Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1996-05-02
1998-04-21
Housel, James C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 4, 435 71, 435 21, 436528, 436435, 436536, G01N 3353, G01N 33544, G01N 33536, C12Q 142
Patent
active
057416540
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates generally to autoantibodies, and more particularly, relates to an immunoassay for the detection of liver-kidney microsomal (LKM) autoantibodies by automated or semi-automated means.
Liver-kidney microsomal (LKM) autoantibodies are known to be associated with inflammatory liver diseases. LKM autoantibodies associated with idiopathic autoimmune chronic active hepatitis (AI-CAH) are termed "LKM-1" autoantibodies. M. P. Manns, in Seminars in Liver Disease, Vol. II, No. 3:205-214 (1991).
The LKM-1 autoantibody has been found to recognize a 50 kilodalton (kDa) protein identified initially as rat liver cytochromes P450 db1 and db2, leading to the development of a method for determining LKM-1 in individuals. M. Gueguen et al., Biochemical and Biophysical Research Communications Vol. 159 (2):542-547 (1989). Recent reports indicate that three P450 cytochromes can be identified as autoantigens in patients with inflammatory liver disease. All three P450 cytochromes (IA2, IID6 and IIC9) are drug metabolizing enzymes recognized by strongly inhibitory autoantibodies. M. P. Manns et al., Archives of Biochemistry and Biophysics Vol. 280 (1):229-232 (1990).
Circulating autoantibodies such as LKM-1 have become important markers for determining the diagnosis of autoimmune hepatitis. The diagnosis of autoimmune hepatitis has become important, since patients suffering from autoimmune hepatitis benefit from treatment with immunosuppressives but not from treatment with interferons, used in the treatment of viral-induced hepatitis. Thus, the differentiation between viral-induced hepatitis and autoimmune hepatitis is important to ensure correct treatment. Recent reports have indicated that patients diagnosed with chronic non-A, non-B viral hepatitis (NANBH) by either exclusionary methods or assays for hepatitis C virus (HCV) and treated with interferon, actually were suffering from autoimmune hepatitis. M. Ruiz-Moreno et al., J. Hep. Hepatol., Vol. 12 (2).:265-266 (1991), and T. Papo et al., Annals of Internal Medicine Vol. 116 (1):51-53 (1992). These recent reports have suggested that a diagnosis of autoimmune hepatitis should at least be considered before beginning interferon therapy, since this therapy is contraindicated for patients suffering from autoimmune hepatitis.
Historically, methods for detection of LKM-1 autoantibodies included indirect fluorescent antibody (IFA) techniques, radioimmunoassay (RIA), electromicroscopy and immunoblotting. M. Manns et al., J. Clin. Lab. Analysis 1:344-352 (1987), Manns et al., Clin. Exp. Immunol. Vol. 57: 600-608 (1984). Recently, enzyme-linked immunosorbent assays (ELSIA) have been reported. M. Gueguen et al., Biochemical and Biophysical Research Communications Vol. 159 (2):542-547 (1989). Traditionally, such ELISAs either have included the purification of the IgG fraction of an LKM-positive reference serum (negative for other autoantibodies), the coating of the IgG fraction to solid surfaces, and the reaction of the so-prepared solid phase with the test sample, or these ELISA assays have attempted to detect antibody to a particular cytochrome. However, while patients diagnosed with autoimmune hepatitis react with the 50 kDa antigen they may not react with a particular cytochrome.
While these known methods have provided researchers with various techniques to determine the presence of LKM autoantibodies, these techniques have been hampered by manual methods and non-standardization and subjective evaluations which make these techniques semiquantitative at best. It would be advantageous to provide an improved immunoassay for autoantibodies to LKM, which method would be quantitative, standardized, highly reproducible and time-saving. Such a standardized immunoassay would be useful not only for differential diagnosis but also for monitoring immunosuppressive therapy of patients diagnosed with autoimmune hepatitis disease.
SUMMARY OF THE INVENTION
The present invention provides an automated method for determining the presence of LKM autoant
REFERENCES:
patent: 4689310 (1987-08-01), Kramer et al.
patent: 5200315 (1993-04-01), Sutton et al.
M. Manns et al., Journal of Clinical Laboratory Analysis, vol. 1(4), 344-352 (1987), No Title.
M. Manns, Seminars in Liver Disease, vol. 11(3), 205-214 (1991) No Title.
M. Manns et al., Clinical and Experimental Immunology, lvol. 57 (3), 600-608 (1984) No Title.
M Ruiz-Moreno et al., Jounal of Hepatology, vol. 12(2), 265-266 (1991) No Title.
M. Gueguen et al., Biochemical and Biophysical Research Communications, vol. 159(2), 542-547 (1989) No Title.
R. Seelig et al., Clinical and Experimental Immunology, vol. 92(3), 373-380, (1993) No Title.
Seelig et al. Anti-LKM-1 Antibodies Determined by use of Recombinant P450 2D6 in Elisa and Western Blot and Their Association with Anti-HCVand HCV-RNA, CLIN. Immuno L. 92(3): 373-380, 1993.
Sigma Chemical Company Catalog, p. 1183--1992.
Braun Hans-Bertram
Michel Gerd
Rohrig Kay
Thome-Kromer Birgit
Abbott Laboratories
Danckers Andreas M.
Housel James C.
Swartz Rodney P.
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