Use of capillary electrophoresis-based competitive immunoassay for a large molecule

A systematic study on the optimization of capillary electrophoresis-based immunoassay (CEIA) was performed using bovine serum albumin (BSA) and monoclonal anti-BSA. The immunocomplex could not be resolved from free BSA or anti-BSA with UV detection. When fluorescein isothiocyanate-labeled BSA (FITC-BSA) was used as tracer, the free and bound FITC-BSA were well separated giving definite peaks with laser induced fluorescence detection. The factors affecting the separation of the free and bound FITC-BSA, including voltage, pH and ionic strength of the running buffer, were systematically analyzed. Competitive CEIAs were demonstrated in uncoated and coated capillaries with whole or Fab fragment of the antibody. The coefficient of variation for the quantification of BSA in coated capillary was less than that in uncoated capillary. This study demonstrated that competitive CEIA could be applied to quantify high-molecular-mass protein in biological fluids.