An enzyme linked immunosorbent assay (ELISA) for the qualitative detection of RF IgA, IgG and IgM isotypes in human serum to aid in diagnosis of rheumatoid arthritis in conjunction with other laboratory and clinical findings.
Microwells are coated with purified rabbit IgG antigen followed by a blocking step to reduce non-specific binding during the assay run. Controls, calibrator and patient sera are incubated in the antigen coated wells to allow specific antibodies present in the serum to bind to the antigen. Unbound antibodies and other serum proteins are removed by washing the microwells. Bound antibodies are detected by adding an enzyme labeled anti-human conjugate to the microwells. Unbound conjugate is removed by washing. Specific enzyme substrate (TMB) is then added to the wells and the presence of antibodies is detected by a color change produced by the conversion of TMB substrate to a colored reaction product. The reaction is stopped and the intensity of the color change, which is proportional to the concentration of antibody, is read by a spectrophotometer at 450 nm. Results are expressed in ELISA Units per milliliter (EU/ml) and reported as positive or negative.