Quote (xaos @ Mon - Nov 23 2009 - 13:54:22)
*THE SHORT VERSION*
US Patent 6111071 - Recombinant fusion protein comprising PDC-E2, BCOADC-E2 and OGDC-E2 and uses thereof
Description
Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
Primary biliary cirrhosis (PBC) is a well-characterized autoimmune liver disease that results in the destruction of intrahepatic bile ducts with progressive inflammatory scarring (Kaplan M. M., Adv Intern Med 32:359-377, 1987). The disease is marked by an autoantibody response to mitochondria, originally identified using immunofluorescence (Berg, P. A. et al., 20 Hepatology 2:123-131, 1986; Frazer, I. H. et al., J. Immunol 135:1739-1745, 1985; Kenna, J. G. et al., J. Immunol. Methods 73:401-413, 1984; Kaplan, M. M. et al, Hepatology 4: 727-730, 1984; Walker, J. G. et al., Lancet 1:827-831, 1965).
A characteristic serologic feature observed in sera from patients with PBC is the presence of high titers of antibodies directed against mitochondrial antigens (AMA) (Mackay I. R., N Engl J Med 1958; 258:185-187; Walker, J. G. et al., Lancet 1965; 1:827-831; Berg, P. A. et al., J Exp Med 1967; 126:277-290; Gershwin M E and Mackay I R. Gastroenterology 1991; 100: 822-833). The major autoantigens recognized by sera from PBC patients have been identified as members of the 2-oxo-acid dehydrogenase complex (2-OADC) family, including the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the E2 subunit of the branched chain 2-oxo-acid dehydrogenase complex (BCOADC-E2), the E2 subunit of the 2-oxo glutarate dehydrogenase complex (OGDC-E2), E1" subunits of PDC and protein X (Yeaman, S. J. et al., Lancet 1988; 1:1067-1070; Van de Water J, et al., J Exp Med 1988; 167:1791-1799; Fussey S P M, et al., Proc Natl Acad Sci USA 1988;85:8654-8658; Fregeau D R, et al., J Immunol 1989;142;3815-3820; Surh C D, et al., Hepatology 1989;10:127-133; Fregeau D R, et al., J Immunol 1990;144:1671-1676; Fregeau D R, et al., Hepatology 1990; 1 1:975-981).
Among these enzyme components, the E2 component of PDC, or dihydrolipoamide dehydrogenase, is the major autoantigen of PBC since the serum samples of the majority of patients (80 to 90%) contain PDC-E2 specific AMA. In addition to PDC-E2, approximately 60% of patients with PBC are also reactive with BCOADC-E2 (Mutimer D J, et al., Hepatology 1989;10:403-407; Leung P S C, et al., Hepatology 1992;15:367-372). Interestingly, 4% to 13% of sera from patients with PBC recognize only the BCOADC-E2 but not the PDC-E2 (Leung P S C, et al., Hepatology 1992;15:367-372; Van de Water J, et al., N Engl J Med 1989;320:1377-1380; Iwayama T, et al., Int Arch Allergy Immunol 1992;99:28-33). The E2 component of OGDC-E2, dihydrolipoamide succinyltransferase, is recognized in 30% to 80% of sera from patients with PBC (Fregeau D R, et al., Hepatology 1990; 1 1:975-981; Mutimer D J, et al., Hepatology 1989;10:403-407; Leung P S C, et al., Inflammatory hepatobiliary cirrhosis. pp. 1429-1443. 1996. In: Clin. Immunology. Principles and Practice. Rich. R. R.(Ed). Mosby. Year book. Inc. St. Louis, Mo., USA). The immunodominant epitopes of PDC-E2 and BCOADC-E2 have been previously mapped to their lipoic acid binding domains (Surh C D, et al., J Immunol 1990;144:3367-3374; Leung P S C, et al., Hepatology 1995; 22:505-513). Although PDC-E2 contains two lipoic acid binding domains, the reactivity of AMA is about 100 times stronger to the inner lipoyl domain (Van de Water J, et al., J Exp Med 1988; 167:1791-1799; Surh C D, et al., Hepatology 1989;10:127-133).
The complementary DNA (cDNA) of PDC-E2, BCOADC-E2 and PDC-E1" have been isolated and used to produce recombinant proteins designated PDC-E2, BCOADC-E2, and PDC-E1", respectively. A strong reactivity of patient AMA to these proteins by immunoblotting and enzyme-linked immunosorbent assays (ELISA) was demonstrated (Leung P S C, et al., Hepatology 1992;15:367-372; Van de Water J, et al., N Engl J Med 1989;320:1377-1380; Gershwin M E, et al., J Immunol 1987;138:3525-353 1; Coppel R L, et al., Proc Natl Acad Sci USA 1988; 85:7317-7321; Griffin T A, et al., J Biol Chem 1988;263:14008-14014; Danner DJ, et al., J Biol Chem 1989;264:7742-7746; Ho L, et al., Proc Natl Acad Sci USA 1989;86:5330-5334; Surh C D, et al., Hepatology 1989;9:63-68; Iwayama T, et al., J Autoimmunity 1991 ;4:769-778).
SUMMARY OF THE INVENTION
The present invention provides a recombinant fusion protein comprising PDC-E2, BCOADC-E2, and OGDC-E2. Further, the invention provides a clone, designated pML-MIT3, that coexpresses the immunodominant epitopes within the three distinct lipoyl domains. The present invention provides the basis for an extremely sensitive and specific diagnostic ELISA for AMA in PBC subjects.
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