Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1993-01-27
1996-03-19
Scheiner, Toni R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
436 64, 436813, 436547, 436548, 435 721, 5303879, 5303882, 5303888, 53038885, 5303897, 530357, 530412, 530417, G01N 3353, G01N 33574
Patent
active
055003478
DESCRIPTION:
BRIEF SUMMARY
The invention concerns a process for the purification of cytokeratin 20, a standard protein material as well as a process for its production and a process for the production of antibodies directed against CK 20 and the use of an antibody which is specific for CK 20 to detect this protein on tissue sections, in tissue homogenates or in body fluids, as well as the use of a standard protein material to detect autoantibodies against CK 20 in blood or serum.
It has been found that the intermediary filament (IF) proteins of the cytokeratin family are effective markers for analyzing the type and state of differentiation of epithelial cells. The epithelial cytokeratins, comprising a family of at least 19 different polypeptides, are expressed in different combinations depending on the course of the cell differentiation. The synthesis of cytokeratins is usually maintained during malignant transformation and this fact could serve as one of the test criteria for epithelial-derived tumours, including tumours of the bladder tract. There was inter alia a need for a reliable detection method which can be easily carried out for determining the location of the primary tumour of metastastic tissue so that effective action can be taken against this primary tumour. The object of the present invention was therefore to provide a way of differentiating various types of tumour cells and detecting the origin of various metastastic tissues which can be carried out easily and as accurately as possible.
A new cytokeratin has now been identified which only occurs in particular cells and thus can serve as a marker to differentiate certain cells and tissues. It has been named cytokeratin 20.
The invention therefore concerns a process for the purification of cytokeratin 20 in which a cytoskeletal fraction from tissues or cells containing CK 20 is prepared, the proteins present therein are separated by gel electrophoresis or/and by chromatography and CK 20 is isolated from the gel or from the chromatography fraction containing the CK 20.
The protein CK 20 has a molecular weight of ca. 46000, an isoelectric point in 9.5 molar urea of ca. 6.1 and is slightly more acidic than non-phosphorylated variants of CK 8. FIG. 1 shows a partial amino acid sequence of CK 20 (denoted IT in this figure; A-D obtained from fragments of CK 20 after digestion with Staphylococcus V8 protease).
According to the present invention it is possible to obtain cytokeratin 20 in such a pure form that it is for example possible to use it to produce specific antibodies. In one embodiment of the present invention the cytoskeletal fraction is produced from duodenal mucosal villi, especially from human tissue. In another embodiment of the invention the cytoskeletal fraction is isolated from culture cells wherein culture cells are preferred which are derived from colon carcinomas, bladder carcinomas or stomach carcinomas. When carrying out a gel electrophoresis in a preferred embodiment of the present invention a first SDS polyacrylamide gel electrophoresis is carried out in a buffer system with an increased salt concentration, subsequently the band containing CK 20 is cut out and the protein is eluted from it and a second polyacrylamide gel electrophoresis is carried out in a buffer system with a lower salt concentration and the now purified CK 20 is isolated from the corresponding band in the gel.
In a further embodiment of the invention, namely separation by chromatography, an anion-exchange chromatography is firstly carried out and then a reversed phase HPLC chromatography. In this case it is in turn preferred that the anion-exchange chromatography is carried out on DEAE cellulose in the presence of urea and using an eluant with a linear gradient of between 0 and 100 mmol/l guanidinium hydrochloride and to subsequently subject the fractions containing CK 20 to HPLC. In this process the fractions containing 38 to 50 mmol/l guanidinium hydrochloride are preferably subjected to HPLC.
The invention furthermore concerns a standard protein material which consists of a reconsti
REFERENCES:
Moll, et al., J. Cell Biol., vol. III, No. 2, pp. 567-580, Aug. 1990.
Moll R., Acta Histochem. Suppl., vol. 41, pp. 117-27, 1991.
Moll et al., "The Catalog of Human Cytokeratins: Patterns of Expression in Normal Epithelia, Tumors and Cultured Cells", Cell, vol. 31, 11-24, (1982).
Gigi et al., "Detection of a Cytokeratin Determinant Common to Diverse Epithelial Cells by a Broadly Cross-Reaction Monclonal Antibody", The EMBO Journal, vol. 1, No. 11, 1429-1437 (1982).
Moll et al., "Cytoskeletal Differences Between Human Neuroendocrine Tumors: A Cytoskeletal Protein of Molecular Weight 46,000 Distinguishes Cutaneous from Pulmonary Neuroendocrine Neoplasms", Differentiation, 30:165-175, (1985).
Moll et al., "Ein neues epitheliales zytoplasmatisches Strukturprotein (46 000 Protein) mit eingeschranktem Expressionsspektrum: Potentieller histodiagnostischer Marker zur Unterscheidung metastatischer Adenokarzinome", Verh. Dtsch. Ges. Path, 526 (1987).
Moll et al., "Cytokeratins in Normal and Malignant Transitional Epithelium", American Journal of Pathology, vol. 132, No. 1 (1988), 123 (5).
Moll, "Cytoskeletal Markers in the Classification of Carcinomas and Their Metastases", Current Communications in Molecular Biology, 139 (1989).
Franke Werner W.
Moll Roland
Progen Biotechnik GmbH
Scheiner Toni R.
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