A SIMPLIFIED WHOLE-BLOOD ENZYME-LINKED-IMMUNOSORBENT-ASSAY (PROTRAC-II) FOR TACROLIMUS (FK506) USING PROTEOLYTIC EXTRACTION IN-PLACE OF ORGANIC-SOLVENTS
G. Macfarlane et al., A SIMPLIFIED WHOLE-BLOOD ENZYME-LINKED-IMMUNOSORBENT-ASSAY (PROTRAC-II) FOR TACROLIMUS (FK506) USING PROTEOLYTIC EXTRACTION IN-PLACE OF ORGANIC-SOLVENTS, Therapeutic drug monitoring, 18(6), 1996, pp. 698-705
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.