The enzyme-linked immunosorbent assay (ELISA) is a specific and highly sensitive method for quantification of cytokines and other analytes in solution. It was first developed in 1971 and has since become one of the most widely used techniques in clinical and research laboratories. The ELISA can be used in a variety of formats from testing only a few individual samples to fully automated high-throughput screening.
The assay involves a specific monoclonal antibody (mAb), which is used to coat a microtiter plate. After addition of the sample, the specific antibody on the plate will capture the protein of interest (e.g. a cytokine). A second mAb, which is used for detection, binds a different epitope on the protein. The detection antibody is labeled with biotin, which allows subsequent binding of a streptavidin-conjugated enzyme. Any unbound reagents are removed by washing. After addition of a substrate, a color reaction will develop that is directly proportional to the amount of protein bound. The concentration of protein in the sample is determined by comparison with a standard curve of known protein concentrations.
Sensitivity: The sensitivity of an ELISA mainly depends on the affinity of the antibodies and on the amplification system used. The detection limits for cytokine ELISAs are typically in the low pg/ml range. The standard range for the assay indicates the upper and lower limits of analyte concentration that can be determined linearly with precision and accuracy.
Accuracy: The accuracy of the cytokine ELISA is commonly established by calibration with an external reference standard. International standards are available from the National Institute for Biological Standards and Control (NIBSC) for calibration of the most widely used cytokine ELISAs.
Analysis of serum and plasma can be hampered by heterophilic antibodies present in the samples.
Heterophilic antibodies found in human serum/plasma are capable of binding to both the capture and detection antibodies used in capture ELISA. Heterophilic antibodies are found in a majority of human individuals and can, by cross-linking the assay antibodies, result in false positive signals.
The ELISA diluent and Assay buffer developed by Mabtech for dilution of samples prevent the heterophilic antibodies from cross-linking the capture and detection antibodies. The cytokine content of serum/plasma samples can therefore be measured without interference by heterophilic antibodies. The lack of interference by heterophilic antibodies has been validated using plasma/serum samples from normal healthy human blood donors. Please note that heterophilic antibody interference in samples from human subjects with various diseases or other conditions has not been assessed.