Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1997-10-17
1999-07-06
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 793, 435975, 436500, 436506, 436518, 436547, G01N 33543, G01N 33564
Patent
active
059196327
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the detection of antibodies in biological fluids with the aid of immunological assays which are carried out with the aid of reagent kits which are produced on an industrial scale and sold by producers of diganostic agents. In particular, the present invention relates to the detection of autoantibodies, in particular autoantibodies against the autoantigens of the thyroid, in which in turn the determination of human autoantibodies against thyroglobulin (hTg) in a human serum or plasma is of primary importance in connection with the present invention.
Thyroglobulin is one of the thyroid autoantigens and is a high molecular weight protein which consists of two identical glycosylated subunits having a molecular weight of about 330 kD each (cf. for example J. Furmaniak and B. Rees Smith in: Autoimmunity, 1990, Vol. 7, pages 63-80). Thyroglobulin is a principle component of the thyroid colloid and is a precursor of the thyroid hormones triiodothyronine (T.sub.3) and thyroxine (T.sub.4). As a preliminary stage to the synthesis of the stated thyroid hormones, unbound circulating iodine or iodide is incorporated in tyrosyl radicals or iodotyrosine radicals of the Tg under catalytic action of the enzyme thyroidperoxidase (TPO). The thyroid hormones T.sub.3 and T.sub.4 are then liberated from the iodinated thyroglobulin, this being effected by hormonal control mechanisms which respond to the content of T.sub.3 or T.sub.4 in the blood.
In the case of thyroid autoimmune diseases, in particular in the case of the destructive Hashimoto's thyroiditis leading to hypothyroidism, in addition to autoantibodies against thyroidperoxidase considerable amounts of autoantibodies against thyroglobulin are detected in the patients' blood (such antibodies are always referred to below as "anti-hTg autoantibodies"). The detection and the quantitative determination of such anti-hTg autoantibodies are thus of considerable importance in the clinical diagnosis of thyroid diseases.
The determination of anti-hTg autoantibodies in patient sera is possible by various techniques, including immunological assays. In a commercially available assay of this type (HENNINGtest.RTM. anti-Tg), prediluted serum samples are incubated in the test tubes simultaneously with a labelled Tg (as tracer) and a protein A suspension. If anti-hTg autoantibodies are present in the sample, they react with the antigen hTg added as the tracer. At the same time, the protein A present in the form of particles of a suspended solid phase unspecifically binds to the Fc subunits of the antibodies, and a sandwich complex is formed. The particles of the solid-phase suspension with all molecules bound thereto are converted into a sediment by centrifuging, and the supernatant containing the unbound tracer is removed. The larger the amount of autoantibodies in the sample, the greater the amount of tracer which is bound. The concentration of the anti-hTg autoantibodies is thus directly proportional to the amount of tracer detectable in the sediment, for example to the radioactivity measurable in the sediment in the case of radiolabelling.
By using the unspecific binding partner protein A, with the aid of which whole classes of IgG antibodies can be bound without distinction, and by the simultaneous use of labelled thyroglobulin as a tracer, all anti-hTg autoantibodies are detected in the assay described. Since it is known that patient sera may also contain anti-hTg autoantibodies which cannot be directly associated with a clinically manifest thyroid autoimmune disease, assays which respond selectively to such anti-hTg autoantibodies which are directly linked with, and are the cause of clinically manifest thyroid diseases, such as, for example, Hashimoto's thyroiditis, would also be of interest per se. Moreover, the fact that the known method described requires a centrifuging step may be regarded as a certain disadvantage of said method. However, another method which works as a competitive assay and, as a coated tube method, manages without a centrifu
REFERENCES:
patent: 4818688 (1989-04-01), Adamich et al.
patent: 5501955 (1996-03-01), Bergman
Copy of PL 162 445 with English translation of Polish patent claims only.
B.R.A.H.M.S. Diagnostica GmbH
Saunders David
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