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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.

(Page 3 of 3)

Mouse Serum

Figure 2. Binding of mAbs and peptide-immunized sera to peptide arrays, plotted as reflectivity changes (change%R) over time as monitored by SPRi. (A) Purified mAbs binding to a peptide array: Arrows show time of addition of anti-148 mAb (blue), PBS wash (gray, open arrowheads), anti-149 mAb (red), and anti-162 mAb (green). “C” suffix indicates immobilized peptides conjugated to ovalbumin. Curves show binding of mAbs to peptides 148 (blue), 149 (red), conjugated 149 (pink), and conjugated 162 (green). For the anti-148 and -149 mAbs, antibody was added first at 10 nM then at 100 nM; association rates increased as they should for the higher mAb concentrations. For anti-162 mAb, only 100 nM antibody was added. (B) Sera binding to a peptide array: Arrows show time of addition of nonimmune sera (gray) and serum from a mouse immunized with peptide 148 conjugated to ovalbumin (green). Curves show average binding to replicate negative control peptide 149 spots (red), immunizing peptide 148 (blue), and ovalbumin (pink) on the array. Inset shows array image at the end of the experiment. Bright spots indicate binding has occurred. Methods: Amine-reactive surfaces were prepared by soaking SpotReady 16 substrates overnight in dithiobis[succinimidyl propionate] then rinsing with ethanol and drying before spotting. Peptides and peptide-ovalbumin conjugates were spotted at ~1 mg/mL and incubated for 60 min. Click Image To Enlarge +

Figure 2. Binding of mAbs and peptide-immunized sera to peptide arrays, plotted as reflectivity changes (change%R) over time as monitored by SPRi. (A) Purified mAbs binding to a peptide array: Arrows show time of addition of anti-148 mAb (blue), PBS wash (gray, open arrowheads), anti-149 mAb (red), and anti-162 mAb (green). “C” suffix indicates immobilized peptides conjugated to ovalbumin. Curves show binding of mAbs to peptides 148 (blue), 149 (red), conjugated 149 (pink), and conjugated 162 (green). For the anti-148 and -149 mAbs, antibody was added first at 10 nM then at 100 nM; association rates increased as they should for the higher mAb concentrations. For anti-162 mAb, only 100 nM antibody was added. (B) Sera binding to a peptide array: Arrows show time of addition of nonimmune sera (gray) and serum from a mouse immunized with peptide 148 conjugated to ovalbumin (green). Curves show average binding to replicate negative control peptide 149 spots (red), immunizing peptide 148 (blue), and ovalbumin (pink) on the array. Inset shows array image at the end of the experiment. Bright spots indicate binding has occurred. Methods: Amine-reactive surfaces were prepared by soaking SpotReady 16 substrates overnight in dithiobis[succinimidyl propionate] then rinsing with ethanol and drying before spotting. Peptides and peptide-ovalbumin conjugates were spotted at ~1 mg/mL and incubated for 60 min.

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.

Conclusion

Valuable information on the characteristics of antibodies in complex analytes can be obtained directly using label-free SPRi analysis of peptide arrays. Distinct rates of association of antibodies in serum are readily distinguished, despite the complexity of the analyte.

Elucidation of the distinct binding properties observed for the same peptide immobilized in opposite orientations, and for peptides arrayed in pure versus conjugated form, underscores the value of the SPRi platform for optimizing the fabrication and analysis of arrays. Moreover, both small peptide probes and much larger protein probes may be spotted and analyzed on the same array, a convenience that greatly extends the versatility of SPRi.