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01.08.2011 | Research article | Ausgabe 4/2011 Open Access

Arthritis Research & Therapy 4/2011

Single-step autoantibody profiling in antiphospholipid syndrome using a multi-line dot assay

Zeitschrift:
Arthritis Research & Therapy > Ausgabe 4/2011
Autoren:
Karl Egerer, Dirk Roggenbuck, Thomas Büttner, Barbara Lehmann, Annushka Kohn, Philipp von Landenberg, Rico Hiemann, Eugen Feist, Gerd-Rüdiger Burmester, Thomas Dörner
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​ar3421) contains supplementary material, which is available to authorized users.
Karl Egerer, Dirk Roggenbuck contributed equally to this work.

Competing interests

Dirk Roggenbuck has a management role and is a shareholder of GA Generic Assays GmbH and Medipan GmbH. Both companies are diagnostic manufacturers. All other authors declare that they have no competing financial interests.

Authors' contributions

KE, DR, TB, AK, RC, and BL carried out the dot assays. EF, PvL G-RB, and TD conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.

Abstract

Introduction

Diagnosis of antiphospholipid syndrome (APS) still remains a laboratory challenge due to the great diversity of antiphospholipid antibodies (aPL) and their significance regarding APS-diagnostic criteria.

Methods

A multi-line dot assay (MLDA) employing phosphatidylserine (PS), phosphatidylinositol (PI), cardiolipin (CL), and beta2-glycoprotein I (β2 GPI) was used to detect aPL, immunoglobulin G (IgG) and immunoglobulin M (IgM) in 85 APS patients, 65 disease controls, and 79 blood donors. For comparison, anti-CL and anti-β2 GPI IgG and IgM were detected by enzyme-linked immunosorbent assay (ELISA).

Results

The level of agreement of both methods was good for anti-CL IgG, moderate for anti-CL IgM, very good for anti-β2 GPI IgG, and moderate for anti-β2 GPI IgM (kappa = 0.641, 0.507, 0.803 and 0.506, respectively). The frequency of observed discrepancies for anti-CL IgG (1.75%), anti-CL IgM (3.93%), anti-β2 GPI IgG (1.75%), and anti-β2 GPI IgM (0.87%) was low (McNemar test, P < 0.05, not-significant, respectively). Sensitivity, specificity, positive (+LR) and negative (-LR) likelihood ratios for at least one positive aPL antibody assessed by ELISA were 58.8%, 95.8%, 14.1, and 0.4, respectively, and for at least three positive aPl IgM and/or one positive aPL IgG by MLDA were 67.1%, 96.5%, 19.3, and 0.3, respectively. The frequency of IgM to PI, PS and CL, and combination of three or more aPL IgM detected by MLDA was significantly higher in APS patients with cerebral transient ischemia (P < 0.05, respectively).

Conclusions

The novel MLDA is a readily available, single-step, sensitive diagnostic tool for the multiplex detection of aPL antibodies in APS and a potential alternative for single aPL antibody testing by ELISA.
Zusatzmaterial
Additional file 1: Characterization of the multi-line dot assay in comparison with ELISA. For evaluation of assay performance, CVs were determined for the four ELISA and the aPL antibody reactivities assessed by MLDA. The anti-CL IgG and IgM ELISAs displayed intra-assay variability ranging from 2.3% to 4.1% and inter-assay variability ranging from 7.4% to 10.3%. The anti- β2 GPI IgG and IgM ELISAs revealed intra-assay variability from 3.3% to 4.5% and inter-assay variability from 5.2% to 6.1%. The intra-assay CVs for a serum reactive with PI, PS, CL, and β2 GPI in the MLDA were 5.2%, 6.8%, 8.3%, and 3.1%, respectively. With respect to ELISA results, the functional assay sensitivity was determined as 3.0 U/ml and 3.5 U/ml for IgG to CL and β2 GPI, respectively, and 2.0 U/ml and 2.5 U/ml for IgM to CL and β2 GPI, respectively. Furthermore, ROC curve analysis revealed the best assay performance for anti-CL IgG antibodies. (DOC 126 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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