Optimization of capillary electrophoresis conditions for a glucagon competitive immunoassay using response surface methodology
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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.
KeywordsCE Response surface methodology Optimization Glucagon Competitive immunoassay
This work was funded by grants from the National Institutes of Health R01 DK080714 and the American Heart Association Greater Southeast Affiliate.
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