In this set of experiments, peptides (derived from shiga-toxin), conjugated peptides, and proteins were all spotted on the same array. The array was fabricated on SpotReady substrates with amine-reactive surfaces; binding of sera to the array was monitored in real time by SPRi.
To confirm the integrity of the method, an array of conjugated and unmodified peptides was exposed to purified mAbs in PBS without blocking (Figure 2A). Each of the three mAbs specifically recognized the peptide used as immunogen, as expected. Peptide 149 was arrayed in both pure and ovalbumin-conjugated form, and stronger binding was observed to the conjugated version. This may reflect superior presentation of the antigen to the analyte when the peptide is conjugated to ovalbumin rather than bound directly to the array surface.
An array was prepared with three peptides and a protein, ovalbumin. For serum analysis, the array was first blocked by exposing it to 5% v/v preimmune serum in PBS for 10 minutes on the instrument. A large nonspecific binding signal was observed on all array elements.
In many SPR instruments, strong serum nonspecific binding signals can occupy most of the linear dynamic range, precluding quantitative analysis of subsequent binding events. GWC’s SPRimager II instrument has a broad 40–70° angle adjustment range that enables the detector to be adjusted back into linear range following such strong binding signals.
Following blocking and readjustment of the angle, serum from a mouse immunized with peptide 148 conjugated to ovalbumin showed specific binding to peptide 148, confirming the success of the immunization scheme (Figure 2). The serum also bound to ovalbumin (Figure 2B). Despite the complexity of the serum analyte, the faster association rate for binding to the pure peptide was readily distinguished from the slower rate of binding to the ovalbumin.