Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1988-09-22
1993-03-23
Ellis, Joan
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 696, 435 698, 435 91, 4351723, 4352351, 4353201, 4352401, 4352523, 435255, 435256, 536 232, 530350, 935 18, 935 31, 935 41, 935 47, 935 58, 935 62, 935 73, 935 81, C12P 2102, C12P 1934, C12N 1500, C12N 700
Patent
active
051963191
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to the identification, cloning and expression of an auto-antigen which is recognised as a target in the characteristic autoantibody response in primary biliary cirrhosis (PBC), and to the use of this protein, fragments thereof or fused polypeptides containing the protein or fragments thereof in diagnostic tests for PBC, and in treatment of patients suffering from PBC.
BACKGROUND OF THE INVENTION
Primary biliary cirrhosis (PBC) is a chronic disease characterised by progressive inflammatory obliteration of the intrahepatic bile ducts. The disease is marked by an autoantibody response to mitochondria.sup.1-4, originally identified using immunofluorescence.sup.5. With the reactant use of immunoblotting, specific proteins have been recognized as targets of the anti-mitochondrial antibodies (AMA) of PBC.sup.2,6,7. In particular, serum antibodies to a 70 kilodalton (kd) protein have been found in greater than 95% of patients with PBC but not in patients with other autoimmune liver diseases,.sup.2,8 ; two other proteins of 45 and 39 kd are less frequently detected in PBC sera.sup.1,2,9. The identity of each of these autoantigens has been unknown, as is the relationship of these antigens to the pathogenesis of the disease. However, the 70 kd antigen is highly conserved in evolution, being present in mammals, yeast and bacteria.sup.10 and it is therefore believed to have an important structural or biological function.sup.2.
SUMMARY OF THE INVENTION
Despite the paucity of data on mechanisms of anti-mitochondrial antibody formation, by enzyme-linked immunosorbent assay (ELISA), clinically more than 95% of patients with PBC can be found to have such anti-mitochondrial antibodies.sup.2,6. When crude mitochondrial antigen preparations are used, subjects with a variety of diseases, including patients with liver diseases other than PBC, certain connective tissue diseases, and drug reactions, and occasionally even healthy individuals, can also be demonstrated to have antibodies to mitochondria. Accordingly, assays using such crude preparations are unable to provide specific diagnosis of PBC. By way of example, German Patent Publication No. 3,237,602 discloses an ELISA for detection and determination of antimitochondrial antibodies in serum using a crude mitochondrial antigen preparation. The lack of specificity of the assay is evident from the suggested use of the assay in the specific diagnosis of disorders such as PBC as well as the cholestatic form of chronic active hepatitis, syphilis (II), drug-induced pseudo lupus erythematodes syndrome, certain primary non-hepatic immunopathies, iproniazed-induced hepatis and side effects of certain medicaments such as beta-receptor blockers. By more vigorous isolation of mitochondrial membranes, the problem of antigenic heterogeneity becomes clearer and has led to definitions of specific mitochondrial antigens based on trypsin sensitivity and location of antigens within inner vs outer mitochondrial membranes. Notwithstanding this, however, the diagnosis of PBC still relies heavily on the demonstration of anti-mitochondrial antibodies by the relatively insensitive procedure of immunofluorescence or by more sensitive, but still relatively nonspecific, methods, including complement fixation, ELISA, and immunoprecipitation.sup.23-28.
The present invention is based on the identification of a cDNA clone derived from a rat liver gene expression library that expresses the 70 kd mitochondrial autoantigen of PBC, (called M2 by some groups of investigators.sup.1,9) and on sequence determination thereof. The sequence is encoded by nuclear and not mitochondrial DNA.
The present invention thus provides the basis of an extremely sensitive and specific diagnostic ELISA for anti-70 kd antibodies found in PBC.
According to a first aspect of the present invention, there is provided a DNA molecule comprising a nucleotide sequence substantially corresponding to all or a portion of the base sequence coding for the 70 kd mitochondrial autoantigen of pri
REFERENCES:
Coppel et al. Proc. Natl. Acad Sci USA vol. 85 pp. 7317-7321.
Levin Science vol. 237 (1987).
Reeck et al. Cell vol. 50 p. 667 (1987).
Young et al. Proc. Natl. Acad. Sci. USA vol. 80 pp. 1194-1198 (1983).
Kemp et al. Proc. Natl. Acad. Sci. USA vol. 80 pp. 3787-3791 (1983).
Frazer et al. J. Immunol. vol. 135 pp. 1739-1745 (1985).
Stemerowicz, et al., "Are Antimitochondrial Antibodies in Primary Billiary Cirrhosis Induced by R(Rough)-Mutants of Enterobacteriaceae?", The Lancet, Nov. 19, 1988, pp. 1166 and 1170.
Stephens, et al., "The Pyruvate Dehydrogenase Complex of Escherichia coli K12, Nucleotide Sequence Encoding the Dihydrolipoamide Acetyltransferase Component" European Journal of Biochemistry, 133, 1983, pp. 481 to 489.
Judy Van de Water, et al., "Detection of Autoantibodies in Recombinant Mitochondrial Proteins in Patients with Primary Billiary Cirrhosis," The New England Journal of Medicine, vol. 320, No. 21, May 25, 1989, pp. 1377 to 1380.
Shelly P. M. Fussey, et al., "Reactivity of Primary Biliary Cirrhosis Sera with Escherichia coli Dihydrolipoamide Acetyltransferase (E2p): Characterization of the Main Immunogenic Region," Proc. National Academy of Sciences USA, vol. 87, May 1990, pp. 3987 to 3991.
Coppel Ross L.
Gershwin Merrill E.
Amrad Corporation Limited
Cooper Iver P.
Ellis Joan
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