Combined RIA technique-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. 

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. 

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. 

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). 

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