Kerkar, Nanda; (2001) Study of cytochrome P4502D6, target of liver kidney microsomal antibody type 1 in autoimmune hepatitis type 2 and liver disease due to hepatitis C virus infection. Doctoral thesis (M.D.), University College London.

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Abstract

Liver Kidney Microsomal antibody type 1 (LKM1) the diagnostic marker of autoimmune hepatitis type 2 (AIH type2) is also found in up to 10% of patients with chronic hepatitis C virus (HCV) infection. Detection of LKM1 is of clinical importance not only in the management of AIH type2 but also in that of patients with LKM1 positive chronic HCV infection because use of interferon can exacerbate disease in this group of patients. Classically, LKM1 is detected by immunofluorescence (IFL), a subjective technique, and has been often mistaken for antimitochondrial antibody. Since at the outset of the present thesis, the molecular target of LKM1 had been identified as cytochrome P4502D6 (CYP2D6), the first aim of this work was to develop an objective and quantitative method for the detection of LKM1. An ELISA using eukaryotically expressed CYP2D6 as antigen was established, its performance being comparable in sensitivity to that of IFL and its results being observer independent. Using the newly established assay a comparative study was performed showing that LKM1 recognises CYP2D6 both in AIH and HCV infection. Since the epitopes on CYP2D6 may differ in the two conditions, the full map of linear epitopes on CYP2D6 was established and a new immunodominant epitope, CYP2D6193-212 was identified. This is recognised by 92% of patients with AIH and 50% of patients with chronic HCV infection. A hexameric sequence within this epitope shares cross-reactive similarities with HCV2985-2990 and with the Cytomegalovirus sequence - CMV130-135. Conformational epitopes on CYP2D6 were also mapped in both AIH and HCV infection using eukaryotically expressed constructs. Nearing the completion of the present work, two commercial assays for the detection of LKM1 have become available and they were compared against the newly established ELISA, an in-house radioligand assay and IFL. Their relative performance is presented and their possible use in the diagnostic laboratory discussed.

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