RIA-Techniques Isotope Dilution

Part—VI has been solely devoted to Miscellaneous Assay Methods wherein radioimmunoassay (RIA) (Chapter 32) has been discussed extensively. Various arms of theoretical aspects viz., hapten determinants and purity importance of antigenic determinants and analysis of competitive antibody binding of isotopically labeled compounds. The applications of RIA in pharmaceutical analysis, such as morphine, hydromorphone and hydrocordone in human plasma clonazepam, flurazepam in human plasma chlordiazepoxide in plasma barbiturates, flunisolide in human plasma have been described elaborately. Lastly, the novel applications of RIA-techniques, combined RIA-technique-isotope dilution and stereospecificity have also been included to highlight the importance of RIA in the analytical armamentarium. 

A most important technique which has been developed as an extension of the isotope dilution principle is that of radioimmunoassay (RIA). Analyses by this method employ substoichiometric amounts of specific binding immuno-chemical reagents for the determination of a wide range of materials (immunogens) which can be made to produce immunological responses in animals such as sheep or rabbits. It is possible to combine the specificity of an immunochemical reaction with the extreme sensitivity of radiotracer detection. Analytical methods based upon these principles have achieved wide applicability in the determination of organic compounds at trace levels. 

The combined RIA-technique and isotope dilution has been successfully developed to estimate SULINDAC along with its two prominent metabolites, namely its sulphone and its sulphide present in the plasma-level as shown in the following chemical structures X and Y. 

Radioimmunoassay (RIA) is a technique based on the formation of antigen-antibody complex. This technique essentially involves the application of isotope dilution analysis. An antigen is typically a protein of molecular weight greater than 10,000 that stimulates the production of antibody in an animal body. The antigen subsequently binds with the antibody. 

A most important technique which has been developed as an extension of the isotope dilution principle is that of radioimmunoassay (RIA). Analyses by this method employ substoichiometric amounts of specific binding immuno-... 

In the isotope-dilution technique it is necessary to isolate, purify, and assay a sample of the substance under study, and this sometimes presents a difficulty. On the other hand, in the radioimmunoassay (RIA) technique it is not necessary to assay the substance, but only to measure the radioactivity. 

A radioimmunological method, developed by Salmon (1), has been selected for routine purposes and has recently been applied to measurements in total blood samples of endotoxin treated animals (2). Being aware of the discrepancies, often reported between radioimmunoassays (RIA) and chemical measurements (3), a reference method using isotope dilution (ID) and mass spectrometry (MS), i.e. selected ion monitoring (SIM) has been worked out in order to support RiA data. The technique of ID-MS may serve as a reference method as it can provide a specific and accurate means for determination in biological fluids. Ihe accuracy of ID-MS is based on the high specifi-... 

Recently Nexo and Hoffmann-Lucke (2011) reported three different methods devised for measuring holoTC, with reasonably similar results. The first method combined ionic precipitation of transcobalamin and measurement of the amount of vitamin B12 trapped in the precipitate (Begley and Hall 1975). Lindemans et al. (1983) improved this method by using antibodies against transcobalamin rather than ionic separation. The second method, proposed by Ulleland et al. (2002), consisted of measuring trapped vitamin B12 by an isotope dilution assay (holoTC radioimmunoassay, RIA). Refsum et al. (2006) used a third technique, a microbiological method, to measure vitamin B12 levels (Nexo and Hoffmann-Lucke 2011). 

As indicated earlier, several immunotechniques are used for the quantification of apo A-I and apo B-100. These techniques are affected differently by the analytical issues discussed previously. RIA and ELISA, for example, are normally used for the determination of analytes present at these very low concentrations (nanograms per miUiliter). Therefore large dilutions (up to 40,000-fold) are required when these techniques are applied to apo A-I or apo B-100 measurement, which can result in a substantial analytical error. In addition, these assays are relatively time consuming and not easily automated, and RIA requires the use of isotopes. 

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