WO1995023972A1 - Essai immunitaire heterogene et son utilisation pour detecter des proteines - Google Patents

Essai immunitaire heterogene et son utilisation pour detecter des proteines Download PDF

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Publication number
WO1995023972A1
WO1995023972A1 PCT/EP1995/000727 EP9500727W WO9523972A1 WO 1995023972 A1 WO1995023972 A1 WO 1995023972A1 EP 9500727 W EP9500727 W EP 9500727W WO 9523972 A1 WO9523972 A1 WO 9523972A1
Authority
WO
WIPO (PCT)
Prior art keywords
counterpart
partner
heterogeneous immunoassay
heterogeneous
binding
Prior art date
Application number
PCT/EP1995/000727
Other languages
German (de)
English (en)
Inventor
Martin Reinecke
Thomas Scheper
Wolfgang NOÉ
Original Assignee
Dr. Karl Thomae Gmbh
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 Dr. Karl Thomae Gmbh filed Critical Dr. Karl Thomae Gmbh
Priority to JP7522691A priority Critical patent/JPH09509745A/ja
Priority to AU26074/95A priority patent/AU701926B2/en
Priority to EP95911276A priority patent/EP0748448A1/fr
Publication of WO1995023972A1 publication Critical patent/WO1995023972A1/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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
    • G01N33/538—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody by sorbent column, particles or resin strip, i.e. sorbent materials
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • RIAs radioimmunoassays
  • ELISAs enzyme linked immunoassays
  • the present invention is therefore based on the task of developing an immunoassay which meets the following minimum requirements:
  • the measuring system (see FIG. 1) is based on a new heterogeneous immunoassay (heterogeneous elution assay). say) according to the principles of flow injection analysis.
  • a partner of a strong binding reaction is irreversibly immobilized on a solid support, which is preferably implemented in a flow-through cartridge.
  • the unlabeled sample is placed in a buffer stream which is constantly flowing through the system and passed through the immobilizate (see FIG. 2).
  • the binding partner binds its specific counterpart from the sample, while all other substances are discharged through the carrier stream. There is a separation between analytes (counterpart) and interfering substances.
  • the bound substances are then discharged by changing from the carrier buffer to an elution buffer. Washing and conditioning steps can be implemented depending on the analysis problem.
  • the discharged substances are then analyzed with a detector, for example, for their concentration or biological properties. Depending on the detector signal and evaluation method, the concentration of the analyte in the sample can be proportional to the peak integral, for example.
  • the cartridge can be cleaned, conditioned or equilibrated before performing a new sample.
  • the present invention thus relates to a heterogeneous immunoassay, which is characterized in that it contains a partner with a strong binding reaction, the partner being irreversibly immobilized on a solid support and the support in a flowing buffer stream It is possible to expose that the counterpart to be determined and bound, after washing, if necessary, is eluted with a suitable buffer and determined with a detector, its use for the determination of proteins and a method for the determination of proteins below Use of the immunoassay according to the invention.
  • Proteins can be determined (the corresponding antibodies are immobilized in the cartridge), immunoglobulins (through immobilized proteins A / G) or other systems with high binding constants such as avidin / biotin or lectins / glycosylated macromolecules.
  • the sensitivity of the immunoassay can be increased by coupling to fluorescent dyes or other markers before or after the cartridge;
  • the measuring range of the immunoassay can be adjusted by varying the injection volume (cumulative effect), which means that very small concentrations can also be measured;
  • samples can be used directly (i.e. undiluted, not conditioned).
  • concentration of the substance to be analyzed is very large, e.g. B. several milligrams per milliliter, can be adjusted upwards by an increased dispersion of the measuring range;
  • the system can be coupled to an autosampler, which enables simple analysis of several hundred samples in succession; the detection can be carried out in the most varied of ways, the determination of the fluorescence of the proteins or of the UV absorption being particularly suitable;
  • Suitable carriers are the usual carriers such as synthetic or natural polymers or glasses, in particular Sepharos 4B, Eupergit, Eurocell ONB and VA epoxy biosynth, the grain size preferably being between 100 and 200 ⁇ m.
  • the binding of the partner of the desired strong binding reaction to the carrier used can be carried out adsorptively, ionically or covalently, but the latter is preferred.
  • the carrier must be activated for this by customary methods. This can be done by means of hydroxy groups on the support by reaction with bifunctional substances such as glutardialdehyde and subsequent binding of the binding partner, after conversion of the hydroxy groups into amino or carboxy groups and subsequent binding of the binding partner by way of example or by way of example Hydroxide groups occur in groups with terminal epoxy functions and subsequent addition of the binding partner, the latter method being preferred.
  • bifunctional substances such as glutardialdehyde and subsequent binding of the binding partner
  • the system was tested for the following analytes: antithrombin III, rt-PA and antibodies of the IgG type.
  • the first two analytes were tested with poly- and monoclonal antibodies, the IgG antibodies with proteins A and G.
  • Sepharose 4B, Eupergit, Eurocell ONB and VA-Epoxy Biosynth were tested as carriers for the rt-PA assay.
  • Detection was carried out in a flow spectrophotometer (excitation at 273 nm, emission measurement at 340 nm).
  • Figure I shows the calibration line for rt-PA with various immobilizates.
  • Figure II shows the influence of the pH value of the elution solution on the protein fluorescence.
  • Figure III shows the long-term stability for the ATIII measurement: the correlation coefficients are very high, even if the slope of the straight line decreases. A simple two-point calibration per measuring day is sufficient to take this effect into account.
  • Figure IV shows the agreement of the measurement results from two different measurement days after a previous two-point calibration.
  • Figure V shows the agreement of the measurement results of the conventional ELISA's and the measurement values of the automated heterogeneous assay.
  • Figure VI shows that interference from foreign proteins can be excluded by the washing step.

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

Abstract

Un essai immunitaire hétérogène se caractérise en ce qu'il contient un partenaire à forte réaction de liaison immobilisé de manière irréversible sur un support solide. Le support peut être exposé à un courant tampon liquide, de sorte que la contrepartie liée non marquée à détecter soit éluée, le cas échéant après un lavage avec un tampon approprié, et détectée par un détecteur. L'invention concerne également l'utilisation de cet essai immunitaire hétérogène.
PCT/EP1995/000727 1994-03-04 1995-02-28 Essai immunitaire heterogene et son utilisation pour detecter des proteines WO1995023972A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP7522691A JPH09509745A (ja) 1994-03-04 1995-02-28 ヘテロジェネアスイムノアッセイ及び蛋白質を検出するためのその使用
AU26074/95A AU701926B2 (en) 1994-03-04 1995-02-28 Heterogeneous immunoassay and the use thereof for detecting proteins
EP95911276A EP0748448A1 (fr) 1994-03-04 1995-02-28 Essai immunitaire heterogene et son utilisation pour detecter des proteines

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4407142.6 1994-03-04
DE19944407142 DE4407142A1 (de) 1994-03-04 1994-03-04 Heterogener Immunassay und dessen Verwendung zur Bestimmung von Proteinen

Publications (1)

Publication Number Publication Date
WO1995023972A1 true WO1995023972A1 (fr) 1995-09-08

Family

ID=6511805

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1995/000727 WO1995023972A1 (fr) 1994-03-04 1995-02-28 Essai immunitaire heterogene et son utilisation pour detecter des proteines

Country Status (6)

Country Link
EP (1) EP0748448A1 (fr)
JP (1) JPH09509745A (fr)
AU (1) AU701926B2 (fr)
CA (1) CA2184714A1 (fr)
DE (1) DE4407142A1 (fr)
WO (1) WO1995023972A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10015448A1 (de) * 2000-03-29 2001-10-11 November Ag Molekulare Medizin Verfahren zum Nachweisen und/oder Quantifizieren des Bindens erster Moleküle an dazu affine zweite Moleküle

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0022005A1 (fr) * 1979-06-21 1981-01-07 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procédé de séparation d'une substance protéinique à partir d'une solution la contenant par filtration d'affinité et application dudit procédé aux dosages enzymatiques
EP0196787A1 (fr) * 1985-02-28 1986-10-08 Trustees of Boston University Procédé de détection des aflatoxines
EP0210107A1 (fr) * 1985-07-26 1987-01-28 Lyonnaise Des Eaux - Dumez Procédé de dosage immunologique en continu de composés organiques dans un fluide en circulation, et dispositif de mise en oeuvre
WO1991016116A1 (fr) * 1990-04-23 1991-10-31 Cellpro Incorporated Dispositif et procede d'immunoselection

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5884869A (en) * 1996-03-18 1999-03-23 Hughes Electronics Corporation Satellite spin vector control with sun sensor

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0022005A1 (fr) * 1979-06-21 1981-01-07 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procédé de séparation d'une substance protéinique à partir d'une solution la contenant par filtration d'affinité et application dudit procédé aux dosages enzymatiques
EP0196787A1 (fr) * 1985-02-28 1986-10-08 Trustees of Boston University Procédé de détection des aflatoxines
EP0210107A1 (fr) * 1985-07-26 1987-01-28 Lyonnaise Des Eaux - Dumez Procédé de dosage immunologique en continu de composés organiques dans un fluide en circulation, et dispositif de mise en oeuvre
WO1991016116A1 (fr) * 1990-04-23 1991-10-31 Cellpro Incorporated Dispositif et procede d'immunoselection

Also Published As

Publication number Publication date
DE4407142A1 (de) 1995-09-07
JPH09509745A (ja) 1997-09-30
AU701926B2 (en) 1999-02-11
AU2607495A (en) 1995-09-18
CA2184714A1 (fr) 1995-09-08
EP0748448A1 (fr) 1996-12-18

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