Preparation of artificial antigen and egg yolk-derived immunoglobulin (IgY) of florfenicol amine for ELISA assay

Abstract

To investigate the feasibility of IgY technology for the immune detection of haptens, a specific egg yolk antibody (IgY) has been developed in order to detect the florfenicol amine (FFA) residues. FFA was conjugated to bovine serum albumin (BSA) by glutaraldehyde (GA) and the conjugate was used to immunize laying chickens. Anti-FFA IgY antibody was purified by PEG-6000 precipitation and identified by SDS-PAGE. The titer of anti-FFA IgY antibody reached a peak of 1:128,000 after three booster injections. Checkerboard titration showed that a 1:800 dilution of anti-FFA IgY could give an optical density (OD) at around 1.0 at 10 μg/mL FFA-OVA coating concentration. An indirect competitive enzyme-linked immunosorbent assay (icELISA) using specific anti-FFA IgY showed that the IC₅₀ value of anti-FFA IgY was 12.30 ng/mL and the regression curve equation was y = −13.71x + 64.95 (R ² = 0.945). The strategy of developing anti-hapten IgY antibody is that it may be further used as a new reagent for an immunoassay of hapten residues.

Keywords:

• ELISA
• florfenicol amine (FFA)
• hapten detection
• yolk immunoglobulin (IgY)

Acknowledgments

This work was supported by the International Cooperation Project of Shaanxi Province Science and Technology Program in 2012 (2012KW-20), the Ministry of Education Scientific Research Foundation for Returned Overseas Experts (K308021101), and the Ministry of Education and State Administration of Foreign Experts Affairs “Overseas Teacher” project (MS2011XBNL057), China.