Rate-nephelometric inhibition immunoassay of phenytoin and phenobarbital.

A proposed rate-nephelometric inhibition immunoassay of phenytoin and phenobarbital in human serum involves the sequential addition to buffer of 42-microL aliquots of sample containing the hapten (drug) of interest, a hapten conjugate (drug-equine apoferritin), and specific antibody to the hapten. The drug and the drug conjugate compete for the binding sites on the antibody. The free hapten-antibody complex is soluble and so does not scatter light, whereas the complex of antibody and drug conjugate is insoluble and thus scatters light. The latter immunoprecipitation is competitively inhibited by free hapten. Thus the higher the concentration of free hapten present, the fewer immunoprecipitin complexes are formed, and the less light scatter. Precision, accuracy, linearity, analytical recovery, and comparison with patients' samples assayed with the DuPont aca were excellent. There was no significant interference from hemolysis, icterus, or lipemia. Many potentially interfering drugs and metabolites were checked for cross reactivity, with negative results. Reaction times range from 30 to 50 s.