Radioimmunoassay of Chlordiazepoxide in Plasma

Chlordiazepoxide is the pioneer member of the 1,4-benzodiazepines to be employed clinically as an antianxiety agent in humans. A number of methods based on extraction processes are available for the assay of this drug, namely spectrofluorometry, polarography and electron-capture GC-technique but RIA measures it directly in the blood without involving extraction and possesses very low sensitivity. 

Synthesis of Immunogen Chlordiazepoxide as suspension inN-methylformamide is treated with HC1 in dioxane to yield a pale-yellow solution. The resulting mixture is cooled to -30 °C and isoamyl nitrite in dioxane is added. The solution is stirred at - 30° to - 40 °C and aqueous ammonium sulfamate is added with continuous stirring. 

The chilled azide solution is added slowly, dropwise with constant vigorous stirring into a solution of bovine-serum albumin. The pH is maintained at 8.0 to 8.7 by the careful addition of NaOH solution. The resulting pale-yellow solution is kept at 4°C for a duration of 36 hours and then dialysed against trimethamine buffer. After further dialysis for two days against distilled water, the immunogen is isolated by lyophilization. 

Immunization and Antibody Production The lypphilized immunogen obtained above is dissolved in normal saline and emulsified with equal volumes of complete Freund s adjuvant into a thick paste. Three New Zealand albino rabbits are immunized with the immunogen-paste through intradermal injections. The process is repeated twice at 2-weeks intervals followed by booster doses at monthly intervals. The antiserum is harvested when the plasma titer value is attained maximum. 

RIA-Procedure The various steps involved in the RIA procedure are enumerated below  

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