TACROLIMUS (FK506) - VALIDATION OF A SENSITIVE ENZYME-LINKED-IMMUNOSORBENT-ASSAY KIT FOR AND APPLICATION TO A CLINICAL PHARMACOKINETIC STUDY

Tacrolimus (FK506) is a macrolide immunosuppressant approved for the p rophylaxis of organ rejection in liver transplant. Immunoassays of low intra-and interday variability and high sensitivity are necessary to adequately characterize terminal elimination phase concentrations in p harmacokinetic studies. A new ELISA kit for the quantitation of tacrol imus in human whole blood has been validated for use in pharmacokineti c studies. Methanol sample extracts were dried and reconstituted in a horseradish peroxidase (HPR)-FK506 conjugate solution. The reconstitut ed samples and mouse anti-FK506 were added to a microplate, precoated with secondary antibody, and incubated. FK506 and the HPR-FK506 conjug ate competed to bind with anti-FK506, which was immobilized by binding to the secondary antibody. Unbound FK506 was washed away, and substra te was added for color development. Once the reaction was stopped with 2 N H2SO4, the plate was read at 450 nm. The linear range was 0.5-60 ng/ml, with a limit of quantitation of 0.5 ng/ml. Interday precision a nd accuracy were less than or equal to 10.4% C.V. and less than or equ al to 3% R.E. for quality control samples. The lack of interference fr om endogenous compounds was established by parallelism and recoveries of FK506 from six lots of control matrix. Cross-reactivity against the metabolites and analogs were not performed because the kit monoclonal antibody was from the same source as Kobayashi et al (I), The utility and sensitivity of the kit present a,good method for the quantitation of tacrolimus in blood from pharmacokinetic studies. The method is ro bust and has been used to assay tacrolimus in several thousand whole b lood samples by multiple analysts.