Homogenous enzyme immunoassay for cyclosporine in whole blood using the EMIT^®2000 cyclosporine specific assay with the COBAS MIRA‐plus analyzer

Abstract

We describe the evaluation of the EMIT^®2000 cyclosporine specific assay kit, an enzyme‐multiplied immunoassay for cyclosporine in whole blood, with a COBAS MIRA‐plus analyzer. The enzyme used for the assay was glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49 G6PDH) from Leuconostoc mesenteroides; the monoclonal antibody is fairly specific for cyclosporine, and is not reactive with most metabolites. The assay principle is based on competitive immunoassay with G6PDH‐labeled cyclosporine and cyclosporine in sample to the anticyclosporine mouse monoclonal antibody binding site. The within‐assay coefficient of variation (CV) of this method was 2.7–4.2% (n = 10) at the levels of 56.2–339.7 μg/L. Day‐to‐day CVs ranged from 4.2–8.1% at the levels of 47.2–350.2 μg/L. The within‐day CVs ranged from 2.0–6.4% at the levels of 43.3–330.5 μg/L. The functional detection limit was 24.9 μg/L. Samples treated with pretreatment reagent were stable at least 5 hr. Calibration was stable at least 10 days. The analytical recovery was 81–109%. The correlation between values obtained with the EMIT^®2000 cyclosporine specific assay kit (y) and fluorescence polarization immunoassay (FPIA) (TDxFLx) (x) was: y = 0.880x – 13.053 μg/L (r = 0.984, Sy/x = 15.968, n = 71) with a mean difference of 31.42 ± 19.89 μg/L ((TDxFLx – EMIT^®2000) ± SD); for the FPIA (AxSYM) (x): y = 0.989 – 4.144 μg/L (r = 0.981, Sy/x = 17.478, n = 71) with a mean difference of 5.56 ± 17.38 μg/L ((AxSYM – EMIT^®2000) ± SD); and for the radioimmunoassay (RIA, CYCLO‐Trac SP) (x): y = 0.893 – 6.764 μg/L (r = 0.993, Sy/x = 10.582, n = 71) with a mean difference of 22.18 ± 14.98 μg/L ((RIA – EMIT^®2000) ± SD) using the Bland‐Altman technique. J. Clin. Lab. Anal. 15:319–323, 2001. © 2001 Wiley‐Liss, Inc.