Radioimmunoassay of Barbiturates

Barbiturates represent a class of sedative and hypnotic drugs employed extensively in medicine. RIA provides a rapid, sensitive specific and reliable means for their determination in plasma levels upto 5 ng without indulging in any type of extraction, filtration or evaporation as required for other conventional analytical methods.  

Synthesis of Immunogen (Hapten) The barbiturate, 5-ally 1-5-(1-carboxyisopropyl) barbituric acid (I) is first converted to 5-allyl-5-(1 -p-n i trophenyloxycarbonylisopropyl) barbituric acid (II) by the interaction of the base with /7-nitrophenol in N, N-dimethylformamide (DMF) as shown below  

The resulting product (II) is subsequently coupled to bovine-serum-albumin in a glycerol-w ater mixture in the presence of dicyclohexylcarbodiimide. The mixture is incubated overnight at 4°C, and the protein-hapten complex is dialysed against distilled water thereby causing its purification. Conjugation of the respective barbiturate to the protein carrier, comparison of the barbiturate BGG-conjugate to control BGG-solution and preparation of 14C-pentobarbital sodium are carried out respectively. 

Preparation of Antiserum The barbiturate-bovine-serum-albumin conjugate is duly emulsified with an equal volume of complete Freund s adjuvant and New Zealand albino rabbits are subsequently im- 

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