DD282003B5 - Solid phase immunoassay for the detection of viral infections - Google Patents

Description

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Field of application of the invention

The invention relates to a solid phase immunoassay for the detection of viral infections. It serves the combined detection of several infectious agents, using the diagnostics of circulating antigen and circulating antibodies. It can be used in the field of human medicine, in clinical chemistry and laboratory diagnostics, in microbiological-virological and clinical-immunological diagnostics. It is also used in hygiene facilities for the elucidation of epidemiological situations concerning the distribution of the pathogens to be detected, as well as in blood donation centers and the pharmaceutical industry in the elimination of pathogen-containing biological fluids or organs.

Characteristic of the known state of the art

Solid phase immunoassay techniques are the most widely used methods for detecting infectious agents (bacteria, viruses). Since the infection with a pathogen in the affected organism causes the generation of specific antibodies, their detection is considered indirect evidence of contact with the corresponding pathogen and applied wherever viral or bacterial antigen due to very low concentration in the sample is not sure can be detected. If the latter occur in the concentrations sufficient for the detection techniques, preference is given to their detection as direct evidence of the infection (review of the literature by Porstmann, T. and G. Porstmann, Dt. Gesundheits 36, 1986-1990 [1981]). Thus, antibody and antigen detection are practiced as Immunoassayvariante side by side in different tests. The constant increase of infectious pathogens which proves to be pathogenic leads to an increasing number of different tests in the diagnosis, but above all to the monitoring of the biological products intended for therapy. So far, at least one test was needed for the detection of a pathogen. It is used in the antigen test of known methods such as the competitive solid phase immunoassay with insolubilized antigen or antibody and the two-site binding assay (Schuurs, A.H.W.M., and B.K. van Weemen: CMn.Chim. Acta 81, 1-40 [1977]). For the detection of specific antibodies, four different methods - antiglobulin or sandwich technique, competition technique, capture technique and bridging technique - have been described (R. Tedder: in JC Gluckman and E. Vilmer (ed.): Acquired Immuno Deficiency Syndrome; Elsevier, Amsterdam, New York, Oxford, Paris, 1986, pp. 173-181).

-2-goal of the invention

It is the object of the invention to detect antigen of bacterial or viral infectious agents as well as specific antibodies to other infectious agents in one and the same approach and thus to reduce the number of test approaches compared to the application in previous test methods.

Explanation of the essence of the invention

The invention has for its object to develop a solid phase immunoassay for the simultaneous detection of antigen and specific antibodies. The object was achieved by co-immobilizing antibodies to be detected antigen and antigen for binding specific antibodies or that antigen is covalently bound to the antibody prior to its immobilization. When an exciter antigen and / or antibody to an antigen of another pathogen is present, either a labeled antibody or a labeled antigen binds; in double infections both bind in parallel. By covalent binding of different peptides as well as their coimmobilization and labeling, specific antibodies against more than one pathogen can be detected simultaneously in one approach. The essence of the invention aims to unite the statements of two or more tests in one, in which case can be dispensed with the exact pathogen diagnosis, if already one of the detected pathogens defined anti-epidemic measures or the blocking of the biological product by itself , According to the invention, this is achieved by combining the two-site binding assay for antigen detection with the bridge antibody technique for antibody detection. For this bacterial or viral antigen (eg purified from viral lysate, recombinant antigen or synthetic peptides) is either co-immobilized with the antibody for binding the additional antigen to be detected by known methods on the solid phase or covalently bound to the antibody, and only this is insolubilized on the solid phase by known methods. In the case of covalently binding peptides to the antibody, its reactivity is hardly affected so that multiple (different) peptides that simulate sequences from proteins of different pathogens (eg, HIV-1 and HIV-2) can be co-immobilized. With the antigen and / or specific antibody present in the sample to be tested, the former is bound by the insolubilized antibody while the latter binds to the co-immobilized or covalently-fixed antigen (Figure 1 and Figure 2). According to known methods, the samples are incubated for 30-120 min at 20-37 ° C and removed after removal by suction nonspecifically bound components by rinsing with washing buffer of known composition. This is followed by incubation with a combination of labeled antibodies and labeled antigen or labeled antibodies of different specificity (Figure 1) or incubation with a labeled antigen-antibody conjugate (covalent binding of antigen to the antibody before or after labeling) via a Period of 30-120 min at 20-37 ⁰ C. After renewed separation of the unbound components, the substrate reaction by known methods or the radioactivity measurement or the determination of the fluorescence intensity.

embodiment

Monoclonal anti-HBsAg IgG (final concentration 10 mg / ml) was probed with a 21-member peptide from the envGen region of HIV-1 (amino acid sequence 586-606) (Wang, JJG, S. Steel, R. Wisniewolski and CY Wang: Proc. Notl Sci. Acad.., USA 83, 6159-6163 [1986]) (final concentration with carbodiimide 5mmolll) in a molar ratio of 1:50 for 120 minutes (at 20 ⁰ C by a known method Talamo, P. C., and E. Haber KF Austen: J. Immunol 101, 333-341 [19681] and subsequently separated from free carbodiimide and unbound peptide by gel filtration over Sephacryl S 200. Part of this conjugate was treated with periodate-activated horseradish peroxidase (final periodate concentration 10 mmol / l, activation time 10 min Separation of free periodate by Sephadex G-25 gel filtration) in the molar ratio of IgG to POD of 1: 4 according to Wilson and Nakane (In: Knapp / Holubar / Wick (ed.): Immunofluorescence and related staining techniques, Eiservier, Amsterdam, 1978, p.215-221) coupled for two hours at room temperature Conjugate stabilization was accomplished by reaction with sodium borohydride (final concentration 5 mmol / L) for 2 hours at 40 ^° C. Excess sodium borohydride was then removed by dialysis against PBS.

Peptide-antibody conjugate was adsorbed on a solid phase (polystyrene microtest plates) at a concentration of 10 μg IgG / ml by a known method (Catt, K. and GW Tregear, Science 158, 1570-1572 [1967]). Subsequently, a serum with a known content of HBsAg and a serum with antibodies to HIV-1 and a mixture of equal parts by volume of both sera was diluted geometrically and incubated in separate wells for 2 h at 37 ⁰ C. As a control, a serum mixture of 10 individual sera containing neither HBsAg nor anti-HIV-1 antibody was geometrically diluted and incubated in the wells under the same conditions. After separation of the unbound reactants, the substrate reaction with o-phenylenediamine and H ₂ O _{2 was carried out} by a known method (T. Porstmann, B. Porstmann, R. Wietschke, R. von Baehr and E. Egger: J. Clin Chem. Clin Biochem. , 23, 41-45 [1985]) and the measurement of the product at 492 nm. Result:

- HBsAg can be detected in this combination test up to a concentration of 1.0 μg / l (Fig. 3)

- anti-HIV antibodies (anti-gp 41) in a positive control serum can be detected in this combination test to a dilution of 1: 1 280% (Figure 4)

- The mixture of the HBsAg-positive and anti-HIV antibody-containing sample has a higher extinction than the corresponding individual samples (additive effect) (Fig. 5)

- the mixture of 10 negative sera does not exceed the limit for the detection of an HBsAg-reactive sample (x Ext. NPS + 0.05 extinction units) or the limit for the detection of an anti-HIV antibody-reactive sample fx Ext NKS + 0.10 extinction units).

Fig. 1 Combination test for the simultaneous detection of antigen and specific antibodies by reactant coimmobilization:

1. Combination test with coimmobilized antigen and antibody and a mixture of labeled antigen and antibody.

2. Combination test with coimmobilized antigen and antibody and a mixture of labeled antibodies of different specificity.

Fig. 2 Combination tests for the simultaneous detection of antigen and specific antibodies by antigen-structure-carrying antigen bodies:

1. Combination test with 1-antigen-structure-carrying immobilized and enzyme-labeled antibody.

2. Combination test with 2-antigen-structure-carrying immobilized and enzyme-labeled antibody.

Fig. 3 Standard curve for hepatitis B surface antigen in the successive two-side immunoassay with insolubilized and

Enzyme-labeled monoclonal anti-HBs antibodies without (O O)

and with coimmobilized and enzyme-labeled HIV1 antigen (···) (Assay Principle 1, Fig. 1).

Fig. 4 Titration of an anti-HIV antibody-containing serum in the antiglobulin (sandwich) technique (D-D) and in the bridge antibody technique with coimmobilized and enzyme-labeled anti-HBs antibodies (· - ·) as well as without coimmobilized and enzyme-labeled antibodies (O-O ) and the reaction of a mixture of 10 anti-HIV antibody-negative sera in the three test variants.

Fig. 5 Extinction of a mixture of 10 normal serums (D), an HBsAg positive serum {Ш), an anti-HIV antibody containing serum (B) and an HBsAg positive serum containing anti-HIV antibodies ().

Claims (7)

claims: 1. solid phase immunoassay for the detection of viral infections, characterized in that it contains co-immobilized antibodies for binding antigen and antigens to be detected as well as antigens for binding of specific antibodies with which different antigens and / or specific antibodies in one batch a) directly as an antigen b) indirectly via induced antibodies
be detected simultaneously. 2. solid phase immunoassay according to claim 1, characterized in that it contains antigen and antigens for binding of specific antibodies which are bound to antibodies covalently to antigen-structure-carrying antibodies. 3. solid phase immunoassay according to claim 1 and 2, characterized in that it contains antigen-structure-carrying antibodies which are optionally immobilized or labeled. 4. solid phase immunoassay according to claim 1 to 3, characterized in that it contains either labeled bound antibody or labeled bound antigen in an existing pathogen antigen and / or antibody to an antigen of another pathogen and both bound in parallel in double function. 5. solid phase immunoassay according to claim 1 to 4, characterized in that it contains peptide as well as different peptides, which are covalently bound to antibodies. 6. solid phase immunoassay according to claim 1 to 5, characterized in that the peptides are protein sequences of pathogens. 7. solid phase immunoassay according to claim 1 to 6, characterized in that the peptides are protein sequences of HIV-1 and HIV-2.