title: A rational route to the development of a competitive capillary electrophoresis immunoassay: assessment of the variables affecting the performances of a competitive capillary electrophoresis immunoassay for human serum albumin.
abstract: Affinity capillary electrophoresis is a powerful analytical tool to extract quantitative information about the binding properties of different interacting systems. The use of LIF detection makes the technique suitable for screening strong binding interactions. The non-equilibrium electrophoretic separations of pre-equilibrated mixtures of ligand and receptor are generally used for such strong molecular interactions allowing the assessment of capillary electrophoresis immunoassays, mostly in competitive formats. As the analytical performances of the assay strongly depend on the preservation of the binding properties during the separation, a rational route to assay development has to be followed to get the best conditions. The paper describes the steps followed to set-up a competitive immunoassay for human serum albumin (HSA) by using a labeled protein (HSA-FITC) and an anti-HSA polyclonal antiserum. A labeling degree of around 1 of the HSA-FITC conjugates is needed to get narrow electrophoretic peak while the titration curve is used to define the optimal antiserum dilution. An antiserum-labeled protein affinity constant of 1.34×10(7) M(-1) was measures in the selected separation conditions. Furthermore, in order to maximize the assay competition between the labeled and unlabelled HSA a short pre-incubation step of the antiserum with the unlabelled HSA (the analyte) was introduced to promote a sharp increase in assay sensitivity.
authors.affiliation: Laboratory of Bioanalytical Chemistry, Department of Chemistry, University of Torino, via Giuria 5, 10125 Torino, Italy. cristina.giovannoli@unito.it
authors.forename: Cristina
authors.initials: C
authors.lastname: Giovannoli
authors.forename: Claudio
authors.initials: C
authors.lastname: Baggiani
authors.forename: Cinzia
authors.initials: C
authors.lastname: Passini
authors.forename: Flavia
authors.initials: F
authors.lastname: Biagioli
authors.forename: Laura
authors.initials: L
authors.lastname: Anfossi
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Giraudi
mesh_terms: D001667:Binding, Competitive; D002138:Calibration; D019075:Electrophoresis, Capillary / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D007106:Immune Sera / Q000737:chemistry / Q000276:immunology; D007118:Immunoassay / Q000379:methods*; D008024:Ligands; D011485:Protein Binding; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000032:analysis*
chemical_list: D005456:Fluorescent Dyes; D007106:Immune Sera; D008024:Ligands; D012709:Serum Albumin; D016650:Fluorescein-5-isothiocyanate