title: Optimization of capillary electrophoresis conditions for a glucagon competitive immunoassay using response surface methodology.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, 95 Chieftan Way, Dittmer Building, Tallahassee, FL 32306, USA.
authors.forename: Anna R
authors.initials: AR
authors.lastname: Lomasney
authors.forename: Christelle
authors.initials: C
authors.lastname: Guillo
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Sidebottom
authors.forename: Michael G
authors.initials: MG
authors.lastname: Roper
mesh_terms: D002138:Calibration; D019075:Electrophoresis, Capillary / Q000379:methods*; D005934:Glucagon / Q000032:analysis*; D007118:Immunoassay / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013499:Surface Properties
chemical_list: D005934:Glucagon