Establishment of a novel radioligand assay using eukaryotically expressed cytochrome P4502D6 for the measurement of liver kidney microsomal type 1 antibody in patients with autoimmune hepatitis and hepatitis C virus infection.
OBJECTIVE
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 in 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.
Ma
Y
Y
Institute of Hepatology, University College London Medical School, UK.
Gregorio
G
G
Gäken
J
J
Muratori
L
L
Bianchi
F B
FB
Mieli-Vergani
G
G
Vergani
D
D
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D001327:Autoimmune Diseases / Q000175:diagnosis* / Q000276:immunology; D015415:Biomarkers; D002648:Child; D002675:Child, Preschool; D003001:Cloning, Molecular; D019389:Cytochrome P-450 CYP2D6 / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology*; D006505:Hepatitis / Q000097:blood / Q000175:diagnosis* / Q000276:immunology*; D006526:Hepatitis C / Q000175:diagnosis* / Q000276:immunology; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008875:Middle Aged; D011863:Radioimmunoassay; D011869:Radioligand Assay; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012016:Reference Values; D012044:Regression Analysis; D015203:Reproducibility of Results; D013270:Stomach / Q000201:enzymology
D001323:Autoantibodies; D015415:Biomarkers; D011994:Recombinant Proteins; C060729:anti-liver kidney microsome antibody; D019389:Cytochrome P-450 CYP2D6