Use of multiple sequential injections of equal volumes to determine the apparent binding constant for antibody-antigen complexes by capillary electrophoresis

Anew modified version of the well-known flow-through partial-filling technique [viz. multiple sequential injection of equal volumes (MSI-EV) of neutral marker, antigen (Ag) and antibody (Ab)] was used to calculate the apparent binding constant (K-a) of monoclonal Ab (mAb) and polyclonal Ab (pAb) to their specific antigens (Ags). Such a constant is very important in immunoassays. The procedure involves the sequential injection of small, identical volumes of a neutral marker (dimethyl sulfoxicle, DMSO), an Ag and an Ab into a capillary column for electrophoresing. The apparent K-a values thus obtained from a Scatchard plot were 0.76 +/- 0.15 mg(-1) mL for the complex of anti-canine Immunoglobulin G (IgG) as mAb and canine IgG as Ag, and 0.79 +/- 0.14 mg(-1) mL for that between anti-human IgG as pAb and human IgG as Ag. These values are of the same order to those provided by indirect competition enzyme-linked immunosorbent assay (ELISA) (viz. 0.42 +/- 0.28 mg(-1) mL for the mAb-Ag complex and 0.81 +/- 0.09 mg(-1) mL for the pAb-Ag complex). The high sensitivity of the MSI-EV-CE technique affords the detection of very low concentrations of Ab. (C) 2009 Elsevier B.V. All rights reserved.