OBJECTIVE: We investigated the mechanism by which the ARCHITECT cyclosporine (CsA) chemiluminescent microparticle immunoassay (CMIA) eliminates cross-reactivity to CsA metabolites AM1 and AM9, despite its use of a monoclonal antibody which shows cross-reactivity in fluorescence polarization immunoassays. DESIGN AND METHODS: The CMIA was accomplished by incubating an extracted blood sample with magnetic microparticles coated with a very low amount of anti-CsA antibody. After a wash step the microparticles were incubated with a chemiluminescent CsA tracer, followed by a second wash step and measurement of chemiluminescence. The reagent concentrations of salt and detergent were optimized to maximize CsA binding and minimize metabolite interference. RESULTS: Elimination of CsA metabolite cross-reactivity was shown using purified metabolites and blood samples containing native CsA metabolites. The CMIA demonstrated precision and sensitivity acceptable for use in a clinical setting. CONCLUSION: We conclude that it is possible to eliminate CsA metabolite immuno-cross-reactivity by careful assay design. Copyright (c) 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
OBJECTIVE: We investigated the mechanism by which the ARCHITECT cyclosporine (CsA) chemiluminescent microparticle immunoassay (CMIA) eliminates cross-reactivity to CsA metabolites AM1 and AM9, despite its use of a monoclonal antibody which shows cross-reactivity in fluorescence polarization immunoassays. DESIGN AND METHODS: The CMIA was accomplished by incubating an extracted blood sample with magnetic microparticles coated with a very low amount of anti-CsA antibody. After a wash step the microparticles were incubated with a chemiluminescent CsA tracer, followed by a second wash step and measurement of chemiluminescence. The reagent concentrations of salt and detergent were optimized to maximize CsA binding and minimize metabolite interference. RESULTS: Elimination of CsA metabolite cross-reactivity was shown using purified metabolites and blood samples containing native CsA metabolites. The CMIA demonstrated precision and sensitivity acceptable for use in a clinical setting. CONCLUSION: We conclude that it is possible to eliminate CsA metabolite immuno-cross-reactivity by careful assay design. Copyright (c) 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.