A Solid Phase Enzyme Immunoassay (ELISA) for the Detection and Quantitation of Anticardiolipin Antibody

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Abstract
A solid-phase enzyme-linked immunoabsorbent assay (ELISA) is described for the detection and quantitation of anticardiolipin antibodies (ACAs) IgG and IgM in sera. In these assays, non-specific binding was controlled by using antigen-negative wells for all serum dilutions tested. Quadruplicate 100-μL serum sam-ples diluted 1:20 for ACA-IgG and 1:40 for ACA-IgM were incubated for two hours, after which alkaline phosphatase-conjugated antihuman IgG or IgM was added. A standard serum was used on each plate to provide reproducibility of the assay. Upper limits of normal for ACA-IgG and IgM were established by testing 161 sera from normal persons. Sixty-one selected pa-tients with SLE were tested; and, from these results, categories of positivity were defined from negative to 4+. All screen-positive sera (≥1+) were assayed in a quantitative ELISA assay for ACAs, using multiple dilutions of the unknowns. These data were fit on a standard curve generated with dilutions of a reference serum on each plate using a computerized data reduction system based on the 2 Plus 2 model. The standard curves were compared with the international standards for IgG and IgM anticardiolipin. The ability to quantitate ACA concentrations allows better definition of positive sera, as well as the opportunity to accurately evaluate and follow this antibody in a variety of patient groups.