Optimization of capillary electrophoresis conditions for a glucagon competitive immunoassay using response surface methodology |
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Authors: | Anna R Lomasney Christelle Guillo Ashley M Sidebottom and Michael G Roper |
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Institution: | (1) Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Dittmer Building, Tallahassee, FL 32306, USA |
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Abstract: | The capillary electrophoresis (CE) conditions for a competitive immunoassay of glucagon were optimized for highest sensitivity
of the immunoassay and resolution of the electrophoretic peaks using a Box–Behnken design. Injection time, voltage ramp time,
and separation voltage were varied between three levels and two responses, bound-to-free (B/F) ratio of the immunoassay peaks and resolution between the peaks, were measured. Analysis of variance was applied to fit
a predictive model, and a desirability function was used to simultaneously optimize both responses. A 10-s injection, 1.6-min
ramp time, and a 22-kV separation voltage were the conditions found when high B/F was given more emphasis than high resolution. To test the model, calibration curves of a glucagon immunoassay were measured
at the optimum and least optimum CE conditions. Optimal conditions increased the sensitivity of the immunoassay by 388% compared
to the least optimum conditions while maintaining adequate resolution.
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Keywords: | CE Response surface methodology Optimization Glucagon Competitive immunoassay |
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