A SIMPLIFIED WHOLE-BLOOD ENZYME-LINKED-IMMUNOSORBENT-ASSAY (PROTRAC-II) FOR TACROLIMUS (FK506) USING PROTEOLYTIC EXTRACTION IN-PLACE OF ORGANIC-SOLVENTS

Tacrolimus (FK506) is a macrolide antibiotic with potent immunosuppres sive properties. FK506 is 10- to 100-fold more potent than cyclosporin A in preventing organ rejection and in toxicity. Extreme inter- and i ntrapatient variability and lack of correlation between drug dosage an d drug blood levels necessitate consistent and reliable therapeutic dr ug monitoring. Previous methods for monitoring drug levels have been l engthy and labor intensive and have required organic solvents for samp le extraction. We have developed a manual enzyme-linked immunosorbent assay (ProTrac II ELISA) that employs standardized reagents and a nono rganic solvent extraction and yields good assay sensitivity and precis ion. The calculated mean sensitivity of the assay was 0.18 ng/ml. Inte rassay precision ranged from 6.3% to 13.1% (coefficient of variation) for FK506 in whole blood (concentration range, 1.0-25.0 ng/ml). Recove ry of the drug from spiked EDTA whole blood was 91-98% over the same c oncentration range. Mean recovery of the drug from clinical samples sp iked with kit standards was 99.5%. Dilutions of high-concentration spi ked EDTA whole blood samples and high-concentration samples from patie nts exhibited linearity of observed versus expected values (y = 0.86x - 1.15; r = 0.99). Comparison of ProTrac II with the INCSTAR ProTrac F K506 ELISA by paired Student's t test showed no statistical difference between the methods (p = 0.46). Comparison of ProTrac II ELISA to the IMx microparticle enzyme immunoassay (MEIA) by linear regression resu lted in a line with a slope of 1.22 (r = 0.91). Analysis by t test res ulted in a p value <0.001, indicating that the absolute values obtaine d in these assays are significantly different. The mean (+/-SD) differ ence in the absolute values was 4.2 +/- 2.6 ng/ml, with the MEIA value s consistently higher. These results indicate that ProTrac Il is a sen sitive, precise ELISA for the determination of tacrolimus in whole blo od; it can be performed in <4 h.