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Label-Free SPRi Analysis of Peptide Arrays

Advanced Platform Finds Applications in Characterizing Antibodies in Complex Samples

• Timothy G. Burland, Ph.D.
• ,
• Voula Kodoyianni, Ph.D.

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Egg Extracts

Figure 1. Binding of egg extracts to peptide arrays, plotted as reflectivity changes (change%R) over time as monitored by SPRi: Peptide arrays were exposed to egg extract from a chicken immunized with peptide B (A) and peptide A (B). Arrows show time of addition of egg extract or PBS. Curves show average binding signals to replicate peptide spots for A1 and A2 (red and pink), D (blue), and B (green). Insets show net binding to immunizing peptides after subtracting average of signals for controls. Methods: Thiol-reactive surfaces were prepared on SpotReady®16 gold chips by overnight incubation in 1 mM amino octane thiol in absolute ethanol followed by activation in 1 mM succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate in PBS pH 7.4. Cysteine-terminated peptides were spotted at 1–4 mg/mL for 60 min. Peptide A1 is from PLA4 (Cys on N-terminus); Peptide A2, same as A1 except Cys on C-terminus; Peptide B is from PLA4; Peptide D is a control from TLR4. For exposure to the array, egg yolks were extracted in acidified PBS (pH 3) and then diluted 1:4,000 with PBS pH 7.4. After exposure to antipeptide B extract, the array was regenerated by washing in 0.1 M glycine pH 2.6, then rinsing with PBS. Click Image To Enlarge +

Figure 1. Binding of egg extracts to peptide arrays, plotted as reflectivity changes (change%R) over time as monitored by SPRi: Peptide arrays were exposed to egg extract from a chicken immunized with peptide B (A) and peptide A (B). Arrows show time of addition of egg extract or PBS. Curves show average binding signals to replicate peptide spots for A1 and A2 (red and pink), D (blue), and B (green). Insets show net binding to immunizing peptides after subtracting average of signals for controls. Methods: Thiol-reactive surfaces were prepared on SpotReady®16 gold chips by overnight incubation in 1 mM amino octane thiol in absolute ethanol followed by activation in 1 mM succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate in PBS pH 7.4. Cysteine-terminated peptides were spotted at 1–4 mg/mL for 60 min. Peptide A1 is from PLA4 (Cys on N-terminus); Peptide A2, same as A1 except Cys on C-terminus; Peptide B is from PLA4; Peptide D is a control from TLR4. For exposure to the array, egg yolks were extracted in acidified PBS (pH 3) and then diluted 1:4,000 with PBS pH 7.4. After exposure to antipeptide B extract, the array was regenerated by washing in 0.1 M glycine pH 2.6, then rinsing with PBS.

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.