Affinity Measurement of Lactoferrin (LF)-Anti-LF Immunoglobulin in Yolk (IgY) Complexes by Competitive Indirect Enzyme-Linked Immunosorbent Assay (CI-ELISA)

YANN-YING TU¹, CHIA-YU MA², SHYUE-BIN HO³, CHAO-CHENG CHEN^4* AND HUNG-MIN CHANG⁵

1. Department of Health and Nutrition, Chia-Nan University of Pharmacy and Science, Tainan 717, Taiwan.
2. Department of Restaurant Management, Northern Taiwan Institute of Science and Technology, Taipei City 112, Taiwan.
3. Department of Tourism and Hospitality Management, Kainan University, Taoyuan 338, Taiwan.
4. Department of Food and Beverage Management, Leader University, Tainan 709, Taiwan.
5. Graduate Institute of Food Science and Technology, National Taiwan University, Taipei 106, Taiwan. PO Box 23-14, Taipei, Taiwan.

(Received: September 6, 2005; Accepted: June 30, 2006)

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.

Key words: dissociation constant, affinity constant, indirect competitive ELISA, immunoglobulin in yolk, lactoferrin