Use of multiple sequential injections of equal volumes to determine the apparent binding constant for antibody-antigen complexes by capillary electrophoresis.
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 (Ka) 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 Ka 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.
Department of Analytical Chemistry, University of Córdoba, Campus of Rabanales, Córdoba E-14071, Spain.
Sara
S
Almeda
Eloy
E
Salinas
Cristina
C
Arce
Angela
A
Moreno
Lourdes
L
Arce
Miguel
M
Valcárcel
D000818:Animals; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D000936:Antigen-Antibody Complex; D004285:Dogs; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G; D011485:Protein Binding / Q000276:immunology; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G