WO1988003649A1 - Process for detecting, identifying and/or quantifying of circulating immune complexes in blood serum - Google Patents

Process for detecting, identifying and/or quantifying of circulating immune complexes in blood serum Download PDF

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Publication number
WO1988003649A1
WO1988003649A1 PCT/NL1987/000034 NL8700034W WO8803649A1 WO 1988003649 A1 WO1988003649 A1 WO 1988003649A1 NL 8700034 W NL8700034 W NL 8700034W WO 8803649 A1 WO8803649 A1 WO 8803649A1
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WO
WIPO (PCT)
Prior art keywords
immune complexes
process according
polyanionic
heparin
detecting
Prior art date
Application number
PCT/NL1987/000034
Other languages
French (fr)
Inventor
Hans Van Dijk
Peter Bloembergen
Original Assignee
Rijksuniversiteit Utrecht
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 Rijksuniversiteit Utrecht filed Critical Rijksuniversiteit Utrecht
Publication of WO1988003649A1 publication Critical patent/WO1988003649A1/en

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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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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

Definitions

  • PROC ⁇ SS FOR DETECTING, IDENTIFYING AND/OR QUANTIFYING OF CIRCULATING IMMUNE COMPLEXES IN BLOOD SERUM.
  • the invention relates to a process for detecting, identifying and/or quantifying of ciculating immune complexes (CIC) in blood serum.
  • CIC ciculating immune complexes
  • CICs are complexes of antigen and antibodies encountered by a growing number of syndromes.
  • CICs are used as one of the primary diagnostics.
  • An inventory is now made of the CICs 1 presence in other syndromes (forms of cancer, various infectious diseases, etc.).
  • the CICs in blood serum can be detected by using polyanionics as universal CIC-binders. It has also been found that by the use of polyanionics the said CICs can be identified individually be the class of antibodies (IgM-, IgG-, IgA- and/or IgE-complexes) . Identification of the CICs based on the antigens present therein is also possible in the aforesaid arrangement. Finally it has been found that the CICs can be quantified by the referred method by the antibody class and possibly antigen as well.
  • the invention relates to a process as mentioned in the preamble characterized by contacting the blood serum to be tested with a polyanionic, by which the immune complexes present in the serum are bound to this substance, and thereafter detecting them by the reagents specific for immune complexes (anti-immune globulines and antigen-specific antisera) .
  • a solid phase technique is used in this process, applying the polyanionic onto a solid support, for example synthetic material.
  • a solid support for example synthetic material.
  • Polyvinyl chloride is a suitable material.
  • heparin-albumin conjugate as polyanionic.
  • a conjugate is known from U.S. patent specification 4 526 714. It is preferred to apply the heparin-co jugate onto the solid support, after applying a polycationic underlayer onto the support.
  • Protamine can be used as an underlayer.
  • high molecular dextran sulfate may successfully be used as polyanionic.
  • Immtine complexes on the polyanionic layer can be detected by using an enzyme-linked immunosorbent. assay (ELISA) arrangement.
  • the invention also relates to a vessel suitable to carry out the process according to the invention and the vessel is characterized in that at least a part of the inner surface is provided with a polyanionic coating, for example heparin-albumin conjugate or high molecular dextran sulfate.
  • a polyanionic coating for example heparin-albumin conjugate or high molecular dextran sulfate.
  • the conjugate is preferably applied onto a layer of the protamine polycationic.
  • a particularly suitable object to carry out the process according to the invention is a microtiter plate with vessel-shaped recesses, the inner surfaces of said vessels being provided with the above mentioned coating.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Rehabilitation Therapy (AREA)
  • Rheumatology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Process for detecting, identifying and/or quantifying of immune complexes in blood serum by contacting the serum with a polyanionic, by which the immune complexes present in the serum are bound to this substance, and thereafter detecting them by the specific reagents for certain, immune complexes.

Description

PROCΞSS FOR DETECTING, IDENTIFYING AND/OR QUANTIFYING OF CIRCULATING IMMUNE COMPLEXES IN BLOOD SERUM.
The invention relates to a process for detecting, identifying and/or quantifying of ciculating immune complexes (CIC) in blood serum.
Said CICs are complexes of antigen and antibodies encountered by a growing number of syndromes. With certain conditions (systemic lupus erythematosus, rheumatoid arthritis, related autoimmune diseases, kidney conditions and bacterial endocarditis) CICs are used as one of the primary diagnostics. An inventory is now made of the CICs1 presence in other syndromes (forms of cancer, various infectious diseases, etc.).
It has now been found that the CICs in blood serum can be detected by using polyanionics as universal CIC-binders. It has also been found that by the use of polyanionics the said CICs can be identified individually be the class of antibodies (IgM-, IgG-, IgA- and/or IgE-complexes) . Identification of the CICs based on the antigens present therein is also possible in the aforesaid arrangement. Finally it has been found that the CICs can be quantified by the referred method by the antibody class and possibly antigen as well. The invention relates to a process as mentioned in the preamble characterized by contacting the blood serum to be tested with a polyanionic, by which the immune complexes present in the serum are bound to this substance, and thereafter detecting them by the reagents specific for immune complexes (anti-immune globulines and antigen-specific antisera) .
Preferably a solid phase technique is used in this process, applying the polyanionic onto a solid support, for example synthetic material. Polyvinyl chloride is a suitable material.
Favourable results are achieved by using a heparin-albumin conjugate as polyanionic. Such a conjugate is known from U.S. patent specification 4 526 714. It is preferred to apply the heparin-co jugate onto the solid support, after applying a polycationic underlayer onto the support.
Protamine can be used as an underlayer. In the process according * to the invention high molecular dextran sulfate may successfully be used as polyanionic. Immtine complexes on the polyanionic layer can be detected by using an enzyme-linked immunosorbent. assay (ELISA) arrangement.
The invention also relates to a vessel suitable to carry out the process according to the invention and the vessel is characterized in that at least a part of the inner surface is provided with a polyanionic coating, for example heparin-albumin conjugate or high molecular dextran sulfate.
In case a heparin-albumin conjugate is present on the bottom of the vessel, the conjugate is preferably applied onto a layer of the protamine polycationic. A particularly suitable object to carry out the process according to the invention is a microtiter plate with vessel-shaped recesses, the inner surfaces of said vessels being provided with the above mentioned coating.
The invention will be further illustrated by the following example that is not limiting in any way.
Example
To a polyvinylchloride microtiter plate comprising 96 flat- bottomed incubation vessels (Costar 2595) per vessel are added 150 μl of a solution of 5 μl protamine sulfate per ml in EDTA- veronal buffer (10 mM EDTA (= ethylene diamine tetraacetic acid) , 150 mM NaCl, 5 mM Veronal, pH = 7.4) . The plate is incubated at 37°C for 1 hour and subsequently washed with EDTA-rinsing buffer (2 mM EDTA, 150 mM NaCl, 0.03% Tween 20, pH = 7.4). Thereafter 150 μl of a solution of 5 μg heparin-humane serum albumin-conjugate per ml EDTA-veronal buffer is added. At this step and the next steps the plate is again incubated at 37 c for 1 hour and washed as after the first step. Subsequently the sera to be tested on CICs are put into the vessels to wit in a quantity and concentration of 100 μl and 1:100 in EDTA-veronal buffer with 0.5% Tween 20. In the next step a peroxydase labeled anti-IgA conjugate (100 μl 1:4000 in EDTA-veronal buffer with 0.5% Tween) is added to the vessels, thereafter in the last step 100 μl colouring peroxydase-substrate (100 μg tetramethyl- benzidine and 150 ml hydrogen peroxyde per ml 0.1 M acetate buffer, pH = 5.5) are added. After an incubation at room temperature for 10 minutes the pH is lowered by 100 μl 2N sulphuric acid. The possible colour reaction is measured at 450 nm in an automatic colorimeter (Titertek Multiskan) . From the result a control value is substracted (vessel without serum added) .

Claims

CLAIMS:
1. Process for detecting, identifying and/or quantifying of circulating immune complexes in blood serum, characterized by contacting the serum with a polyanionic, by which the immune complexes present in the serum are bound to this substance, and thereafter detecting them by the specific reagents for certain immune complexes-
2. Process according to claim 1, characterized in that the polyanionic is applied onto a solid support.
3. Process according to claim 2, characterized in that a heparin-albumin conjugate is used as polyanionic.
4. Process according to clai 3, characterized in that the heparin-conjugate is applied onto a solid support, applying an underlayer of a polycationic, for example protamine.
5. Process according to one of the preceding claims, characterized in that the immune complex is detected by an enzyme-linked immunosorbent assay (ELISA) ,
6. Process according to claim 5, characterized in that horse¬ radish peroxydase is used as enzyme and tetramethylbenzidine and hydrogen peroxide are used as underlayer.
7. Vessel suitable for the process according to the invention, characterized in that at least a part of the inner surface is coated with a polyanionic.
8. Vessel according to claim 7, characterized in that the coating consists of a heparin-albumin conjugate or high molecular dextran sulfate.
9. Vessel according to claim 8, characterized in that the heparin-albumin conjugate is applied onto an underlayer of polycationic, for example protamine.
10. Microtiter plate with vessel-shaped recesses according to one of the claims 7, 8 or 9.
PCT/NL1987/000034 1986-11-12 1987-11-12 Process for detecting, identifying and/or quantifying of circulating immune complexes in blood serum WO1988003649A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL8602869A NL8602869A (en) 1986-11-12 1986-11-12 METHOD FOR DETECTING, IDENTIFYING AND / OR QUANTIFYING CIRCULATING IMMUNE COMPLEXES IN BLOOD SERUM
NL8602869 1986-11-12

Publications (1)

Publication Number Publication Date
WO1988003649A1 true WO1988003649A1 (en) 1988-05-19

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PCT/NL1987/000034 WO1988003649A1 (en) 1986-11-12 1987-11-12 Process for detecting, identifying and/or quantifying of circulating immune complexes in blood serum

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NL (1) NL8602869A (en)
WO (1) WO1988003649A1 (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2320553A1 (en) * 1975-08-06 1977-03-04 Riedel De Haen Ag DIAGNOSIS AGENT FOR THE DETECTION OF BLOOD AND OTHER SUBSTANCES WITH PEROXIDATIVE EFFECT IN BODY FLUIDS
NL8001972A (en) * 1980-04-03 1981-11-02 Akzo Nv Peroxidase determn. in enzyme immunoassay - using tetra:alkyl-benzidine as chromogenic hydrogen donor
WO1982002773A1 (en) * 1981-02-05 1982-08-19 Icl Scient Polystyrene latex reagents and related methods for use in immunological diagnostic procedures
EP0080109A1 (en) * 1981-11-19 1983-06-01 New York Blood Center, Inc. Sensitive immunoassays of antigens or antibodies sequestered with immune complexes
WO1983003475A1 (en) * 1982-03-31 1983-10-13 Biostar Med Prod Method of performing an immuno-assay, article for use therein and method for making such article
EP0141343A2 (en) * 1983-10-26 1985-05-15 Roche Diagnostics GmbH Process for the determination of low-density lipoproteins and reagent therefor
US4526714A (en) * 1982-12-13 1985-07-02 Cordis Europa N.V. Conjugates of anticoagulant and protein

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2320553A1 (en) * 1975-08-06 1977-03-04 Riedel De Haen Ag DIAGNOSIS AGENT FOR THE DETECTION OF BLOOD AND OTHER SUBSTANCES WITH PEROXIDATIVE EFFECT IN BODY FLUIDS
NL8001972A (en) * 1980-04-03 1981-11-02 Akzo Nv Peroxidase determn. in enzyme immunoassay - using tetra:alkyl-benzidine as chromogenic hydrogen donor
WO1982002773A1 (en) * 1981-02-05 1982-08-19 Icl Scient Polystyrene latex reagents and related methods for use in immunological diagnostic procedures
EP0080109A1 (en) * 1981-11-19 1983-06-01 New York Blood Center, Inc. Sensitive immunoassays of antigens or antibodies sequestered with immune complexes
WO1983003475A1 (en) * 1982-03-31 1983-10-13 Biostar Med Prod Method of performing an immuno-assay, article for use therein and method for making such article
US4526714A (en) * 1982-12-13 1985-07-02 Cordis Europa N.V. Conjugates of anticoagulant and protein
EP0141343A2 (en) * 1983-10-26 1985-05-15 Roche Diagnostics GmbH Process for the determination of low-density lipoproteins and reagent therefor

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NL8602869A (en) 1988-06-01
AU8323587A (en) 1988-06-01

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