RIA Kits

Our radioimmunoassay (RIA) kits and accessories represent the recognized gold standard of highly sensitive, reproducible immunodetection for metabolic targets such as insulin, adiponectin and ghrelin. We offer over 30 species-specific assays, covering targets for human, mouse, rat, porcine, canine, bovine and primate research.

We continue to focus on new products that are key research tools for metabolic disease. For example, RIAs have been developed for ghrelin, resistin, adiponectin, growth hormones and PYY. Our portfolio has been expanded with several new RIAs including intact and total proinsulin, c-peptide, rat/mouse insulin and sensitive leptin. Our “classic” products include a human insulin-specific RIA that does not cross-react with the circulating forms of proinsulin, leptin RIAs for most species, and our premier RIA for rat and mouse insulin.

Methodology

Radioimmunoassay is based on the antigen-antibody reaction in which tracer amounts of the radio-labeled antigen competes with endogenous antigen for limited binding sites of the specific antibody against the same antigen.

RIA Components

In principle, radiolabeled antigen should be similar in bioactivity and/or immunoreactivity of the native antigen. The most commonly used radioisotope in RIA is 125-I, although other beta-emitting isotopes such as C14 and H3 have also been used. Usually, high specific activity radiolabeled (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 radiolabeled 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.

RIA Validation

After the initial development of the radioimmunoassay for any analyte, the assay method has to be validated for accurate determination of the analyte in the biological fluid. It should meet the criteria of sensitivity, specificity, precision, recovery, and linearity & dilution.

Sensitivity is defined as the minimal detection limit which can be accurately measured above the background. The assay sensitivity can be improved by decreasing the amount of radiolabeled analyte and/or antibody. Sensitivity can also be improved by disequilibrium incubation format in which radiolabeled antigen is added after initial incubation of antigen and antibody.

The RIA method developed should be specific for the analyte in question, i.e. other analytes should not cross-react with the antibody. If any cross-reactivity is observed with other analyte(s), selection of a different antibody is advised or the antibody needs to be purified from the cross-reacting analyte(s) by affinity chromatography.

Precision is the inter- and intra-assay reproducibility of quality controls with concentrations at the low, medium and high range of the standard concentrations.

Recovery of the exogenously added analyte in different concentrations to the plasma, serum or any other biological fluid should be close (80–100 %) of the amount added for recovery. Recovery of the exogenously added analyte suggests that other sample components are not interfering with the antigen-antibody reaction.

Similarly, linearity and dilution experiments suggest that the antigen in the sample is behaving similar to the native standard.

These experiments are performed by serial dilution of either basal serum and plasma samples or exogenously spiked serum and plasma samples. Sample dilution curves will be parallel to the standard curves if the analyte measured is similar to the standard used and no other sample component is interfering with the antigen-antibody reaction. If recovery and linearity & dilution studies are not optimal, the appropriate matrix should be substituted. Usually, the proper matrix is the serum or plasma or any biological fluid, but devoid of the native antigen. Assay matrix devoid of native antigen is prepared by treating the serum or plasma from the same species with charcoal or other adsorbent.