Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-08-04
2003-03-25
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
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Reexamination Certificate
active
06537760
ABSTRACT:
The present invention relates to a competitive receptor binding assay for the determination of TSH receptor autoantibodies which occur in autoimmune diseases of the thyroid, in particular in Graves' disease.
It is known that many diseases in which the thyroid is involved are autoimmune diseases in which autoantibodies against molecular structures of the thyroid are formed and, in conjunction with the disease, begin to act as autoantigens. The most important known autoantigens of the thyroid are thyroglobulin (Tg), thyroid peroxidase (TPO) and in particular the TSH receptor (TSHr) (cf. Furmaniak J. et al., Autoimmunity 1990, Vol. 7, pages 63-80).
The TSH receptor is a receptor which is localized in the thyroid membrane and to which the hormone TSH (thyroid-stimulating hormone or thyrotropin) secreted by the pituitary gland binds and thus triggers the secretion of the actual thyroid hormone, in particular thyroxine. The TSH receptor belongs to the receptor family, comprising the G-protein-coupled glycoprotein receptors having a large amino terminal extracellular domain, to which the LH/CG and the FSH receptor also belong. The chemical structure of the TSH receptor, i.e. the sequence of the DNA coding for it and the amino acid sequence, derivable therefrom, of the receptor itself, was elucidated at the end of 1989 (cf. Libert F. et al., Biochem. Biophys. Res. Commun. 165: 1250-1255; Nagayama Y. et al., Biochem. Biophys. Res. Commun. 165: 1184-1190; cf. also EP-A-0433509 and WO-A-91/09121; and WO-A-91/09137; WO-A-91/10735 and WO-A-91/03483; furthermore, Yuji Nagayama & Basil Rapoport in: Molecular Endocrinology, Vol. 6 No. 2, pages 145-156, and the literature cited therein).
It is generally known that stimulating autoantibodies play a role in the thyroid autoimmune disease known as Graves' disease, said autoantibodies being formed against the TSH receptor and interacting with it so that the thyroid is stimulated, which manifests itself as hyperthyroidism. The determination of autoantibodies against the TSH receptor is thus of considerable clinical importance for the diagnosis of Graves' disease.
Apart from assay methods in which experimental animals or special cell cultures play a role and which are now in particular of historical interest (cf. Schumm-Draeger et al., Akt. Endokr. Stoffw. 10 (1989), pages 90-102), it has been possible to date to determine TSH receptor autoantibodies essentially according to two basic methods (cf. Morgenthaler N. G. et al., Exp Clin Endocrinol Diabetes 104 (1996) Suppl 4, pages 56-59): In cell stimulation tests, the presence of stimulating TSH receptor autoantibodies, which are frequently referred to in the literature with the abbreviation TSI (TSI=thyroid stimulating immunoglobulins), manifests itself by the fact that specific functions of suitable cells which have natural or recombinant TSH receptors in their cell membrane and come into contact with a sample containing autoantibodies are triggered or enhanced by stimulation, in particular the formation of cAMP (cyclic adenosine monophosphate). In these tests also referred to as bioassays, the stimulating effect is selectively measured, but the measurement is extremely complicated and therefore not very suitable for routine clinical diagnostics.
Alternatively, autoantibodies can also be determined using competitive receptor binding assays, in particular radio receptor assays, for example with the use of the TRAK-Assay® from B.R.A.H.M.S Diagnostica GmbH. For the determination of TSH receptor autoantibodies by this conventional method, the autoantibodies to be determined and originating from a serum sample are allowed to compete in the liquid phase with a radiolabelled bovine TSH competitor for the binding sites of a detergent-solubilized porcine TSH receptor (cf. Southgate, K. et al., Clin. Endocrinol. (Oxford) 20, 539-541 (1984); Matsuba T. et al., J. Biochem. 118, pages 265-270 (1995); EP 719 858 A2; product information on the TRAK-Assay® from B.R.A.H.M.S Diagnostica GmbH). To determine the labelled TSH bound to the receptor preparation, after incubation is complete the TSH receptor is separated from the liquid phase using a precipitation reagent and a subsequent centrifuging step. The receptor-bound labelled TSH is determined by measuring the radioactivity bound in the sediment. Since the determination is based on competition between labelled TSH and the autoantibodies to be determined for common binding sites of the TSH receptor, all those autoantibodies, and only those antibodies, which actually compete with TSH are determined in this method. Such competing autoantibodies capable of inhibiting the TSH binding are also referred to in the literature as TBII (TBII=thyrbtropin-binding inhibitory immunoglobulin), and the extent of their activity is also stated as a percentage so-called TBII activity.
It has long been known that heterogeneous autoantibody populations of different compositions are formed in autoimmune diseases of the thyroid. The stimulating autoantibodies and the autoantibodies competing with TSH are only partly identical, i.e. there are stimulating autoantibodies which do not compete with TSH and there are also autoantibodies competing with TSH which do not have a stimulating effect. In addition, autoantibodies which neither have a stimulating effect nor compete with TSH may also be present (cf. for example Ludgate M. et al., Mol. Cell. Endocrinol. 73 (1990), R13-R18; Filetti S. et al., J. Clin. Endocrinol. Metab. 72, pages 1096-1101, 1991; Morgenthaler N. G. et al., Exp Clin Endocrinol Diabetes 104 (1996) Suppl 4, pages 56-59, and literature cited therein). As a consequence of this, autoantibodies are detectable with the aid of radio receptor assays only in about 80% to 90% of patients suffering from Graves' disease (cf. for example Rationelle Diagnostik in der Endokrinologie [Rational Diagnostics in Endocrinology], Thieme Verlag, page 49, paragraph: TSH Rezeptorautoantikörper (TSH-RAK) [TSH receptor autoantibodies (TSH-RAB)]; or Ropars A. et al., Cell. Immunol. 161, pages 262-269 (1995); Ohmori M. et al., Biochem. Biophys. Res. Commun. 174, No. 1 (1991), pages 399-403; Endo T. et al., Biochem. Biophys. Res. Commun. 181, No. 3 (1991), pages 1035-1041; Gupta M. K., Clin. Biochem. 25, pages 193-199 (1992)). Since the failure to detect a part of the autoantibodies occurring in Graves' disease is due to the measuring principle of the competitive radio receptor assays to date, it has already been proposed, in spite of considerable complexity, to carry out a supplementary bioassay measurement for determining stimulating TSI autoantibodies if a discrepancy between the clinical picture of a patient suffering from Graves' disease and the result of the determination of competing TBII autoantibodies is evident (Derwahl M. et al., Exp. Clin. Endocrinol. 100 (1992), pages 75-79).
Apart from the limited clinical value described, the competitive radio receptor assays known to date for the detection of TSH receptor autoantibodies have fundamental disadvantages of a practical nature which are due to the fact that the binding capability of TSH receptor preparations is generally very sensitive to changes in the receptor or in the biomolecule bound by it. The binding of biomolecules of a peptide or protein nature, for example of hormones or autoantibodies, to receptors is as a rule very complex, and the formation of a specific bond between receptor and biomolecule is very much more sensitive to structural changes, in particular in the receptor, than is the case with a conventional antigen/antibody binding pair, which is the basis of most immunoassays in which the receptors play no role. Attempts to immobilize and/or to label the TSH receptor led as a rule to structural changes which greatly impaired the functionality of the receptor. As a consequence of this, many basic assay types which are available in immunoassays utilizing an antibody/antigen bond, in particular those in which immobilized binding partners are used and, at the end of
Bergmann Andreas
Struck Joachim
B.R.A.H.M.S. Aktiengesellschaft
Chin Christopher L.
Cook Lisa V.
Pillsbury & Winthrop LLP
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