Next-Generation Autoantibody Testing by Combination of Screening and Confirmation-the CytoBead® Technology

Abstract

Occurrence of autoantibodies (autoAbs) is a hallmark of autoimmune diseases, and the analysis thereof is an essential part in the diagnosis of organ-specific autoimmune and systemic autoimmune rheumatic diseases (SARD), especially connective tissue diseases (CTDs). Due to the appearance of autoAb profiles in SARD patients and the complexity of the corresponding serological diagnosis, different diagnostic strategies have been suggested for appropriate autoAb testing. Thus, evolving assay techniques and the continuous discovery of novel autoantigens have greatly influenced the development of these strategies. Antinuclear antibody (ANA) analysis by indirect immunofluorescence (IIF) on tissue and later cellular substrates was one of the first tests introduced into clinical routine and is still an indispensable tool for CTD serology. Thus, screening for ANA by IIF is recommended to be followed by confirmatory testing of positive findings employing different assay techniques. Given the continuous growth in the demand for autoAb testing, IIF has been challenged as the standard method for ANA and other autoAb analyses due to lacking automation, standardization, modern data management, and human bias in IIF pattern interpretation. To address these limitations of autoAb testing, the CytoBead® technique has been introduced recently which enables automated interpretation of cell-based IIF and quantitative autoAb multiplexing by addressable microbead immunoassays in one reaction environment. Thus, autoAb screening and confirmatory testing can be combined for the first time. The present review discusses the history of autoAb assay techniques in this context and gives an overview and outlook of the recent progress in emerging technologies.

Keywords: Autoimmune disease; Digital fluorescence; Indirect immunofluorescence; Multiplex diagnostics; Second-generation autoantibody testing.

Fig. 1

Evolving autoantibody (autoAb) testing and strategies for the serological diagnosis of systemic autoimmune rheumatic diseases. ANA antinuclear antibody, autoAb autoantibody, CIE counterimmunoelectrophoresis, D/LIA dot/line immunoassay, ELISA enzyme-linked immunosorbent assay, ENA extractable nuclear antigen, IB immunoblot/westernblot, ID/DRID immunodiffusion/double radial immunodiffusion, IIF indirect immunofluorescence, IP immunoprecipitation, MIA microbead immunoassay, RIP radioimmunoprecipitation

Fig. 2

Multiplexing strategy of CytoBead® technology exemplified for CytoBead® ANA assay. Combination of ANA screening with HEp-2 cells (middle part) and anti-ENA testing with antigen-coated microbeads (peripheral parts I–IV) in one reaction environment. Example of an ANA positive serum with positive homogeneous fluorescence pattern on HEp-2 cells and positive signal on dsDNA-coated microbeads presented as green fluorescence halo (small red microbeads in part III). ANA antinuclear antibody, CENP centromere protein, Da Dalton, dsDNA double-stranded DNA, ENA extractable nuclear antigen, hom homogeneous, RNP ribonuclear protein, Scl-70 DNA-Topoisomerase I, Sm Smith, SS Sjögren-Syndrome, (+) positive, (−) negative

Fig. 3

CytoBead® assays for the detection of a antinuclear antibodies (ANA) with CytoBead® ANA assay, b antineutrophil cytoplasmic autoantibodies (ANCA) with CytoBead® ANCA assay, and c celiac disease (CD)-specific (auto)antibodies (auto/Abs) with CytoBead® CeliAK assay. Matching principle of specific fluorescence patterns on HEp-2 cells (a), neutrophil granulocytes (b), and esophagus tissue (c) with positive reactions of antigen-coated microbeads immobilized in peripheral compartments. CENP centromere protein, Da Dalton, dsDNA double-stranded DNA, EmA endomysial antibody, GBM glomerular basement membrane, MPO myeloperoxidase, PR3 proteinase 3, RNP ribonuclear protein, Scl-70 DNA-Topoisomerase I, Sm Smith, SS Sjögren-Syndrome, (+) positive, (−) negative