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Radioimmunoassay (RIA)

Radioimmunoassay (RIA) was the first of many immunoassay techniques developed to analyze nano molar and pico molar concentrations of the hormones in biological fluids. Berson and Yallow of Albert Einstein School of Medicine initially described the method to determine insulin levels in human plasma in early sixties.

Millipore’s 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 and primate research.

Millipore continues 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.

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. In principle, radio-labeled antigen should be similar in bio-activity and/or immunoreactivity of the native antigen.

Most commonly used radio-isotope in RIA used is 125-I although other beat emitting 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.

In radioimmunoassay, a fixed concentration of radio-labeled antigen in trace amounts is incubated with a constant amount of antiserum such that the total antigen binding sites on the antibody are limited such that the only 30–50% of the total radio-labeled antigen may be bound in the absence of the antigen. When unlabeled antigen, either as standard or test sample, is added to this system, there is competition between radio-labeled antigen and unlabeled antigen for the limited constant number of binding sites on the antibody. The amount of radio-labeled antigen bound to antibody decreases as the concentration of unlabeled antigen increases. Following optimal incubation condition e.g. buffer, pH, time and temperature, radio-labeled antigen bound to antibody is separated from unbound radio-labeled antigen.

Several separation techniques such as double antibody, charcoal, cellulose, chromatography, solid phase can be applied to separate bound and free radio-labeled antigen. Double antibody technique combined with polyethylene glycol is most frequently used for this purpose. After separating antibody-bound radio-labeled antigen from free radio-labeled antigen, the bound or free fraction is counted a g counter with appropriate settings for the particular radioisotope.

Thereafter, a calibration or standard curve is generated with a set of known concentrations of the unlabeled standards and from this curve the amount of antigen in unknown samples can then be calculated. Thus, the four basic necessities for developing a radioimmunoassay are: a specific antiserum to the antigen to be measured, the availability of a radioactive labeled form of the antigen, a method whereby antibody-bound tracer can be separated from the unbound tracer, and finally, an instrument to count radioactivity.

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 and 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 radio-labeled 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 samples 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.

Although, radioimmunoassay is the oldest and still a widely used immunoassay technique, it continues to offer distinct advantages in terms of their simplicity, sensitivity and handling and efficacy.

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