Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-04-20
1996-03-26
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 792, 435 794, 435 795, 435 28, 436503, 436506, 436518, G01N 33564, G01N 33543
Patent
active
055019552
DESCRIPTION:
BRIEF SUMMARY
The invention relates to an immunological assay method for the determination of antibodies in biological fluids, in particular of autoantibodies, the detection of which permits the diagnosis of an autoimmune disease.
Various immunological assay methods play a very important role in medical diagnostics. In addition to those assay methods aimed at the qualitative and/or quantitative determination of antigens or haptens, for example hormones, there are also many assay methods of this type for the determination of antibodies in biological fluids, in particular human sera.
Antibodies are proteins which are designated as immunoglobulins (Ig) and which are formed by the body as a reaction to an antigen. Since antigens normally have many antigenic determinants, antibodies are polyclonal and therefore represent a population of proteins having different binding properties relative to the antigen against which they are directed. They are normally formed to counteract exogenous antigens in order to protect the body from substances which have corresponding antigenic determinants. If the immune system of the body incorrectly recognises certain endogenous cells or cell structures as being exogenous, however, antibodies can also be formed against antigenic determinants of endogenous elements. Such endogenous elements are then designated as autoantigens, and the antibodies formed to counteract them are designated as autoantibodies.
Known assay methods for antibodies realise, in one form or another, various basic principles, two of which are shown schematically, for example, at the top of column 3 of U.S. Pat. No. B1 3,654,090. According to a variant which corresponds to the classical radioimmunoassay (RIA), a deficiency of an immobilised antigen is used, and a labelled form of the antibody to be determined is added in a known amount to the sample to be investigated. Information about the presence or concentration of the required antibody can be obtained from the degree of binding of the labelled antibody to the immobilised antigen. The antigen is required in highly pure form for this test based on the competition principle.
According to a second principle, a known amount of the antibody to be determined or of a suitable derivative thereof is immobilised on a solid substrate, and the antibody present in the sample to be investigated and the immobilised antibody are then allowed to compete for a labelled antigen added to the reaction system. The presence or amount of the antibody to be determined is obtained from the reduction in the binding of the labelled antigen to the immobilised antibody and therefore to the solid phase.
In the last-mentioned method of determination, a labelled form of the associated highly pure antigen is required, and the antibody to be determined and the immobilised antibody must be present in amounts such that effective competition can occur between the immobilised antibody and the antibody in the sample to be investigated, the fact that affinities to the labelled antigen may not be completely identical being taken into account.
According to a further principle, in a procedure analogous to the sandwich test well known for antigen determination an excess of an antigen, usually in immobilised form, is first taken, by means of which the total amount of the antibody to be determined is bound, and, by a subsequent second immunological reaction with a second labelled "antigen", against the antibody bound in the first step, the latter is labelled with formation of a sandwich-like immune complex. The second "antigen" is frequently an anti-antibody (double antibody method), or it is used for labelling the labelled so-called protein A, a protein which is obtained from bacteria and binds unspecifically to many IgG antibodies. In this method, the amounts of antigen required are such that the binding capacity is sufficient for binding all antibodies present in the sample. If larger amounts of the antibodies to be determined are expected, the samples must therefore generally be highly diluted before they can be u
REFERENCES:
Harlow et al, 1988. Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor pp. 567-569, 579-583, 591.
Ruf et al, 1988. Novel routine assay of thyroperoxidase autoantibodies. Clin Chem 34:2231-34.
Schardt et al, 1982 An enzyme-linked immunoassay for thyroid microsomal antibodies. J Immunol Meth 55:155-68.
Ruf et al, 1989. Relationship between immunological structure and biochemical properties of human thyroid peroxidase. Endocrinol. 125:1211-18.
Seradyn, Inc., 1988. Microparticle immunoassay techniques. Seradyn, Inc. pp. 4, 26-27.
Oellerich, 1984. Enzyme-immunoassay. A review. J Clin Chem Clin Biochem 22:895-904.
B.R.A.H.M.S. Diagnostica GmbH
Grun James L.
Scheiner Toni R.
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