DD297254A5 - Solid phase immunoassay and test kit for determination of rheumatoid factors - Google Patents

Abstract

The invention relates to a solid phase immunoassay and a test kit for the determination of rheumatoid factors * which are autoantibodies against IgG. The determination method is characterized in that human thermo-aggregated IgG is bound to a solid phase and reacted in this form with the sample to be examined. The bound RF are detected enzymatically with a conjugate of human warm aggregated IgG and horseradish peroxidase. Areas of application include medical diagnostics (for rheumatoid arthritis and other autoimmune diseases) and experimental medical research (determination of RF in cell culture supernatants) {solid phase immunoassay; Test kit; Rheumatoid factors; Autoantibody; IgG; IgG peroxidase; enzyme immunoassay; Medicine; diagnostics; Research; Rheumatoid arthritis}

Description

Field of application of the invention

The invention relates to a solid phase immunoassay and a test kit for the determination of rheumatoid factors (RP). RF are autoantibodies to the body's own Immunoglobulln Q (IgQ). The field of application is medical diagnostics, in particular for the detection of rheumatoid arthritis (RA) and differentiation from other diseases of the rheumatic type. An application also results for experimental medical research (determination of RF in cell culture supernatants).

Characteristic of the known state of the art

RA is a systemic autoimmune disease characterized by the presence of certain autoantibodies to IgQ. These autoantibodies were termed RF (Fudenberg, H.H. and H.Q. Kunkel: J. Exp. Mod. 114 [1961], 167). RF are not specific for RA, but are an important criterion in the diagnosis of this disease. They also have prognostic significance. Therefore, an RF test is always carried out if RA is suspected or the disease is monitored. For this purpose, agglutination assays, z. The latex floxatlon test (Singer, JM and L. PIoU: Am J. Mod 21 (1956), 688-982) or the hemagglutination test (Waaler, E .: Acta Path, Mcroblol, Scand. 17 [1940] 172- 188).

The solid phase immunoassays for RF are predominantly constructed as follows: On the solid phase is IgG from human, rabbit or other species. Upon incubation of the serum samples on these solid phases, the RF (anti-IgQ class IgM) bind. After a washing procedure, incubation with a labeled antibody specific for IgM as an indicator reagent is then carried out. Possibly bound IgM, which do not belong to the RF, are therefore indicated in the subsequent substrate reaction (Palosuo, T. and K. Aho: Mod. Biol. 61 (1983), 203-207). The anti-IgM used must be strictly class specific; they must not react with the IgQ of the solid phases. This can only be achieved by a complicated purification and absorption procedure or by the use of monoclonal anti-IgM. A simpler and significantly cheaper test variant results from the following:

The IgM-RF are pentavalent molecules. Therefore, after binding to the solid-phase IgG, they still have free valencies to react with other IgQ molecules. The latter can therefore be used as an indicator reagent instead of the anti-IgM if they are labeled. Two corresponding assays with IgG-glucose oxidase or IgG-biotin have been described (Maldini, R. et al .: J. Immunol Moth 2 (1978), 25-34; Zrein, M. et al .: J. Immunol. Meth. 87 (1986), 229-237). Lukowsky, A. et al. (Z. Klin. Med. 41 (1986), 923-926) developed a test according to this principle with IgG peroxydase as an indicator. In this case, fermentation-regulated rabbit IgQ was used. However, the use of canine IgQ is disadvantageous when compared to human IgG.

Human IgG Is More Sensitive and More Suitable for the Detection of RF In Solid Phase Assays (Pope, R. and S. McDuffy: J. Lab. CI In. Med. 97 (1981), 842-853).

The patent literature describes enzyme immunoassays for RF. According to EP 353306 (analogous to WO 89/06800) the use of a special solid phase for the immunoassays (methacrylate) as well as immune complexes of albumin and anti-albumin IgQ, the latter of the rabbit, is protected as an RF binding partner. EP 323785 refers exclusively to RF of class IgQ. Here, the claim extends to the type of solid phase antigen used (SchaMgG).

Object of the invention

The aim of the invention is to provide medical diagnostics with a method and set of test instruments for determining RF in serum or in other samples.

Explanation of the essence of the invention It is the object of the invention to develop a solid phase immunoassay and a test kit which can be used for the detection

of autoantibodies to IgQ, i. against RF. According to the invention, the object has been achieved by binding human immunoglobulin IgQ as an antigen to a solid phase and using a sample to be investigated

Reaction is brought. In the presence of RF, they bind to a solid phase. In a second reaction, the Indicator reaction, bind RF human-regulated IgG: the latter contains covalently bound horseradish Peroxydase as a marker enzyme. Thus, the detection of RF is based on the principle of an enzyme-immunological two-site binding assay. A

advantageous variant of eniymmlmmunologlschen Nachwelses is to run both reactions simultaneously.

The complex of the RF and the IgQ peroxydase is determined by means of a color reaction with the usual substrates, such. B.

o-phenylenediamine and hydrogen peroxide, visualized. The color reaction produced is quantified by a photometer or purely qualitatively, i. H. visually, determined. The color intensity correlates with the RF concentration in the sample.

A positive finding from RF is an important indication of the presence of RA and is considered one of the criteria for diagnosis

This disease (Johnson, P. M. and W. P. Faulk: CI In Immunol Immunopathol 6 (1976), 414-430).

Surprisingly, sl-.h has shown that human warm-agglomerated IgQ can be used simultaneously as a solid-phase antigen and,

labeled with peroxydase, is useful as an indicator reagent for RF detection. This means that otherwise for rheumatoid factor ·

Enzyme immunoassays required enzyme-labeled specific anti-IgM antibodies are no longer needed. Surprisingly, the possibility of a simultaneous test procedure continued to emerge. This can be the Incubate the sample to be tested and complete the reaction with the indicator reagent in one step. With the human IgG as solid phase antigen, sera are recognized as positive by RA patients, who in an analogous test with Rabbit IgQ did not appear. The test kit for the detection of RF consists of:

a) solid r-hare, such as microtiter plates and tubes made of polyethylenes, and the attached (absorptive or covalent, usually absorptive) human heat-aggregated IgQ,

b) human heat-aggregated IgQ covalently linked to horseradish peroxidase for detection of bound RF,

c) the buffers commonly used in enzyme immunoassays, such as. PBS / Tween,

d) dilution media for the samples and the conjugate,

e) substrates for the color reaction of the IgQ-Peioxidasekonjugates (reagent for the peroxidase reaction),

f) stop reagents to complete the color reaction and

g) control materials, such as. B. positive or negative control sera or other RF standards.

AusfQhrungsbelsplele

1. Methodology

Human IgQ purified by known methods, such as ammonium sulfate prepitiation, Tone Exchange Chromatography

and gel filtration-is homogenized by heating to 63 ° C. for 20 min (concentration: 10 g / l) and dissolving in 0.1 mol / l of carbonate buffer, pH 9.5, in a concentration of 10 mg / l, followed by wetting of polystyrene. Microtiter plates (cavity volume 400μΙ, wetting volume 50μΙ per well) at room temperature (22-25 ⁰ C) for 16-24h.

The cavities are subsequently filtered off with suction and rinsed twice with a washing buffer. This buffer can z. B. following Composition have:

0.01 mol / l phosphate buffer, pH 7.2, 0, 15, moi / l NsCt, 0.1% (v / v) Tween 20 (.PBS / Tween "). Prepared accordingly

Microtiter plates can be used immediately welter or at -20 "C for up to several months for later use

be kept.

2. Examples

A) Qualitative determination of RF activities in suspected RA: The sera to be examined are in the o.a. Medium (PBS / Tween) diluted 1: 100, e.g. B. 10μΙ serum to 1 ml with PBS-Tween

refilled. From the thus diluted serum sample add 50μΙ to the IgG '' coated plates (s.o.) and incubate for 90 min

Room temperature. The aspiration of the serum samples is followed, for example, by washing with PBS / Tween (see above). After that takes place

a further incubation for 90 min at room temperature with 50 μΙ per well of aggregated IgG-peroxidase conjugate. The latter was prepared from the aggreglerten, human IgQ and horseradish peroxidase according to known instructions by means of sodium periodate (Lukowsky et al .: Z. med Lab.diagn., 27 (1986), 191-197).

The final concentration of the conjugate should be 0.5 mg / l, and the dilution medium should be PBS / Tween. To At the end of the second incubation, the conjugate is aspirated off and the plates are again washed ζ times with PBS / Tween to

separate unbound reactants. This is followed by the substrate reaction with eii em for enzyme immunoassays

Peroxydase conjugates commonly used dye. Is particularly suitable z. As an '(eaktlon with o-phenylenediamine and Hydrogen peroxide as substrates and sulfuric acid as stop reagent (Porstmann, B. et al .: J. CI In. Chem., Clin. Biochem

(19811.435-439).

For the latter case, all cavities are measured bichromatically at 492 and 690 nm for the evaluation. Results auo

If the test has an absorbance value that exceeds three times the blank value plus the methodological variance, then the examined serum contains RF. A screonlng examination with visual evaluation is possible.

B) Inclusion of a reference curve for the quantitative determination of RF

The determination of the sera to be examined is carried out as described under A). In addition, a standard curve with a commercial RF standard or its own secondary standard is included. The standard should be diluted to give values in the range between 0.1 and 5 IU / ml. At a standard of e.g. 50 IU / ml should then be the lowest dilution about 1:10, the highest dilution about 1: 500. From at least β measuring points, the reference curves must be constructed manually or with the aid of a computer program for ELISA evaluation and the serum values read therefrom. The method enables the tracking of concentration or activity changes in the patient.

C) Detection of RF in cell culture supernatants

A corresponding Nachwels is significant for a number of experimental issues and can be done by the said method. In this case, the supernatants are to be treated in principle as the sera (see A)), except for the dilution: At low expected synthesis performance is not diluted, otherwise recommends an initial dilution of 1: 6 to 1:10. A quantitative determination is possible according to 2.

Claims (5)

A solid phase immunoassay for the determination of rheumatoid factors (RF), characterized in that human immuneaglobulin immunoglobulin Q (IgQ) is bound to a solid phase, is reacted with the sample to be tested, and immunologically bound RF with human IgG peroxydase heat conjugate conjugate as Indicator reagent are detected enzyme-immunologically. A solid phase immunoassay according to claim 1, characterized in that the IgG peroxydase conjugate is obtained by covalent attachment of human heat aggregated IgQ to horseradish peroxidase. 3. Festphaseniminunoassay according to claim 1 and 2, characterized in that the detection of RF according to the principle of an enzyme-immunological two-sided binding assay. 4. solid phase immunoassay according to claim 1 to 3, characterized in that the RF detection is carried out in the successive test with separate RF-Blndung and indicator reaction or in a simultaneous test with concurrent RF-Blndung and indicator reaction. 5. Test kit for the determination of rheumatoid factors, characterized in that it consists of a) solid phases such as microtiter plates and tubes of polystyrene loaded with human heat-aggregated IgG, b) human heat-aggregated IgO-peroxidase conjugate, c) buffering, d) dilution media for the samples and the conjugate, e) substrates for the color reaction, f) stop reagents to complete the color reaction and g) control materials, such as. B positive or negative control sera or other RF standards.