Short Communication
A novel assay for detecting antibodies to cytochrome P4502D6, the molecular target of liver kidney microsomal antibody type 1

https://doi.org/10.1016/S0022-1759(98)00213-0Get rights and content

Abstract

Liver Kidney Microsomal type 1 (LKM1) antibody, the diagnostic marker of autoimmune hepatitis type 2, is also found in a proportion of patients with hepatitis C virus infection (HCV). It is detected conventionally by the subjective immunofluorescence technique. Our aim was to establish a simple and objective enzyme-linked immunosorbent assay (ELISA) that measures antibodies to cytochrome P4502D6 (CYP2D6), the target of LKM1. An indirect ELISA using eukaryotically expressed CYP2D6 was designed. Absorbance values obtained against a reference microsomal preparation were subtracted from those obtained against a microsomal preparation over-expressing CYP2D6, thus removing the non-CYP2D6-specific reaction. Sera from 51 LKM1 positive patients (21 autoimmune hepatitis and 30 with HCV infection), 111 LKM1 negative patients with chronic liver disease (including 20 with HCV infection) and 43 healthy controls were tested. Of 51 patients positive by immunofluorescence, 48 were also positive by ELISA while all the 154 LKM1 negative subjects were also negative by ELISA. There was a high degree of association between IFL and ELISA as demonstrated by a kappa reliability value of 0.96. The absorbance values by ELISA correlated with immunofluorescence LKM1 titres both in autoimmune hepatitis (r=0.74, p<0.001) and HCV infection (r=0.67, p<0.001). The simple, objective ELISA described has the potential to replace the standard immunofluorescence technique.

Introduction

Liver kidney microsomal antibody type 1 (LKM1) is the hallmark of a form of autoimmune hepatitis (AIH) referred to as type 2 AIH or LKM1 positive AIH (Homberg et al., 1987). LKM1 autoantibody was discovered by Rizzetto et al. (1973)who, using an indirect immunofluorescence (IFL) technique, described its characteristic pattern staining hepatocytes and proximal renal tubules (Rizzetto et al., 1973). The antigen targeted by LKM1 has been identified as cytochrome P4502D6 (CYP2D6), a protein of 50 kDa located primarily in the smooth endoplasmic reticulum (Alvarez et al., 1985; Manns et al., 1989). LKM1 has also been described in up to 10% of patients with hepatitis C virus (HCV) infection (Vergani and Mieli-Vergani, 1993; Cassani et al., 1997). A major antibody epitope of this enzyme was shown to span a 33 aminoacid region on CYP2D6 (Manns et al., 1991). The observation that the HCV polyprotein has sequence homology with CYP2D6 has prompted Manns et al. to propose that the LKM1 seen in patients with evidence of exposure to HCV infection arises through a mechanism of molecular mimicry.

In the routine diagnostic laboratory, LKM1 is detected by IFL using rat liver and kidney tissue as substrate. This technique is subjective and requires experienced personnel. Following the identification of CYP2D6 as the target of LKM1, there have been numerous attempts to establish assays using the isolated CYP2D6 molecule—expressed in a prokaryotic system such as Escherichia coli—as the target of LKM1 autoantibodies. When we ourselves used the enzyme expressed prokaryotically in an immunoblot technique, a differential reactivity to CYP2D6 was found in patients with AIH 2 (73%) or HCV infection (27%), even though they had identical autoantibody titres in IFL (Ma et al., 1994). This suggests that LKM1 in HCV infection recognizes epitopes or antigens different from those targeted in AIH. Since many proteins synthesized in bacteria fold incorrectly or insufficiently, eukaryotic systems in which mammalian proteins are expressed in mammalian cells should be used. These allow post-translational modifications such as accurate disulphide bond formation, oligomerization or specific protein cleavage, processes that are not performed by bacterial cells. One such assay, using the eukaryotically expressed cytochrome developed in our laboratory, has unambiguously shown that CYP2D6 is a major target antigen in both AIH and HCV (Ma et al., 1997). In this highly sensitive and specific radioligand assay, [35S]methionine-labelled recombinant CYP2D6 is used for the quantitative detection of LKM1. The assay, however, is laborious requiring the in-house preparation of the radiolabelled target antigen and appears to be unsuitable for a busy, clinical diagnostic laboratory.

The aim of the present study was to establish a simple, quantitative and objective assay, using eukaryotically expressed CYP2D6 as the target antigen. We report here an enzyme-linked immunosorbent assay (ELISA) that has the potential to replace the standard IFL technique.

Section snippets

Materials and methods

Sera from 51 patients positive for LKM1 by IFL were studied (Table 1). Twenty one had classical LKM1 positive AIH diagnosed according to international criteria (Johnson and McFarlane, 1993) while 30 had evidence of chronic liver disease due to HCV infection with positive anti-HCV antibody tested by second-generation RIBA test (Ortho Diagnostics, Raritan, NJ, USA). Of these, 17 patients tested were found to be positive for HCV RNA (Amplicor, Hoffmann La Roche, Basel, Switzerland). Sera from 111

Results

A test sample was considered positive when the subtracted absorbance (absorbance of `experimental' microsome−absorbance of `control' microsome) was greater than 0.063, this representing 3SD above the mean in the 43 healthy controls. The value 0.063 also corresponded to the highest absorbance value in the healthy control group. Table 2 gives a summary of the absorbance values obtained against `experimental' and `control' microsomes as well as the subtracted absorbance.

Of the 51 patients who were

Discussion

We describe an objective assay for the quantitation of antibodies to CYP2D6, the target of LKM1. The enzyme-linked immunosorbent assay (ELISA) is simple, specific and reflects accurately the titres of LKM1 as detected by immunofluorescence (IFL). It uses as antigen the eukaryotically expressed cytochrome. Not only are the preparatory steps easy to reproduce, but also both reagents and equipment required are readily available. Most importantly, the assay provides a quantitative assessment and

Acknowledgements

We would like to thank Dr. Paul Cheeseman for his assistance with the statistical analysis. Nanda Kerkar is the holder of a Scholarship from the Royal College of Physicians, London, UK, funded by Children Nationwide Medical Research Fund, 8 Dorset Square, London NW1 6PU, UK. Giorgina Mieli-Vergani and Yun Ma are supported by the Children's Liver Disease Foundation, 138 Digbeth, Birmingham B5 6DR, UK. Munther Hussain is supported by Children Nationwide Medical Research Fund.

References (22)

  • J.C. Homberg et al.

    Chronic active hepatitis associated with anti-liver/kidney microsome antibody type 1: a second type of `autoimmune' hepatitis

    Hepatology

    (1987)
  • Cited by (14)

    • Interferon alpha and auto-immunity

      2002, Revue de Medecine Interne
    • Auto-antibodies in hepatitis C

      1999, Biomedicine and Pharmacotherapy
    View all citing articles on Scopus
    View full text