title: 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 microg/L. Day-to-day CVs ranged from 4.2-8.1% at the levels of 47.2-350.2 microg/L. The within-day CVs ranged from 2.0-6.4% at the levels of 43.3-330.5 microg/L. The functional detection limit was 24.9 microg/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 microg/L (r = 0.984, Sy/x = 15.968, n = 71) with a mean difference of 31.42 +/- 19.89 microg/L ((TDxFLx - EMIT 2000) +/- SD); for the FPIA (AxSYM) (x): y = 0.989 - 4.144 microg/L (r = 0.981, Sy/x = 17.478, n = 71) with a mean difference of 5.56 +/- 17.38 microg/L ((AxSYM - EMIT 2000) +/- SD); and for the radioimmunoassay (RIA, CYCLO-Trac SP) (x): y = 0.893 - 6.764 microg/L (r = 0.993, Sy/x = 10.582, n = 71) with a mean difference of 22.18 +/- 14.98 microg/L ((RIA - EMIT 2000) +/- SD) using the Bland-Altman technique.
authors.affiliation: Laboratory for Clinical Investigation, Osaka University Hospital, Suita, Osaka, Japan. skimura@hp-lab.med.osaka-u.ac.jp
authors.forename: S
authors.initials: S
authors.lastname: Kimura
authors.forename: S
authors.initials: S
authors.lastname: Iyama
authors.forename: Y
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Y
authors.initials: Y
authors.lastname: Kanakura
mesh_terms: D002851:Chromatography, High Pressure Liquid; D016572:Cyclosporine / Q000097:blood*; D017248:Enzyme Multiplied Immunoassay Technique / Q000295:instrumentation*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D016014:Linear Models
chemical_list: D007166:Immunosuppressive Agents; D016572:Cyclosporine; D005954:Glucosephosphate Dehydrogenase