Figure 4.

Inoculation of chickens with α-peptides 4a, 4b, ent-4a, or α/β-peptides 5, 6a, 6b, or 6d conjugated to bovine γ-globulin with adjuvant stimulates production of peptide-specific responses (“anti-peptide” IgY antibodies). Association of antibody with immobilized peptide was evaluated only between an antibody and the peptide injected to stimulate production of that antibody. In all panels, the indicated peptides were conjugated to ovalbumin and were immobilized using a 1:100 dilution of the solution produced from the conjugation reaction. (a, b) Indirect ELISA analysis of IgY antibodies induced from injection of conjugated peptides 4a-b, ent-4a, 5 and 6a–d. The x-axis represents the dilution of the egg yolk used. “FCA” data points represent the binding of antibodies from chickens injected only with Freund’s complete adjuvant (FCA) to the indicated immobilized peptide, which serve as negative controls. Panel a displays data generated from a single experiment, carried out separately from the single experiment performed to generate data in panel b. Data points in panels a and b represent mean ± SD, with each condition run in duplicate. (c) Competitive ELISA analysis of peptides in solution (peptide competitor) inhibiting the binding of egg yolk derived-antibodies to immobilized peptides. IC₅₀ values were estimated manually as the concentration of peptide needed to eliminate approximately 50% of the signal (absorption at 405 nm) resulting from complex formation between IgY antibodies and immobilized peptide. All competition ELISA curves are presented in Supporting Information Figure S4.