Development of enzyme immunoassay for Platonin (NK 19)

A sensitive and specific enzyme immunoassay for an immunomodulator PLATONIN (NK 19) has been developed and applied to the evaluation of pharmacokinetics in human and rats. NK 19 analog, succinyl NK 19 has been synthesized and coupled to bovine serum albumin. This conjugate is immunogenic and when injected into rabbits antibodies of high affinity for NK 19 were obtained. Some light decomposed products of Platonin were of lower cross-reactivity with these antibodies. For competitive reactions, these antibodies were incubated with a mixture of succinyl NK 2900-beta-D-galactosidase conjugate and standard NK 19 or sample. Then, antibody-bound enzyme-hapten were separated from free one using anti-rabbit IgG immobilized on polystyrene balls. Activity of the enzyme on the solid phase was fluorometrically determined. The present immunoassay allows detection of as low as 10 pg of NK 19 in the biological fluids using 50 microliters of the samples. The accuracy and reproducibility were also quite satisfactory. By use of this assay, plasma levels of NK 19 after a single oral administration of 5 mg or 10 mg/body to human volunteers or intravenous injection of 2 micrograms/kg to rats could be determined.