Non-radioactive antigen

The non-radioactive antigen (A) and radioactive antigen (A ) are indistinguishable chemically i.e., both of them are identical chemically,... 

Since, both the radioactive and non-radioactive antigens (haptens) are more or less chemically and immunochemically the same, they will eventually compete for the limited number of antibody sites available thus, the amount of radioactivity that ultimately combines with the antibody will be an inverse function of the amount of unlabelled hapten competing for these sites,... 

Figure 8-33. Precipitation of a standard radioactive antigen in the presence of increasing amounts of non-radioactive competitor. A curve such as this is required for standardization of radioimmunoassays.

The earliest immunoassays made use of radioactively labeled antigens or antibodies. These analytes are referred to as radiolabeled immunoassays. Antibody binding sites are extremely specific and to retain this specifity the best option would be to replace a non-radioactive isotope in the tracer molecule by its radioisotope (e.g., replace hydrogen by H). However when the substitution is made in a part of the molecule away from the antibody binding site, the choice of radioisotope can be governed by other considerations, such as half-life, availability, high activity, and radiochemical purity. 

Radioiminunoassay (RIA) is a highly sophisticated technique and can detect extremely small amounts of non-radioactive material. It can achieve this even if the mixture contains huge amounts of other materials in which the Investigator is not interested. With the development of methods for labeling antigens to a high specific activity, very low concentrations (10- g/ml) can be detected easily. 

Humphrey and Keller (1970) found that the synthetic antigen TGAL, extensively iodinated to very high specific activity (1400 [xCi (jig ) was unable to elicit an antibody response in mice, whereas TGAL substituted with non-radioactive iodine could do so. 

The non-competitive assay uses an excess of antibody. Different approaches to detect the bound antigen have been developed, the most common use an antibody, in excess, coupled to a solid phase. The bound antigen is then detected with a second antibody labeled in a way that aids detection (e.g., radioactive, fluorophore, etc.). The amount of antigen in the sample is then directly proportional to the amount of labeled antibody captured on the solid phase. 

Precipitation techniques can be categorized into two general classes, non-specific and specific. The nonspecific separations involve the addition of a salt or solvent that decreases the solubility of the antigen-antibody complex under conditions that do not affect the free-labeled antigen. After addition, the immune complexes can be precipitated by centrifugation and the radioactivity in either the supernatant solution or the precipitate can be measured. Examples of precipitation reagents used in immunoassays include alcohol, ammonium sulfate, polyethylene, and dioxane. Care must be taken to avoid coprecipitation of the unbound label. 

The method is based on competitive binding in which a certain quantity of antigens in the sample marked with an isotope competes with the antibodies for a limited number of specific binding sites. Both the isotope marked antigens and the non-marked ones are linked to the antibodies forming complexes of antigen-antibody. The concentration of the antigens in the sample is inversely proportional to the radioactivity of the complexes. 

Antigen-antibody reactions occur in aqueous solution. Therefore, after separating "free" and "bound" radioactivity in a radioimmunoassay, one must frequently measure the radioactivity of an aliquot of an aqueous solution. Here we are faced with the problem of dispersing water, a polar solvent, in a non-polar toluene or xylene based scintillator system. To minimize quenching problems, especially serious when counting weak beta particles such as those emitted by tritium, it is best if a homogenous mixture of the sample and liquid scintil-lant results. That is, two-phase systems should be avoided if possible. 

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