Immunological studies on delayed hypersensitivity to phenytoin--I. A new method for preparing phenytoin antiserum

The present study was initiated to produce an antiserum to phenytoin with high specificity and sensitivity which would be suitable not only for determination of blood phenytoin concentration but also for induction of a hypersensitivity reaction to phenytoin in experimental animals. p-Aminophenytoin was synthesized and identified by means of IR, 1H-NMR and mass spectroscopy. BSA-phenytoin conjugate was prepared by using p-aminophenytoin, BSA and, as a coupling reagent, glutaraldehyde. Satisfactory response to immunization was achieved at a 9.8:1 molar ratio of p-aminophenytoin to BSA. The antiserum obtained from rabbits immunized with BSA-phenytoin conjugate exhibited practically no cross-reactivity with either phenytoin metabolites or other anti-epileptic drugs, indicating that this antiserum provides sufficiently high specificity. In our experiments, the lower limit for detecting phenytoin was 2 ng using RIA, whereas 200 ng was the minimum amount detectable by HPLC. Thus, by a difference of two orders of magnitude, the present RIA method shows a much higher sensitivity than that of HPLC, though we found a good correlation of simultaneous determinations of serum phenytoin between the two methods. Reproducibility of phenytoin determination in plasma was confirmed by calculating the coefficient of variance. The values were less than 10%.