Immunoassay for determining the avidity of immunoglobulins

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage

Reexamination Certificate

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C435S007100, C435S007220, C435S007320, C435S962000, C436S501000

Reexamination Certificate

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06372426

ABSTRACT:

The present invention relates to a method and a diagnostic aid for the qualitative or quantitative detection of antibodies and for determining their avidity. This makes it possible to diagnose the early phase of viral, bacterial or parasitic infections. The diagnostic aid according to the invention is particularly suitable for automated processing in large analytical laboratories.
Immunoassays are frequently employed, because of their particularly good specificity and sensitivity, for detecting immunoglobulins in serum and plasma samples for medical diagnostic purposes. In addition, immunoassays are distinguished by being simple to use.
In the case of acute infections, the diagnosis is mainly based on the detection of IgM antibodies. However, cases repeatedly arise in laboratory practice where it is not possible to distinguish reliably between fresh, primary infection and one which has completed its course or has been reactivated without additional investigations. However, it is known that, for example, additional measurement of the avidity of IgG antibodies which are specific for particular viruses, bacteria or parasites makes it possible to assess serological situations which, without this additional measurement, allow no, or only an insufficiently accurate, statement to be made about the time of infection.
Determinations of the avidity of immunoglobulins are carried out in various assay systems, for example in protein-denaturing immunoassays, which are disclosed to the skilled worker inter alia in the following publications: J. Schubert et al. (1996), J. Lab. Med. 20 (12): 713-717; J. J. Gray (1995), J. Virol. Methods 52: 95-104; J. Polanec et al. (1994), J. Clin. Lab. Analysis 8: 16-21; H. O. Kangro et al. (1991), J. Med. Virol. 33: 100-105. Examples of commonly used denaturing substances are urea, diethylamines, guanidines, thiocyanates.
Determination of avidity has already been employed successfully in the diagnosis of viral and nonviral causes of infection, for example Epstein-Barr virus (EBV), rubella virus, cytomegalovirus (CMV), hantavirus, parvovirus B19, varicella zoster virus (VZV), human herpesvirus type 6 (HHV-6), hepatitis C virus (HCV), respiratory syncytial virus (RSV), herpes simplex virus type 1 or 2 (HSV-1/-2) and Toxoplasma gondii. Nevertheless, this method has not become widely used in analytical laboratories because of, amongst other things, technical problems. Three reasons are essentially responsible for this:
1. The substance which is mostly used, urea, is, at the concentration recommended by the authors, on the point of crystallization, which results in frequent blockage of all pipette tips and tubes of equipment for automatic or partly automatic processing of immunoassays (immunoassay processors).
2. If urea solutions of lower molarity (for example 5 to 6 M) are used, the validity of the method is considerably reduced.
3. The range stated in the literature to be marginal or impossible to evaluate for the method is too large.
There has thus been a need to improve known methods for determining the avidity of antibodies. In particular, the applicability to automated immunoassay processes was in need of improvement.
Surprisingly, it has been found within the scope of the present invention that the problems described above can be solved by the possibility of employing the substance, which has not previously been. used in protein-denaturing immunoassays, urea-hydrogen peroxide (obtainable, for example, from Carl Roth GmbH+Co Karlsruhe: Article number 7641.1), as protein-denaturing reagent. This substance surprisingly has the property of being as effective in a protein-denaturing immunoassay even at a low molar concentration (approximately 2.5 to 6.5 M) as a high-molar (approximately 6 to 8 M) urea solution. The present invention therefore relates to a method for the qualitative or quantitative detection of an antibody, in which this antibody is brought into contact with the antigen against which it is directed so that immune complexes are able to form, and in which the reaction mixture is brought into contact with a protein-denaturing agent which destabilizes immune complexes containing antibodies of low avidity, while immune complexes containing antibodies of higher avidity are substantially retained, and in which the extent of the binding of the antibody to the antigen is determined by a method known to the skilled worker, wherein the protein-denaturing agent is urea-hydrogen peroxide.
The present invention additionally relates to a method for determining the avidity of an antibody, in which the antibody is brought into contact in a first and a second mixture independently of one another with the antigen against which the antibody is directed so that immune complexes are able to form, and in which one of the two mixtures is brought into contact with a protein-denaturing agent which destabilizes immune complexes containing antibodies of low avidity, while immune complexes containing antibodies of higher avidity are substantially retained, and in which the extent of the binding of the antibody to the antigen in both samples is determined independently of one another by a method known to the skilled worker, and where the avidity of the antibody is revealed by the ratio of the extent of the antigen-antibody bindings in the first and the second mixture, wherein the protein-denaturing agent is urea-hydrogen peroxide.
A preferred method of this type is one in which the antigen is brought into contact, in a form bound to a solid phase, with the antibody, and subsequently washed with a buffer solution containing urea-hydrogen peroxide. Examples of preferred methods of this type include the enzyme immunoassay, radioimmunoassay, Western blot or immunofluorescence assay. However, the skilled worker is aware of other immunoassay systems which can be carried out straightforwardly according to the invention using urea-hydrogen peroxide by means of the present is description.
The abovementioned methods are particularly suitable according to the invention for detecting diagnostically relevant antibodies in body fluids. Antibodies of this type may be directed against viruses, bacteria or parasites such as, for example, EBV, rubella virus, CMV, hantavirus, parvovirus B19, VZV, HHV 6, HBV, HCV, HIV, RSV, HSV-1, HSV-2 or Toxoplasma gondii.
The determination of avidity can advantageously be carried out using commercially obtainable immunoassays (for example ELISA), partly or fully automatically. Another advantage of the present invention is the very good interpretability of the results.
The novel method for determining the avidity of antibodies is particularly suitable for differentiating fresh (i.e. only recently occurring) infections from older (i.e. less recent) infections. In particular, the present invention relates to methods for detecting an acute rubella virus infection, or one which has recently completed its course, a first CMV infection, first EBV infection, first HSV infection or Toxoplasma gondii infection.
The present invention additionally relates to diagnostic aids (diagnostic reagents, assay kits) which are suitable for application of the novel methods described. Diagnostic aids of these types are produced in a manner known per se to the skilled worker, based on the present description of the invention.


REFERENCES:
patent: 5183901 (1993-02-01), Login et al.
patent: 5206385 (1993-04-01), Login et al.
patent: 5512659 (1996-04-01), Ullman et al.
patent: 5679537 (1997-10-01), Newkirk
patent: 5830634 (1998-11-01), Brust et al.
patent: 90/02202 (1990-03-01), None
patent: 97/09619 (1997-03-01), None
Zusammenfassung, Patent Abstracts of Japan, vol. 012, No. 088 (P-678), Mar. 23, 1988 & JP 62 220865 A (Yatoron :kk), Sep. 29, 1987.
Behring Diagnostics GMBH, Order NO OSEW, Marburg, Germany.
J. Schubert, et al., “Avidity Determination in Epstein-Barr Virus Diagnosis—a Comparison of Immunofluorescence Assay and ELISA”,J. Lab Med., vol. 20, No. 12, pp. 713-717, 1996.
J.J. Gray, “Avidityof EBV VCA-specific IgG antibodies: distinction between recent primary infecti

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