Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1991-07-23
1994-02-22
Kepplinger, Esther L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 75, 435 794, 43524027, 436 64, 53038885, 5303911, 5303913, 530828, G01N 33574, C12N 1506, C07K 1528
Patent
active
052886140
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a method for the detection of malignant diseases and a suitable reagent therefor.
In clinical diagnostics the search for an indicator substance which can indicate malignant diseases has already been going on for a long time. Hopes have been placed on CEA (carcinoembryonic antigen) and TPA (tissue polypeptide antigen) as tumour markers with a broad organ specificity. Meanwhile, however, it has turned out that both proteins could not fulfill these expectations. The application of CEA has in the meantime been reduced mainly to therapeutic monitoring e.g. of colorectal carcinomas while TPA has only gained acceptance for a few indications in therapeutic monitoring because of its lack of specificity and sensitivity. Very many other proteins have been tested as indicators for malignant diseases but they were all unsatisfactory.
The cytokeratin (CK) class has also been associated with tumours. In the form of intermediary filament proteins they are components of the cytoskeleton of epithelial cells. Nineteen cytokeratins are known of which the cytokeratins 1 to 8 are denoted basic and the cytokeratins 9 to 19 acidic cytokeratins. The cytokeratins can aggregate in the cell to form tetramers. Each tetramer consists of two basic and two acidic cytokeratin molecules. Filaments are produced by the linear aggregation of tetramers. Intact cytokeratin molecules are water-insoluble as integral components of the intermediary filaments of epithelial cells. The complexity and composition of cytokeratins is different in the various epithelial tissues i.e. epithelial cells have cytokeratin compositions which are typical for the tissue. However, up to now nothing has been known about whether malignant diseases correlate with the occurrence of cytokeratins in body fluids.
It was thus the object of the present invention to provide a method with which the presence of a malignant disease can be diagnosed.
This object is achieved by a method for the detection of malignant diseases which is characterized in that the sample of a body fluid is incubated with at least two receptors R.sub.1 and R.sub.2 in which a signal change is produced by binding of at least one of the receptors R.sub.1 and R.sub.2 to the substance to be detected in the sample solution and in which one of the two receptors contains a monoclonal antibody which binds to the amino acid sequence 291 to 335 of cytokeratin 19 and the other receptor contains a monoclonal antibody which binds to the amino acid sequence 346 to 367 of cytokeratin 19 and the signal change in the sample caused by the binding is determined.
Surprisingly it was established that fragments of cytokeratin 19 are formed primarily in epithelial tumour tissues which have at most the amino acid sequence 219 to 367, but at least the sequence 311 to 359, of the complete cytokeratin 19 and have epitopes on the amino acid sequences 291 to 335 and 346 to 367 which can specifically bind to the aforementioned monoclonal antibodies or to derivatives thereof and which, in addition, can be found in body fluids. Cytokeratin 19 (CK 19) has an amino acid chain consisting of 400 amino acids. The sequence is described in Stasiak, P. C. et al., J.Invest.Dermatol. 92 (1989) 707-716, in particular on page 712 and is divided into 3 domains 1A, 1B and 2. The fragments which are to be detected according to the present invention originate from domain 2 (helix 2). It was found that these CK 19 fragments which bind specifically to both of the above-mentioned antibodies are detectable in particular in bronchial, breast, stomach, biliary tract, liver and colon carcinomas. These fragments could not as a rule be detected in patients with inflammatory epithelial diseases of the respective tissues.
The monoclonal antibodies which are formed by the cell lines ECACC 89112803 (binds to the sequence 291 to 335, preferably to the sequence 311 to 335) and ECACC 89112804 (binds to the amino acid sequence 346 to 367, preferably to 346 to 359) are preferably used for the invention.
Other monoclonal antibodies (mAB) which b
REFERENCES:
patent: 4727021 (1988-02-01), Cote et al.
patent: 4775620 (1988-10-01), Cardiff et al.
Stasiak et al, J. Invest. Dermatol., 92(5):707-716 (May 1989).
Wiedmann et al, Gastroenterology 96 (5 Part 2) A673 (May 1989).
Karsten et al, Eur. J. Cancer Clin. Oncol., 21:6 733-740 (1985).
Lerner, Nature, 299:592-596 (Oct. 14, 1982).
Suter et al, Immunology Letters, 13:313-316 (1986).
Sundstrom et al, J. Histochem. Cytochem., 37(12):1845-1854 (Dec. 1989).
Bodenmuller Heinz
Dessauer Andreas
Bidwell Carol E.
Boehringer Mannheim GmbH
Kepplinger Esther L.
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