Abstract
Competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was employed to perform the affinity measurement of dissociation constant (Kd) and affinity constants (Ka) for bovine milk lactoferrin (LF) and IgY (immunoglobulin in yolk) specific against LF using antisera from rabbit and hen as references. In liquid phase equilibrium measurements by CI-ELISA, the elevation in antigen level in antigen-antibody mixtures decreased the ELISA values, suggesting the increased competition of free LF in solution with that coated on plate for LF-specific IgY purified by immunoaffinity chromatography (purified IgY). From the Klotz plots of the binding of LF to purified IgY, Kd and Ka were determined to be about 2.6 × 10-8 M and 0.5 × 108 M-1, respectively. The Kd values of 1500-fold diluted crude IgY, diluted sera from hen and rabbit were determined to be very close to that of purified IgY, revealing that CI-ELISA under liquid phase equilibrium by CI-ELISA was appropriate for the affinity measurement of IgY samples.
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