ANA HEp-2 Screen ELISA

Intended Use

ANA HEp-2 Screen ELISA is a solid phase enzyme immunoassay for the combined qualitative detection of IgG antibodies against HEp2 cells in human serum. Each well is coated with lysed HEp2 cells. The test collectively detects, in one well, total ANAs against double stranded DNA (dsDNA), histones, SS-A (Ro), SS-B (La), Sm, snRNP/Sm, Scl-70, PM-Scl, Jo-1 and centromeric antigens along with sera positive for HEp2 immunofluorescence test (IFT).

The assay is a tool in the diagnosis of systemic rheumatic diseases like systemic lupus erythematosus (SLE), mixed connective tissue diseases (MCTD), scleroderma, Sjögren`s syndrome, polymyositis and dermatomyositis.

Clinical Application and Principle of the Assay

Anti-nuclear antibodies (ANA) directed against a variety of nuclear and cytoplasmic antigens occur in high frequency in systemic rheumatic diseases and thus are an important tool for the differential diagnosis. For instance, SS-A (Ro) and SS-B (La) antibodies are associated with SLE and Sjögren’s syndrome (SS), anti-dsDNA and anti-Sm antibodies with SLE, antihistone antibodies with SLE and drug-induced lupus, anti-RNP antibodies with mixed connective tissue disease (MCTD) and SLE, anti-Scl-70 antibodies with scleroderma (progressive systemic sclerosis [PSS]), anti-Jo-1 antibodies with polymyositis and dermatomyositis and anti-centromere antibodies with CREST syndrome.

Indirect immunofluorescence test (IFT) on eucaryotic cells like HeLa and HEp2 has been the established method for the detection of ANAs. Although the IFT is a sensitive test, it is laborious when testing large numbers of patient samples and is subject to errors from human interpretation and from variability in fluorescent microscope. The ELISA test system is an excellent alternative to the IFT for screening patient`s serum for the presence of ANAs of clinical significance. Single antibody specificities have to be determined by more specific testing using ELISAs employing the specific target antigens for a simple and reliable differentiation of ANAs.

Principle of the test

Serum samples diluted 1:101 are incubated in the microplates coated with the specific antigen. Patient´s antibodies, if present in the specimen, bind to the antigen. The unbound fraction is washed off in the following step. Afterwards anti-human immunoglobulins conjugated to horseradish peroxidase (conjugate) are incubated and react with the antigen-antibody complex of the samples in the microplates. Unbound conjugate is washed off in the following step. Addition of TMB-substrate generates an enzymatic colorimetric (blue) reaction, which is stopped by diluted acid (color changes to yellow). The intensity of color formation from the chromogen is a function of the amount of conjugate bound to the antigen-antibody complex and this is proportional to the initial concentration of the respective antibodies in the patient sample.