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Keywords:

  • HEp-2 cell;
  • multiplex;
  • microbead-based immunoassay;
  • autoantibodies

Abstract

Advances in immunofluorescence assay development paved the way for the simultaneous detection of several antibodies in one sample, for the serological diagnosis of systemic rheumatic diseases. Standardized automated screening of such antibodies can be achieved by HEp-2 cell-based indirect immunofluorescence (IIF) using a multicolor fluorescence imaging technical platform. To create a common platform for both screening and specific antibody assessment, multiplex measurement of antibodies using fluorescence-coded immobilized microbeads was employed on the same platform. The multicolor fluorescence detection system VideoScan (AKLIDES®) was used for the fluorescence analysis of a multiplex microbead-based immunoassay (MIA). First, immunoglobulin G (IgG) was covalently coupled to one microbead population in duplicate and in three independent experiments. The coupled IgG was detected by a Cy™5-conjugated secondary antibody. Thus, intra- and interassay coefficients of variation (CV) were obtained. Second, a multiplex determination of antinuclear autoantibodies (ANA) to Scl-70, Sm, dsDNA, SS-A (Ro60), CENP-B, and La/SS-B by solid-phase MIA was investigated, using 72 sera from patients with autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis (SS). The reproducibility study revealed intra-assay CVs ranging from 3.2% to 9.9%, and interassay CVs ranging from 9.6% to 14.7%. The detection of Scl-70-, Sm-, CENP-B-, and La/SS-B-ANA with MIA showed very good agreement with the ELISA results (kappa = 1.0). The resulting relative sensitivities and specificities for Scl-70-, Sm-, CENP-B-, dsDNA-, and La/SS-B-ANA were 100%, respectively, with the exception of dsDNA (specificity 97%). Multiplex detection by immobilized fluorescence-coded microbeads using multicolor fluorescence is a reliable method for the assessment of rheumatic-disease-specific antibodies. Multicolor fluorescence analyses with pattern detection algorithms provide a common platform technique for both the screening of ANA by cell-based IIF and specific antibody assessment by multiplex detection. © 2011 International Society for Advancement of Cytometry