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.

Utility, promise, and limitations of liquid chromatography‐mass spectrometry‐based therapeutic drug monitoring in precision medicine

“…Automated analyzers were developed using these techniques and became commercially available. 126 , 127 , 128 …”

“…Then immunoassays were converted into high‐throughput assays such as enzyme multiplied immunoassay (EMIA), fluorescence polarization fluoroimmunoassay (FPFIA), and CLIA. Automated analyzers were developed using these techniques and became commercially available 126–128 …”

“…Automated analyzers were developed using these techniques and became commercially available. [126][127][128] Immunoassays are still widely used for TDM (see Figure 2), because they can be automated and are easy to perform, but the quality, purity, selectivity, and specificity of antibodies used are a critical part of the assay development process. 129 Being antibody based, immunoassays can suffer from interferences, lack of sensitivity, and are not readily available for every drug.…”

Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances

“…These kits are used with the Viva automated analyser or with automated analysers from different manufacturers (54). Other homogenous EMIA were developed for measuring the concentration of thyroxine (126), triiodothyronine (127), theophylline (128), and cyclosporine (129) in biological fluids (Table 4).…”