An array with replicates of four cys-terminated peptides was fabricated on thiol-reactive SpotReady® gold substrates; egg yolk extracts from chickens immunized with peptides were then exposed to the array and binding was monitored in real time using GWC Technologies’ SPRimager®II label-free array reader (Figure 1).
The array was first exposed to egg extracts made just seven days after immunization with peptide A (a segment of the PLA4 protein). As no antibodies had yet been generated, no specific binding was observed, but exposing the array to the egg extract served to block most nonspecific sites.
Next, the array was exposed to egg extract from chickens hyperimmunized with peptide B, a different segment of the PLA4 polypeptide. Significant binding of extracts was observed only to peptide B, confirming the presence of antipeptide B antibodies. The low level of binding to the other three peptides is considered nonspecific and was subtracted from the peptide B binding signals to generate corrected curves (Figure 1A inset).
The peptide array was regenerated, then exposed to egg extract made seven days after a third boost with peptide A. Strong antibody binding was observed to both peptide A probes (Figure 1B, A1 and A2 have the Cys-spacer sequence on opposite ends). The observed association rate for binding to peptide A1 was faster than for A2 (3.1 x 10-2 sec-1 vs 2.2 x 10-2 sec-1) as judged by fitting the data to standard models for simple bimolecular interactions.