Multiplex assessment of non‐organ‐specific autoantibodies with a novel microbead‐based immunoassay

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 1 ) 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 TM 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 5 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 Key termsHEp-2 cell; multiplex; microbead-based immunoassay; autoantibodies ANTINUCLEAR antibodies (ANA) to extractable nuclear antigens (ENA) are hallmarks in the serological diagnosis of systemic rheumatic diseases (1). The detection of ANA by indirect immunofluorescence (IIF) employing HEp-2 cells as a multiple antigen source has been established as the ''gold standard'' in routine diagnostics (2). However, advances in assay development and recombinant technology have paved the way for the detection of ANA to individual ENA, such as double-stranded DNA (dsDNA), improving the diagnostic power of ANA testing. The growing variety of ANA found in different systemic rheumatic diseases has generated the need for innovative techniques to overcome the shortcomings of single ENA detection, with regard to cost and time taken to obtain results (3). Hence, multiplexed platforms have been ...

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“…The VideoScan technology implemented in the Aklides system (GA Generic Assays GmbH, Dahlewitz, Germany) is a versatile fluorescence microscope imaging technology for the analysis of fluorescent objects (27,(29)(30)(31). The VideoScan instrument was automatically guided to each well of a 96-well plate, where it focused on microbeads or cell monolayer surfaces and took images, which were saved as .bmp files.…”

Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing

“…Fluorescent microbeads were covalently coated with SS-B, CENP-B, Sm, RNP-Sm, dsDNA, Scl-70, SS-A/Ro60, and SS-A/Ro52, respectively, as described elsewhere [21]. Further, glass slides with multi-compartment wells were employed to immobilize HEp-2 cells in the central compartment ( Figure 1).…”

“…For semi-quantitative analysis, the intensities of the fluorescence halos can be readily detected by standard fluorescence microscopy. Instead, the signal intensity of the halo can be quantified and simultaneously located to the appropriate microbead population by digital fluorescence reading [21]. This provides the basis for automatic simultaneous interpretation of ANA on HEp-2 cells and their confirmation on antigen-coated microbeads.…”

“…Our group has pioneered the use of digital fluorescence microscopy in autoAb testing by developing an automated interpretation technique providing standardization of autoAb interpretation by IIF including ANA pattern reading [18][19][20]. Furthermore, we set up a multiplex IIF assay for the simultaneous detection of autoAbs employing fluorescent microbeads with chemically activated surfaces for autoantigen immobilization [21]. Merging of both techniques provides the unique opportunity to perform ANA screening and multiplex confirmatory testing within one test.…”

Clinical Cell Cycle Analysis Revisited

“…It is worth noting that this equipment is not a cytometer from its origin but has been developed as the first reliable automated system for analysis of systemic autoantibodies (4,5).…”