Differential detection of nuclear envelope autoantibodies in primary biliary cirrhosis using routine and alternative methods

Abstract

Background: Detection of autoantibodies giving nuclear rim pattern by immunofluorescence (anti-nuclear envelope antibodies - ANEA) in sera from patients with primary biliary cirrhosis (PBC) is a useful tool for the diagnosis and prognosis of the disease. Differences in the prevalence of ANEA in PBC sera so far reported have been attributed to the methodology used for the detection as well as to ethnic/geographical variations. Therefore, we evaluated the prevalence of ANEA in sera of Greek patients with PBC by using methods widely used by clinical laboratories and a combination of techniques and materials.

Methods: We screened 103 sera by immunoblotting on nuclear envelopes and indirect immunofluorescence (IIF) using cells and purified nuclei. Reactivities against specific autoantigens were assessed using purified proteins, ELISA, immunoprecipitation and mass spectrometry.

Results: We found higher prevalence of ANEA when sera were assayed by IIF on purified nuclei or cultured cells (50%) compared to Hep2 commercially available slides (15%). Anti-gp210 antibodies were identified in 22.3% and 33% of sera using ELISA for the C-terminal of gp210 or both ELISA and immunoprecipitation, respectively. Immunoblotting on nuclear envelopes revealed that immunoreactivity for the 210 kDa zone is related to anti-gp210 antibodies (p < 0.0001). Moreover, we found that sera had antibodies for lamins A (6.8%), B (1%) and C (1%) and LBR (8.7%), whereas none at all had detectable anti-p62 antibodies.

Conclusions: The prevalence of ANEA or anti-gp210 antibodies is under-estimated in PBC sera which are analyzed by conventional commercially available IIF or ELISA, respectively. Therefore, new substrates for IIF and ELISA should be included by clinical laboratories in the analysis of ANEA in autoimmune sera.

Figure 1

Prevalence of ANEA. Histograms showing the percentage of PBC sera containing ANEA of different Ig class as determined by IIF on Hep2 slides, HeLa cells, rat liver nuclei (RLN) and either substrate. "IgG Total" or "IgM Total" represent the sum of sera which had ANEA of both IgG and IgM antibodies (IgG+IgM) and those with either IgG class alone or IgM class alone, respectively.

Figure 2

Validation of substrates used in IIF for the detection of ANEA. Hep2 slides, HeLa and HepG2 cells fixed with formaldehyde either 4% (upper panel) or 1% (lower panel) and purified nuclei from HepG2 cells and rat liver were incubated with various PBC sera (s54, s32, s16, s189 and s201) and secondary anti-human FITC-labeled IgG antibodies, as described in Materials and Methods.

Figure 3

Purity of isolated nuclei from HEpG2 cells, HeLa cells and rat liver.

Figure 4

Purity of nuclear envelopes isolated from rat liver. (A) Reactivity of PBC sera (s) as detected by IIF on HeLa cells and nuclei isolated from rat liver. Note the cytoplasmic pattern of fluorescence and the absence of staining on nuclei. The same PBC sera were tested (B) for anti-mitochondrial autoantibodies by dot blotting (mitochondria profile blot, Alphadia Diagnostic Products, Wavre, Belgium) and (C) on nuclear envelopes from rat liver by immunoblotting. Note for all sera the presence of anti-mitochondrial autoantibodies and the absence of reactivity on nuclear envelopes.

Figure 5

Detection of ANEA of IgG and IgM class by IIF. Commercially available slides with Hep2 cells (Hep2 cells), cultured HeLa cells (HeLa cells) and purified nuclei from rat liver (R.L. nuclei) were incubated with various PBC sera and secondary anti-human FITC-labeled IgG and IgM antibodies, as described in Materials and Methods.

Figure 6

Detection of ANEA by immunoblotting. Apparent molecular mass of nuclear envelope proteins from rat liver which are recognized by autoantibodies comprised in selected PBC sera.

Figure 7

Prevalence of anti-gp210 autoantibodies. On the top the reactivity by immunoblotting of the 35 different sera (s) with a protein zone of approximately 210 kDa is shown. The presence (+) or the absence (-) of autoantibodies against gp210 in these sera is assayed by ELISA and immunoprecipitation followed by immunoblotting. For details see text and Figure 8.

Figure 8

Identification of specific ANEA by biochemical approaches. (I) Identification of anti-gp210 autoantibodies. Immunoprecipitation (IP) of gp210 and immunoblotting (Blot) using IgG (IgG) and IgM (IgM) secondary antibodies. (II) Identification of autoantibodies directed against lamins A/C (LmA/C) and B (LmB) by immunoblotting (Blot) using purified proteins (SDS-PAGE). (III) Identification of anti-LBR autoantibodies by immunoblotting (Blot) using purified recombinant LBR (SDS-PAGE). "d.p." denotes characteristic proteolytic products of LBR that are produced after expression in bacteria. Sera negative (s33, s201) and highly (s211, s21) or moderately (s196) positive for LBR are shown. (IV) Identification of anti-p62 autoantibodies.

Figure 9

Prevalence of autoantibodies for lamins and LBR. On the top the reactivity by immunoblotting of the 39 different sera (s) with a protein zone of molecular mass between 55 and 75 kDa is shown. The presence (+) or the absence (-) of autoantibodies against lamins A, B, C and LBR in these sera is assayed by immunoblotting using purified proteins. "+l" denotes low titer for anti-LBR antibodies.