary binding member, one may calculate the amount of uncomplexed labeled binding member or complexed binding member or both, so as to calculate the amount of analyte present in the sample.
  • the amount of label in the faster moving zone By calculating the amount of label in the faster moving zone, one can directly determine the amount of analyte in the sample.
  • An apparatus for carrying out the subject invention is extensively described in U.S. Patent No. 5,104,512, which patent is incorporated herein by reference. While this apparatus has many advantages for monitoring the electrophoresis in real time, other apparatuses will also find application which allow for appropriate detection of the movement of the bands.
  • the reagents comprise: buffer solution, Tris/glycine, 7.5 M, pH 8.8; anti-human IgG-antibody (LAB) , either Texas red (TRITC) or Fluorescein (FI C) labeled; human IgG (AG) as AG-reference for positive control; agarose gel, 2% T in buffer solution pH 8.8.
  • LAB anti-human IgG-antibody
  • FI C Fluorescein
  • All dilutions are carried out with buffer solution, pH 8.8.
  • a 1 + 4 dilution of antibody (LAB-dil) is prepared and 100 ⁇ l of a human serum sample is combined with 100 ⁇ l of (LAB-dil) in an Eppendorf vial.
  • the negative control replaces the sample with 100 ⁇ l of distilled water, while the positive control employs 100 ⁇ l of AG-reference (100% binding) .
  • the contents of the vials are mixed thoroughly without foaming in an incubator for 15 min. at 37°C. At the end of this time, the vials are centrifuged or 2 min. at 15,000 rpm to remove any precipitated material.
  • the subject methodology provides for numerous advantages. No staining or fixation steps are required and evaluation is performed in real time. One need only evaluate the fluorescent emitting zones, thus ensuring that only antibody (LAB) and antigen-antibody complexes (AGAB-CX) are evaluated. One need only determine the amount of unbound antibody, the fastest moving zone, in the present example the most cathodic zone. One can also determine the AGAB-CX- zone, particularly where a plurality of complexes are determined, so as to quantitate each of the complexes. Total antigen concentration (AG) can be calculated by decrease of free LAB concentration against the LAB concentration of the negative control.
  • the concentration of insoluble immuno complexes is obtained by the difference between the sum of LAB plus AGAB-CX- concentrations obtained for the sample and the LAB- concentration in the negative control.
  • the positive control uses a reference for the maximum for the AGAB-CX- concentration obtainable in accordance with the protocol. Samples having AGAB-CX-concentrations equal to the positive control require a repeat test with a higher dilution of the sample.
  • the concentration range for the test evaluation may cover three to six orders of magnitude without a requirement for additional dilution steps prior to the immuno reaction.
  • the method can use standard equipment in a novel and efficient manner to substantially expand the information that can be obtained in a single electrophoresis.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention se rapporte à la quantification d'analytes par électrophorèse sur gel, de façon à pouvoir analyser simultanément une pluralité d'échantillons. En combinant un analyte avec son élément de liaison complémentaire marqué et en séparant par électrophorèse le complexe de l'élément de liaison complémentaire marqué non lié, on peut calculer la quantité d'analyte contenu dans l'échantillon. Une pluralité d'échantillons espacés peuvent être placés sur une seule et même ligne, de façon à accroître considérablement le rendement de chaque plaque de gel.
PCT/US1993/009701 1992-10-14 1993-10-07 Quantification par electrophorese de complexes de liaison specifiques WO1994009185A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US96052892A 1992-10-14 1992-10-14
US07/960,528 1992-10-14

Publications (2)

Publication Number Publication Date
WO1994009185A1 true WO1994009185A1 (fr) 1994-04-28
WO1994009185B1 WO1994009185B1 (fr) 1994-06-09

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0666476A1 (fr) * 1994-01-14 1995-08-09 Sebia Dispositif pour l'immunofixation sur un support (plaque) unique, d'échantillons différents
WO1999034203A1 (fr) * 1997-12-24 1999-07-08 Cetek Corporation Procede electrophoretique capillaire permettant de detecter des ligands se liant a une cible et de determiner leurs affinites
WO2000079260A1 (fr) * 1999-06-24 2000-12-28 Cetek Corporation Procede d'electrophorese capillaire destine au criblage de ligands d'affinite utilisant un ligand competitif detectable
WO2001053817A2 (fr) * 2000-01-19 2001-07-26 Mosaic Technologies Procedes et dispositifs permettant de deceler et d'extraire un acide nucleique
GB2374423A (en) * 2001-02-14 2002-10-16 Zetatronics Ltd A method for detecting the prescence of cells or other biological entities in a fluid sample
US6837977B1 (en) 1999-06-24 2005-01-04 Cetek Corporation Capillary electrophoresis method for screening for affinity ligands using a detectable competitive ligand

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5055415A (en) * 1987-04-01 1991-10-08 Hitachi, Ltd. Method for immunoassay procedure for the detection of antigen by reacting with antibody which increases its electrical charge and electrophoretic mobility
US5137609A (en) * 1992-01-31 1992-08-11 Biometric Imaging Inc. Differential separation assay

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5055415A (en) * 1987-04-01 1991-10-08 Hitachi, Ltd. Method for immunoassay procedure for the detection of antigen by reacting with antibody which increases its electrical charge and electrophoretic mobility
US5137609A (en) * 1992-01-31 1992-08-11 Biometric Imaging Inc. Differential separation assay

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0666476A1 (fr) * 1994-01-14 1995-08-09 Sebia Dispositif pour l'immunofixation sur un support (plaque) unique, d'échantillons différents
WO1999034203A1 (fr) * 1997-12-24 1999-07-08 Cetek Corporation Procede electrophoretique capillaire permettant de detecter des ligands se liant a une cible et de determiner leurs affinites
US6524866B1 (en) 1997-12-24 2003-02-25 Cetek Corporation Capillary electrophoretic method to detect target-binding ligands and to determine their relative affinities
WO2000079260A1 (fr) * 1999-06-24 2000-12-28 Cetek Corporation Procede d'electrophorese capillaire destine au criblage de ligands d'affinite utilisant un ligand competitif detectable
US6837977B1 (en) 1999-06-24 2005-01-04 Cetek Corporation Capillary electrophoresis method for screening for affinity ligands using a detectable competitive ligand
WO2001053817A2 (fr) * 2000-01-19 2001-07-26 Mosaic Technologies Procedes et dispositifs permettant de deceler et d'extraire un acide nucleique
WO2001053817A3 (fr) * 2000-01-19 2002-02-07 Mosaic Technologies Procedes et dispositifs permettant de deceler et d'extraire un acide nucleique
GB2374423A (en) * 2001-02-14 2002-10-16 Zetatronics Ltd A method for detecting the prescence of cells or other biological entities in a fluid sample

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