Production of avian immunoglobulin Y (IgY) against the linear epitope E2EP3 of chikungunya virus

Abstract

Utilization of Immunoglobulin Y (IgY), instead of mammalian antibodies, in diagnostics has become increasingly popular due to non-invasiveness, non-reactiveness to rheumatoid factors and human anti-mammalian antibody, simple isolation process, and large yield. This study produced Immunoglobulin Y antibody against the synthetic linear epitope E2EP3 of Chikungunya virus and evaluated its specificity against the antigen. Twenty-eight-week-old White Leghorn hens were immunized with E2EP3 synthetic peptide by intramuscular injection followed by two boosters. Eggs were collected 1 week before and 13 weeks after primary immunization. IgY was isolated from egg yolks by delipidation and sodium sulfate precipitation and then pooled on a weekly basis. Pooled IgY isolates were characterized by native and Sodium Dodecyl Sulfate (SDS) - Polyacrylamide Gel Electrophoresis (PAGE) for presence and purity and by Radial Immunodiffusion Assay (RIA) and indirect ELISA for binding activity. E2EP3-specific IgY was purified by affinity chromatography and its binding affinity and specificity were determined by indirect ELISA. Native PAGE confirmed that the molecular weight of the isolated IgY was 165 kDa, while SDS-PAGE showed the presence of the heavy (60 kDa ) and light (28 kDa) chains of IgY. In RIA, precipitin lines were observed between the antigen (E2EP3) and pooled IgY isolates from 4 – 11 weeks after primary immunization, which indicate antigen-IgY binding. In a series of E2EP3 antigen dilution, indirect ELISA showed that the putative E2EP3-specific IgY was sensitive to the E2EP3 antigen solution at 10 μg/ml. The putative E2EP3-specific IgY from 4 – 13 weeks after primary immunization did not exhibit cross-reactivity against the Dengue virus specific DEN-1 synthetic peptide. Therefore, the Chikungunya virus E2EP3-specific IgY obtained in this study, with no cross-reactivity against a Dengue virus synthetic peptide, can be further developed into a cheap and simple diagnostic tool for Chikungunya infection at onset.