Measurement of anti-cardiolipin antibodies by an enzyme-linked immunosorbent assay (ELISA): standardization and quantitation of results

Abstract

We describe the development of a simple and highly sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for measuring IgG and IgM anticardiolipin antibodies (ACA). Microtitre plates were coated with cardiolipin at a concentration of 45 micrograms/ml by evaporation under nitrogen. Non-specific binding of diluted sera was eliminated by blocking of plates with 10% fetal calf serum in phosphate buffered saline (PBS/FCS) for 2 h. Then sera (100 microliters) at a dilution of 1:100 were incubated in the wells for 1 h. Affinity purified goat anti-human IgG or IgM (100 microliters) at a concentration of 1 microgram/ml was subsequently added and allowed to incubate for 1 h; detection of ACA was achieved using an alkaline phosphatase conjugated rabbit anti-goat IgG reagent by reading the colorimetric yield at 405 nm after incubation with substrate. Reference serum pools were established to study reproducibility of the assay throughout its sensitivity range, and Standard curves were established. The quantitative normal range was 0-9.0 Anticardiolipin ELISA Units (AEU) for IgG and 0-8.0 (AEU) for IgM-ACA. A strong correlation was found between the ELISA and radioimmunoassay methods for measuring ACA of both IgG and IgM classes. Results from 65 patients with systemic lupus erythematosus (SLE) and 45 patients with seropositive rheumatoid arthritis are also reported. The advantages of the ELISA method for quantitative determination of ACA levels, should make it a useful and reliable method for clinical and experimental monitoring of patients with SLE and associated autoimmune disorders.