Creative BioMart provides Radioimmunoassay (RIA) that uses antibodies to detect and quantitate the amount of antigen (analyte) in a sample. This assay is typically very sensitive and specific. It is possible to detect as low as a few picograms of analyte in the experimental tube when using antibodies of high affinity (Kd = 10-8 - 10-11 M). The basic principle of radioimmunoassay is competitive binding, where a radioactive antigen ("tracer") competes with a non-radioactive antigen for a fixed number of antibody or receptor binding sites. When unlabeled antigen from standards or samples and a fixed amount of tracer (labeled antigen) are allowed to react with a constant and limiting amount of antibody, decreasing amounts of tracer are bound to the antibody as the amount of unlabeled antigen is increased.
125-I labels are usually applied although other isotopes such as C14 and H3 have also been used. Usually, high specific activity radio-labeled (125-I) antigen is prepared by iodination of the pure antigen on its tyrosine residue(s) by chloramine-T or peroxidase methods and then separating the radio-labeled antigen from free-isotope by gel-filtration or HPLC. Other important components of RIA are the specific antibody against the antigen and pure antigen for use as the standard or calibrator.