IL-1ra ELISA: reduction and alkylation of synovial fluid eliminates interference by IgM rheumatoid factors

IL-1 and a specific receptor antagonist of IL-1, IL-1ra, may play important roles in the pathophysiology of rheumatoid arthritis and in other types of inflammatory synovitis. Measurement of IL-1ra in synovial fluids and in other body fluids may lead to a greater understanding of its possible activity as a modulator of the immune and inflammatory systems in vivo. Therefore, a modified sandwich ELISA was developed to measure IL-1ra protein concentration in synovial fluids. The antibodies used in this ELISA were polyclonal and derived from rabbits hyperimmunized with human recombinant IL-1ra. IgM rheumatoid factors within synovial fluid resulted in false elevation of determined IL-1ra by the sandwich ELISA through binding of the primary and secondary antibodies. Reduction and alkylation of synovial fluid samples before application to the ELISA plate eliminated the interference caused by greater than or equal to 2000 micrograms/ml IgM rheumatoid factor (latex agglutination titer of 1/5.120). This ELISA was specific for IL-1ra; there was no detection of IL-1 alpha, IL-1 beta, or lysozyme. The sensitivity of this ELISA was less than 200 pg/ml, making it a useful assay for the accurate measurement of synovial fluid IL-1ra protein concentration.