Elsevier

Clinica Chimica Acta

Volume 413, Issues 15–16, 16 August 2012, Pages 1211-1216
Clinica Chimica Acta

Diagnostic and clinical utility of antibodies against the nuclear body promyelocytic leukaemia and Sp100 antigens in patients with primary biliary cirrhosis

https://doi.org/10.1016/j.cca.2012.03.020Get rights and content

Abstract

Background

The lack of an immunoassay that detects antibodies to promyelocytic leukaemia (PML) protein, the primary biliary cirrhosis (PBC)-specific multiple nuclear dot (MND) antigen, has prompted us to develop a line immunoassay (LIA) for the simultaneous detection of PML and Sp100 MND-specific autoantibodies.

Methods

PML and Sp100 were expressed in Escherichia coli, and analysed by SDS-PAGE and immunoblotting using a monoclonal antibody and MALDI-ToF fingerprinting. A quantitative PML and Sp100 LIA were developed and testing was performed in 150 anti-mitochondrial antibody (AMA) positive, 20 AMA-PBCs and 130 controls.

Results

Thirty-five (23%) of 150 AMA + PBCs (18 anti-MND +) were anti-PML + (12%) or anti-Sp100 + (20%), 10 being anti-PML +/Sp100+, 5 single anti-PML + and 20 single anti-Sp100+. Six (30%, 5 anti-MND +) AMA-PBCs were anti-PML + or Sp100+. Only 2 (1.7%) pathological controls were anti-PML + and/or anti-Sp100+. Levels of anti-PML correlated with those of anti-Sp100 (R = 0.64, p < 0.0001). The autoantibody profile largely remained unchanged over a 10 year-follow up (52 patients, 352 samples). Anti-PML, Sp100 or MND-reactive PBCs were younger and had longer disease duration than the seronegative (p = 0.06, for both). Anti-Sp100 levels correlated with the Mayo risk score (r = 0.63, p = 0.01). Anti-PML +/Sp100 + patients had more advanced disease compared to patients negative for anti-PML/Sp100 (p = 0.04).

Conclusion

The new line immunoassay offers a robust and accurate method for the detection of clinically-relevant PBC-specific anti-MND antibodies.

Highlights

► Nuclear body sp100 autoantibodies are diagnostic markers of primary biliary cirrhosis (PBC). ► A robust assay for the testing of nuclear body PML autoantibodies is missing. ► A new line immunoassay simultaneously detecting PML and sp100 autoantibodies has been developed. ► PML/sp100 autoantibody positive PBCs have more advanced disease compared to seronegative. ► Multi-parametric testing of nuclear autoantibodies is a useful immunodiagnostic tool in PBC.

Introduction

Primary biliary cirrhosis (PBC) is an immune-mediated cholestatic liver disease characterized by progressive destruction of intrahepatic bile ducts and the presence of disease-specific anti-mitochondrial (AMA) and anti-nuclear antibodies (ANA) [1], [2], [3], [4]. The most frequent PBC-specific immunofluorescence (IFL) pattern of ANA is that of multiple nuclear dot (MND) found in 20–50% of PBC patients but in less than 3% of patients with other chronic liver or extra-hepatic autoimmune diseases [5], [6], [7], [8], [9], [10].

The major target antigens of anti-MND antibodies have been recognized as Sp100 and promyelocytic leukaemia (PML) nuclear body (NB) proteins [3], [6], [11], [12], [13], [14]. While the diagnostic and clinical relevance of anti-Sp100 antibodies in PBC has been studied extensively in recent years, that of anti-PML is poorly defined [8], [9], [15], [16], [17], [18], [19], [20]. This is due to the fact that anti-PML testing was restricted to essentially one research laboratory, and therefore data for the use of this assay for routine use has been limited [13], [14].

In the mid 1990s, Will and colleagues identified PML as an anti-MND specific autoantigen in patients with PBC and screened a large number of patients and controls by IFL using cells over-expressing PML [14]. These investigators showed that anti-PML antibodies were present in 19% of AMA positive PBC cases, and that the great majority of the anti-PML seropositive cases were simultaneously reactive to Sp100 [13], [15]. Similar data were obtained by a French study in a limited number of PBC samples [21]. Of clinical relevance, patients with reactivity to both Sp100 and PML progressed faster compared to seronegative cases, suggesting that the simultaneous presence of the two autoantibodies may be of prognostic significance [14].

Due to the scarcity of data on anti-PML reactivity and the lack of a readily available anti-PML assay, we developed a line immunoassay (LIA) for the determination of this autoantibody and placed the Sp100 autoantigen on the same membrane to allow the simultaneous detection of anti-Sp100 antibodies. We then studied the prevalence and clinical significance of both autoantibodies in a well-defined population of 150 AMA-positive consecutive PBC patients from UK, and 20 PBC cases negative for AMA by IFL, LIA and Western blotting. The specificity of the autoantibody reactivity in 127 pathological and 23 healthy controls was also tested. Finally, we assessed the behaviour of these autoantibodies over time, in a subset of the 150 PBC patients followed up to 10 years.

Section snippets

Subjects

The study included 150 PBC patients positive for AMA by IFL and reactive with at least one of the two antigens (M2 or the recombinant hybrid BPO [22]) and 20 PBC patients negative for AMA. The AMA-positive serum samples had previously been used in a series of studies investigating the role of microbial/self mimicry in the pathogenesis of PBC and demographical, laboratory, histological and clinical details have also been reported previously [23], [24], [25].

Among the 150 AMA positive PBC

Preparation of recombinant proteins

DNA fragments coding for the PML and Sp100, respectively, were ligated with prokaryotic expression vectors and expressed in E. coli. The proteins, purified by metal chelate affinity chromatography, migrated according to their calculated masses when separated by SDS-PAGE. Identity and full length of both recombinant proteins were verified by MALDI-ToF fingerprinting. In the case of PML several N-terminal fragments were present. The antigens consistently reacted with the monoclonal antibody

Discussion

In the present study, we report the development of a line immunoassay for the routine determination of PML autoantibodies, in conjunction with those against Sp100 which are both associated with the immunofluorescent multiple nuclear dot pattern. We showed that PML and Sp100 autoantibodies are highly specific for PBC, being present in approximately 23% of AMA-positive PBC patients but in less than 2% of the pathological and none of the normal controls. Anti-PML antibodies are not as prevalent as

Conclusions

The new anti-PML/Sp100 line system offers a robust, reliable, sensitive and highly-specific method for the detection of antigen-specific, nuclear body reactivities. The ability to test for anti-PML and anti-Sp100 antibodies in an observer-independent manner will help us to design larger studies in order to determine the diagnostic, clinical and pathogenic significance of these autoantibodies for PBC.

Conflict of interest

LK is an employee of EUROIMMUN; WM, TS and CP are employees and shareholders of EUROIMMUN.

    Abbreviations

    AIH

    autoimmune hepatitis

    BCOADC

    branched-chain 2-oxo-acid dehydrogenase complex

    BPO

    fusion protein of E2 lipoyl domains of BCOADC, PDC, and OGDC

    ELISA

    enzyme-linked immunosorbent assay

    FITC

    fluorescein-isothiocyanate

    HBV

    hepatitis B virus

    HCV

    hepatitis C virus

    IFL

    immunofluorescence

    LIA

    line immunoassay

    MND

    multiple nuclear dot

    OD

    optical density

    OGDC

    2-oxo-glutarate dehydrogenase complex

    PBC

    primary biliary cirrhosis

    PDC

    pyruvate

Acknowledgements

MGM is supported by a Sheila Sherlock Entry-Lever Research Fellowship from the European Association for the Study of Liver (EASL); DPB is a recipient of a CLS award from the Higher Education Funding Council for England.

Part of this work has been presented as a poster (#678), entitled ‘Diagnostic and clinical application of a new line immunoassay that simultaneously detects the multiple nuclear dot specific antigens in primary biliary cirrhosis’ by MG. Mytilinaiou et al. at the 44th Annual

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