Establishment of a novel radioligand assay using eukaryotically expressed cytchrome P4502D6 for the measurement of liver kidney microsomal type 1 antibdy in patients with autoimmune hepatitis and hepatitis C virus infection

Abstract

Background/Aims: Liver kidney microsomal type 1 antibody (LKM1) is the diagnostic marker of autoimmune hepatitis (AIH) type 2 and is also found in patients with hepatitis C virus (HCV) infection. Cytochrome P4502D6 (CYP2D6) is the documented target antigen of LKM1 in AIH, but not in HCV infection. To compare the reactivity in the two conditions, we established a radioligand assay using eukaryotically expressed CYP2D6 as target. Methods: A 1.2-kb human CYP2D6 cDNA was isolated from a human liver cDNA library and subcloned into an vitro transcription vector pSP64 Poly(A). Recombinant CYP2D6 was then produced by in vitro transcription/translation, metabolically labelled with 35S methionine and used in the immunoprecipitation assay. Antibodies that bound radiolabelled CYP2D6 were immunoprecipitated and their levels assessed as cpm. Sera from 50 LKM1-positive patients (26 with AIH; 24 with HCV infection ), 128 LKM1-negative patients and 57 normal controls were tested. Results: Reactivity to 35S labelled CYP2D6 was observed in all LKM1-positive sera from patients with AIH and HCV infection, but in none of the controls. The cpm in both conditions were significantly higher than in normal controls ( p<0.0001), and were correlated with the immunofluorescence titres of LKM1 ( r 0.87, p<0.001 and r=0.64, p<0.001 for AIH and HCV infection, respectively.). Reactivity to 35S labelled CYP2D6 was inhibited by addition of an excess of eukaryotically expressed CYP2D6. Conclusions: CYP2D6 is a major target antigen of both AIH and HCV infection. The novel radioligand assay is highly sensitive and specific.