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

Abstract A new 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 sulfoxide, 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.