Determination of the association constant for slow phosphoserine-anti-phosphoserine interaction kinetics by capillary zone electrophoresis

Electrophoresis
S Lin, S M Hsu

Abstract

Capillary zone electrophoresis (CZE) was used to study the interaction of a monoclonal anti-phosphoserine antibody with its antigen. A model system that allows the determination of the real binding constant in a solution based on the change in peak areas at different concentrations of phosphoserine has been used for studying slow monoclonal antibody-antigen interaction kinetics involving low-molecular-weight antigens. CZE was applied in preincubation experiments. The slow interaction kinetics led to band broadening and resulted in far lower efficiency of the separation of complexed antibody from unbound antibody. However, when the run-to-run reproducibility of free phosphoserine was examined, it was found that it can be recovered quantitatively under electrophoresis conditions. On the basis of measurement of peak areas at different phosphoserine concentrations, the association constant was estimated (Ka = 5.21 X 10[5] M[-1]) and shown to be in close agreement with that obtained by equilibrium dialysis (Ka = 4.65 X 10[5] M[-1]). As long as the antigen participating in the interaction can be detected and recovered quantitatively in the CZE system, the method is generally useful for the study of monoclonal antibody-antigen interac...Continue Reading

References

Aug 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·M F KnauerC G Glabe
May 1, 1991·Journal of Biochemical and Biophysical Methods·H KajiwaraK Oono
Oct 7, 1994·Journal of Chromatography. a·N H Heegaard
Jun 15, 1994·Analytical Chemistry·C A Monnig, R T Kennedy

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Citations

May 4, 2005·Analytical Biochemistry·Shiming Lin, Su-Ming Hsu
May 30, 2006·Electrophoresis·Jesper Ostergaard, Niels H H Heegaard

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