**3.4 Efficiency of the regeneration step**

As mentioned above, a potential decrease in the number of binding sites available on the probe could be due to a partially inefficient regeneration process. In these conditions, still bound Ab would lead to a progressive increase in baseline level. To assess this point, we analyzed SPR signal after each regeneration step.

Results presented in Fig. 4 demonstrate that there is no significant increase in the SPR signals measured after each regeneration, attesting that no Ab stays behind the washing step. Therefore, the loss of signal observed for the specific peptide/Ab binding cannot be attributed to incomplete regeneration of the chip.

#### **3.5 Influence of the saturation process**

Probe accessibility is a key parameter to ensure a good interaction with injected ligands. This point is especially important when the probe is a small molecule (peptide). Indeed, the molecules used to saturate the chip surface to avoid non-specific interactions could impact on epitope accessibility, either by masking the binding sites or by interfering with peptide conformation through molecular interactions. Furthermore, the conformation of the peptide can evolve during the experiment, affecting the epitope reactivity. Moreover, an incomplete saturation could lead to a progressive fouling of the surface and, thus, to a gradual loss of available binding sites. To saturate chip surface, we usually use non-immune serum (NIS, 1/25 dilution) which leads to non-specific adsorption of many proteins on chip surface, probably predominantly albumin (MW = 65800 Da) as it represents about 60% of total

Fig. 3. Influence of the grafting process on the evolution of SPR signal (change in reflectivity = R). C131 peptide was immobilized in triplicate on the gold chip surface via either pyrrole electropolymerization or diazonium electrodeposition. Successive injections of NIS (1/50) with periodical injections of anti-C131 serum (1/200) were performed, followed by HCl-

As mentioned above, a potential decrease in the number of binding sites available on the probe could be due to a partially inefficient regeneration process. In these conditions, still bound Ab would lead to a progressive increase in baseline level. To assess this point, we

Results presented in Fig. 4 demonstrate that there is no significant increase in the SPR signals measured after each regeneration, attesting that no Ab stays behind the washing step. Therefore, the loss of signal observed for the specific peptide/Ab binding cannot be

Probe accessibility is a key parameter to ensure a good interaction with injected ligands. This point is especially important when the probe is a small molecule (peptide). Indeed, the molecules used to saturate the chip surface to avoid non-specific interactions could impact on epitope accessibility, either by masking the binding sites or by interfering with peptide conformation through molecular interactions. Furthermore, the conformation of the peptide can evolve during the experiment, affecting the epitope reactivity. Moreover, an incomplete saturation could lead to a progressive fouling of the surface and, thus, to a gradual loss of available binding sites. To saturate chip surface, we usually use non-immune serum (NIS, 1/25 dilution) which leads to non-specific adsorption of many proteins on chip surface, probably predominantly albumin (MW = 65800 Da) as it represents about 60% of total

Glycine regeneration.

**3.4 Efficiency of the regeneration step** 

analyzed SPR signal after each regeneration step.

attributed to incomplete regeneration of the chip.

**3.5 Influence of the saturation process** 

Fig. 4. Efficiency of the regeneration process. C131 peptide was immobilized in triplicate on the gold chip surface via pyrrole electropolymerization. Successive injections of NIS (1/50) with periodical injections of anti-C131 serum (1/200) were performed, followed by HCl-Glycine regeneration. SPR signal was quantified after anti-C131 injection and after the regeneration step.

Fig. 5. Influence of the saturation process and of the peptide sequence on SPR signal loss. C131, C20 and Ova75 peptides were immobilized in triplicate on the gold chip surface via pyrrole electropolymerization. Saturation of the chip surface was ensured using either NIS or PLL-PEG or PVP. Successive injections of anti-C131, anti-C20 and anti-Ova75 serums (1/50) were performed, followed by HCl-Glycine regeneration. SPR signal loss after 74 injections.

Stability of Peptide in Microarrays: A Challenge for High-Throughput Screening 207

Altogether, these results indicate that the saturation process impacts on the amplitude of the signal loss while the shape of the signal decay curve is more likely dependant on the peptide. The impact of the saturation process may be related to the physico-chemical properties (hydrophibicity, charge, etc) of both the saturating molecules and the peptides.

Until now, our analyses were always performed using the same Ab for a given Ag, which did not allow checking the influence of the peptide from that of the Ab. In the next experiment, we compare the signal obtained on C131 and C20 spots after injections of anti-C131 and anti-C20 serums. In each case (anti-C131 and anti-C20) two different serums issued from the same rabbit, but collected at two different days (D=39 and D=66) were

Fig. 7. Influence of the ligand on SPR signal loss. C131 and C20 peptides were immobilized in triplicate on the gold chip surface via pyrrole electropolymerization. Saturation of the chip surface was ensured using NIS. Successive injections of anti-C20 and anti-C131 serums (1/50) were performed, followed by HCl-Glycine regeneration and signal loss after 74 injections was quantified. In each case (anti-C131 and anti-C20) two different serums issued from the same rabbit, but collected at two different days (D=39 and D=66) were injected.

These results could be related to a difference in the affinity of the ligands for the probe. Indeed, it is well known that Ab affinity usually increases gradually during the immune response, which is referred to affinity maturation process (Berek & Ziegner, 1993). Thus, it is likely that D66 sample contains anti-C20 Ab with higher affinity for C20 than D39. But we cannot exclude that, despite the small size of the antigen (20aa), the epitopes recognized by D66 Ab differ from those recognized by D39 Ab, which would impact on the overall

characteristics of peptide/Ab interaction.

tested. As observed in Fig.7, the second sample led to a more stable signal.

**3.6 Influence of the ligand** 

serum proteins. To determine whether the saturation process is involved in the loss of chip efficiency, we performed the same type of experiments with two anti-fouling molecules: Poly (L-Lysine)-PolyEthyleneGlycol (PLL-PEG) and Polyvinylpyrrolidone (PVP) instead of NIS (0.5mg/mL and 1% p/v respectively). Signal evolution was analyzed in each case. As shown in Fig. 5, signal loss on C131 spot is similar, whatever the saturation process used. As interactions between peptide and anti-fouling molecules could also depend on the physico-chemical characteristics of the peptide, we wondered whether signal loss depends on the grafted probe. Two others peptides (C20 and Ova75) were immobilized on the chip and their corresponding rabbit anti-serums were injected in the same conditions than anti-C131.

As shown in Fig. 5, signal loss depends both upon saturation process and grafted peptide sequence. However, the general shape of the signal decay curve seems to be related with the peptide (Fig. 6).

Fig. 6. Evolution of the SPR signal along the experiment. C131, C20 and Ova75 peptides were immobilized in triplicate on the gold chip surface via pyrrole electropolymerization. Saturation of the chip surface was ensured using either NIS or PLL-PEG or PVP. Successive injections of anti-C131, anti-C20 and anti-Ova75 serums (1/50) were performed, followed by HCl-Glycine regeneration. Remaining SPR signal obtained A) on the different spots upon anti-C131, anti-C20 and anti-Ova injections on a chip saturated with PLL-PEG B) on C131 spots after anti-C131 injection on chip saturated with NIS or PLL-PEG or PVP C) on C20 spots after anti-C20 injection on chip saturated with NIS or PLL-PEG or PVP D) on Ova75 spots after anti-Ova injection on chip saturated with NIS or PLL-PEG or PVP.

Altogether, these results indicate that the saturation process impacts on the amplitude of the signal loss while the shape of the signal decay curve is more likely dependant on the peptide. The impact of the saturation process may be related to the physico-chemical properties (hydrophibicity, charge, etc) of both the saturating molecules and the peptides.
