Application of radioimmunoassay to pharmacology

The radioimmunoassay, hased on specific antigen‐antihody hinding, was applied to the measurement of drug concentrations. To elicit antihody formation to drugs (substances usually of low molecular weight), the drug was coniugated to a Zarge molecule, such as a protein or synthetic polymer to form an immunogen. Three morphine derivatives linked to hovine serum alhumin hy an amide hond were used to immunize rah hits with the morphine‐protein coniugate (0.10 mg). After periodic iniections of the antigen into the footpads of the animals, and as soon as high titers of antihodies had heen formed, the animals were hled. Antihodies of high titer and affinity were incuhated in a medium of varying concentrations of unknown drug and laheled drug at 4° C, until equililJrium was achieved. Following incuhation, the laheled drug that exists hoth as free and honnd to a,ntihody was then separated by a variety of procedures, e.g., microfiltration, anti‐gamma globulin, chromatoelectrophoresis, salt precipitation, and coated charcoal. The data ohtained hy precipitating the antihody‐ligand complex yielded the following information: (1) the concentration required to inhihit 50% of the hinding of laheled drug to antibody indicates the senstivity of the method; (2) it also indicates to some extent the specificity of the antibody generated by the immunogens employed to evoke its production, as exemplified hy the fact that 3‐0‐carhoxymethylmorphine coniugate recognizes normorphine equally as weil as rrwrphine. The factors that enhance or reduce the sensitivity of the method are discussed.