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Technical Briefs |
Laboratoire de Biochimie et d’Immunopathologie, Centre Hospitalier de Luxembourg, Rue Barblé 4, L-1210 Luxembourg, Luxembourg
a author for correspondence: fax 00352-457794, e-mail schmit.patrick{at}chl.lu
Primary biliary cirrhosis (PBC) is a chronic disease characterized by portal inflammation and necrosis of small intrahepatic bile ductules (1). PBC is an irreversible condition, and destruction of the bile ductules leads to progressive cholestasis and fibrosis, and may eventually lead to the development of cirrhosis. PBC is most likely an autoimmune disorder (2), and anti-mitochondrial antibody (anti-M2) has been a diagnostically very useful marker: several studies have reported a positivity rate for anti-M2 of >95% in biopsy-confirmed PBC patients (3). The major mitochondrial antigen was identified as the 74-kDa E2 subunit of the pyruvate dehydrogenase complex (PDC), a member of the 2-oxoacid dehydrogenase complex family (4). The traditional technique for the detection of anti-M2 is immunofluorescence (5), but recently new serological assays, such as ELISA, immunoblotting, and enzyme inhibition (EI), have been developed (6)(7). A miniaturized EI assay for performance on microtiter plates has already been described (8)(9). In this study we evaluated a new commercially available and completely automated EI assay manufactured by Trace Scientific (Victoria, Australia) and compared it to the immunofluorescence and ELISA techniques performed in our laboratory.
The TRACE enzymatic procedure is a unique method based on the PDC
inhibitory properties of the principal anti-74-kDa antibody. PDC
catalyzes the following reaction:
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With a selected serum presenting a moderate degree of inhibitory activity close to the cutoff value of 70% of residual PDC activity, replicate testing for intraassay variation (n = 10) gave a mean (SD) inhibition of activity of 76.1% (1.7%) with a CV of 2.2%, and replicate testing for interassay variation (10 determinations performed on 10 days with a new calibration for each day) gave a mean (SD) inhibition of activity of 74.5% (2.4%) with a CV of 3.2%.
Our ELISA method used polystyrene microtiter plates (Nunc Maxisorp) coated with porcine heart pyruvate dehydrogenase (cat. no. P-7032; Sigma) at 1.56 mU per well in 0.01 mol/L bicarbonate buffer, pH 9.6. Plates were left overnight at 4 °C, washed twice with distilled water, and blocked with SuperBlock (Pierce) for 5 min at room temperature. Duplicate samples for each of the sera were diluted 1:100 in phosphate-buffered saline, pH 7.4, with 1 mL/L Tween 20; 100 µL was dispensed per well and incubated for 1 h under constant shaking at room temperature. Plates were washed three times in 150 mmol/L NaCl, 10 mmol/L Tris, 1 mL/L Tween 20, pH 7.4, and antibody binding was detected with 100 µL of peroxidase-labeled rabbit anti-human IgG (cat. no. P0406; Dako) and anti-human IgM (cat. no. P322; Dako) diluted 1:5000 in SuperBlock containing 0.5 mL/L Tween 20 and incubated for 1 h at room temperature under constant shaking. The reactivity was visualized with ABTS (Roche-Boehringer Mannheim) at 405 nm in a microplate reader (MIOS; Dynatech). The absorbance cutoff value of the assay (mean + 3 SD) was established on a blood donor population (n = 70), and a reference curve using an anti-M2-positive PBC serum was established to assign arbitrary units (AU), with 10 AU being the cutoff value. With a selected serum presenting a weakly positive value, replicate testing for intraassay variation (n = 10) gave a mean (SD) of 50.6 AU (6.3 AU) with a CV of 12%, and replicate testing for interassay variation (10 determinations performed on 10 days with a calibration for each day) gave a mean (SD) of 48.8 AU (7.3 AU) with a CV of 15%.
Indirect immunofluorescence (IIF) for anti-M2 was performed on 5-µm cryostat sections from rat liver, kidney, and stomach (Sanofi Diagnostics Pasteur). The screening dilution of the sera was 1:20 in phosphate-buffered saline with 20 g/L bovine serum albumin. Fluorescein isothiocyanate-labeled anti-human IgG H + L (Scimedx) was used as a second antibody.
Our clinically well-characterized patient population was composed of 23
histologically demonstrated PBC patients, 20 cases of other autoimmune
hepatic disorders (autoimmune hepatitis type I and II), 40 cases of
non-autoimmune hepatic disorders (hepatitis B and C), and 32 healthy
control subjects. Blood was drawn after an overnight fast and allowed
to clot at room temperature; serum was obtained by centrifugation at
1500g for 10 min and was stored at -24 °C until analyses
were performed. All PBC samples were positive with the TRACE EI assay,
the ELISA, and the immunofluorescence technique. The discordant results
(Table 1
) obtained between the EI assay and the ELISA may be
explained by the fact that the EI assay specifically measures the
inhibitory properties of the 74-kDa E2 subunit of the PDC, whereas we
used in our ELISA the commercially available purified intact PDC for
coating, a preparation known to contain minor amounts of 2-oxoacid
dehydrogenase complex enzymes other than PDC. Moreover, antibodies that
bind PDC are not necessarily inhibitory. It has already been reported
that with ELISA techniques, the choice among recombinant PDC-E2
(10), commercially purified PDC (11), and
in-house-purified PDC (12) could influence the results
obtained for anti-M2 antibodies.
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We also performed a comparative study of the three techniques (IIF,
ELISA, and EI) with 70 samples that were referred to our laboratory for
diagnostic testing by immunofluorescence and gave a specific anti-M2
staining pattern. Available clinical data for these 70 samples showed
19 patients (27%) for whom PBC could be established as the diagnosis,
2 (3%) with another liver disease (1 acute hepatitis and 1 chronic
ethylism), and 49 (70%) for whom there was no clinical evidence of
liver disease. The high percentage of sera from patients with no
evidence of liver disease presenting a typical anti-M2 pattern in IIF
may be surprising, but similar data have already been presented in a
larger study (13). The long asymptomatic phase in PBC is
probably the main reason for this situation because in 33 of these 49
cases (67%), the anti-M2 pattern observed in immunofluorescence could
be confirmed by a positive result obtained by the ELISA and EI
techniques (Fig. 1
). In seven cases (14%), only the ELISA gave a positive result,
illustrating the difference in performance of the ELISA and the EI
assay already described above. In the remaining nine cases (18%) of
serum from patients with no evidence of liver disease presenting an
anti-M2 pattern, neither the ELISA nor the EI assay gave a positive
result, confirming that the immunofluorescence assay identifies
mitochondrial reactants other than PDC.
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In conclusion, our study on a limited number of samples showed that the TRACE EI assay, run on a COBAS Mira automated analyzer, is a very promising test for the determination of anti-M2 antibodies, having the advantage of few procedural steps, rapid turnaround time, nonsubjective read-out, and low costs. The analytical performance was better than that obtained for the ELISA. All of the PBC patients in our populations were correctly detected by the EI assay, and the specificity of the assay was higher than with IIF and ELISA using commercially purified PDC. We feel that the TRACE EI assay is a valuable tool that could provide considerable help in the interpretation of anti-M2-positive patterns in IIF and could replace labor-intensive ELISA testing for anti-M2 antibodies.
Acknowledgments
This study was supported in part by Trace Scientific (Victoria, Australia).
References
The following articles in journals at HighWire Press have cited this article:
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  F. Mor, M. Izak, and I. R. Cohen Identification of Aldolase as a Target Antigen in Alzheimer's Disease J. Immunol., September 1, 2005; 175(5): 3439 - 3445. [Abstract] [Full Text] [PDF]
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