**3.6 Influence of the ligand**

206 Electropolymerization

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

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

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.

the same conditions than anti-C131.

peptide (Fig. 6).

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

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.

Stability of Peptide in Microarrays: A Challenge for High-Throughput Screening 209

**3.7 Is there a change in the affinity of the ligand (Ab) for the probe (peptide) between** 

Affinity is a key parameter in Ab-Ag interaction which could impact on the signal observed upon Ab binding. Actually, Ab binding on the grafted peptides was quantified by measuring the change in reflectivity obtained after washing, a step during which ligands can dissociate, accordingly to their affinity for the probes. Moreover, it is well known that Ag conformation influences Ab binding (Fieser et al., 1987). Thus, a modification of peptide conformation during the experiment could lead to a change in the Ag-Ab affinity. As SPRi technique allows label-free and real-time detection of biomolecular interactions, it gives access to affinity parameters. But, as ligands in our experiments consisted in polyclonal antibodies, the dissociation curves obtained during the washing step after serum injection correspond to a mean value resulting from all the individual dissociation constants of the various Ab. In this case, it is not possible to determine classical affinity parameters, but the slope of the dissociation curve is representative of the overall affinity. In order to analyze the evolution of peptide/Ab affinity during an experiment, the slopes of the dissociation

As shown in Fig. 8B, the slope of the curve corresponding to the dissociation of Ab-peptide complexes decreased during the experiment. Thus the diminution of the quantity of Ab bound to the peptides (signal loss) is associated with an increase in the affinity of the

These results suggest that successive injection/regeneration cycles lead to a slight conformational change in the probe. At the beginning of the experiment, peptide conformation is suitable for the binding of different Ab with a large range of affinity. As the experiment progress, Ab having lower affinity for the peptide can no longer bind, due to

It is well known that the stability of protein is enhanced by various molecules among which polyols (Lee & Kim, 2002, Vagenende et al., 2009). In the aim of reducing conformational change in the grafted probes, glycerol (0.1%) was added to both the running buffer and the regeneration solution. As shown in Fig. 9, the presence of glycerol in the running solutions improved the signal stability in the case of Ova75, but not in the case of C131. It seems that

As injected samples are complex biological mediums (serums), we wondered if some peptide degradation could occur, due to protease activities. So, we performed the same experiments after addition of protease inhibitors in the samples (cocktail set VII, CalbBiochem, 1/100). We did not observe any improvement of chip stability (Fig. 9), suggesting that probes proteolysis was not responsible for the loss of signal during the

Various strategies can be implemented to improve the quality of the data resulting from samples screening on a peptide chip. First, addition of glycerol in the solutions can improve peptide stability, depending on the peptide (§3.8). Second, we observed that signal loss was usually biphasic, with a rapid drop during the first injections followed by a slighter decrease

curves were calculated using the last 6 min of washing (Fig. 8A).

interaction, whatever the probe/ligand pair used or the saturation process.

the protective effect of glycerol occurs only on the less stable peptides.

**the first and the last injection?** 

modification in peptide conformation.

experiments.

**3.8 Effect of glycerol and protease inhibitors** 

**3.9 Improvement possibilities of the system** 

Fig. 8. Ab-peptide binding affinity. 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 PVP or PLL-PEG. Successive injections of anti-C131, anti-C20 and anti-Ova serums (1/50) were performed, followed by HCl-Glycine regeneration. A) Sensorgramms obtained on C20 spots after the third and the 78th injection of anti-C20 serum (PLL-PEG saturation). Insert: detail of the dissociation curves. B) Quantification of antibody dissociation for the different peptides, from the slope of the dissociation curves.

Fig. 8. Ab-peptide binding affinity. 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 PVP or PLL-PEG. Successive injections of anti-C131,

regeneration. A) Sensorgramms obtained on C20 spots after the third and the 78th injection of anti-C20 serum (PLL-PEG saturation). Insert: detail of the dissociation curves. B) Quantification of antibody dissociation for the different peptides, from the slope of the

anti-C20 and anti-Ova serums (1/50) were performed, followed by HCl-Glycine

dissociation curves.

### **3.7 Is there a change in the affinity of the ligand (Ab) for the probe (peptide) between the first and the last injection?**

Affinity is a key parameter in Ab-Ag interaction which could impact on the signal observed upon Ab binding. Actually, Ab binding on the grafted peptides was quantified by measuring the change in reflectivity obtained after washing, a step during which ligands can dissociate, accordingly to their affinity for the probes. Moreover, it is well known that Ag conformation influences Ab binding (Fieser et al., 1987). Thus, a modification of peptide conformation during the experiment could lead to a change in the Ag-Ab affinity. As SPRi technique allows label-free and real-time detection of biomolecular interactions, it gives access to affinity parameters. But, as ligands in our experiments consisted in polyclonal antibodies, the dissociation curves obtained during the washing step after serum injection correspond to a mean value resulting from all the individual dissociation constants of the various Ab. In this case, it is not possible to determine classical affinity parameters, but the slope of the dissociation curve is representative of the overall affinity. In order to analyze the evolution of peptide/Ab affinity during an experiment, the slopes of the dissociation curves were calculated using the last 6 min of washing (Fig. 8A).

As shown in Fig. 8B, the slope of the curve corresponding to the dissociation of Ab-peptide complexes decreased during the experiment. Thus the diminution of the quantity of Ab bound to the peptides (signal loss) is associated with an increase in the affinity of the interaction, whatever the probe/ligand pair used or the saturation process.

These results suggest that successive injection/regeneration cycles lead to a slight conformational change in the probe. At the beginning of the experiment, peptide conformation is suitable for the binding of different Ab with a large range of affinity. As the experiment progress, Ab having lower affinity for the peptide can no longer bind, due to modification in peptide conformation.

### **3.8 Effect of glycerol and protease inhibitors**

It is well known that the stability of protein is enhanced by various molecules among which polyols (Lee & Kim, 2002, Vagenende et al., 2009). In the aim of reducing conformational change in the grafted probes, glycerol (0.1%) was added to both the running buffer and the regeneration solution. As shown in Fig. 9, the presence of glycerol in the running solutions improved the signal stability in the case of Ova75, but not in the case of C131. It seems that the protective effect of glycerol occurs only on the less stable peptides.

As injected samples are complex biological mediums (serums), we wondered if some peptide degradation could occur, due to protease activities. So, we performed the same experiments after addition of protease inhibitors in the samples (cocktail set VII, CalbBiochem, 1/100). We did not observe any improvement of chip stability (Fig. 9), suggesting that probes proteolysis was not responsible for the loss of signal during the experiments.

#### **3.9 Improvement possibilities of the system**

Various strategies can be implemented to improve the quality of the data resulting from samples screening on a peptide chip. First, addition of glycerol in the solutions can improve peptide stability, depending on the peptide (§3.8). Second, we observed that signal loss was usually biphasic, with a rapid drop during the first injections followed by a slighter decrease

Stability of Peptide in Microarrays: A Challenge for High-Throughput Screening 211

Fig. 10. SPR signal correction: 14 peptides were immobilized in triplicate on gold chips surface via pyrrole electropolymerization. Saturation of the chip surface was ensured using NIS and glycerol was added to running solutions. Successive injections of serums from HDV infected or healthy donors (1/50) were performed, followed by HCl-Glycine regeneration. Rabbit anti-C131 serum was periodically injected. A) Remaining signal obtained on C131 spot upon anti-C131 serum injections. B) Standard deviation of the SPR signal obtained before and after signal correction, for three independent sets of injections.

upon the subsequent injections. Thus we suggest performing 10 – 12 blank injections/regeneration cycles before sample analysis to limit the conformational change between the first and the last sample of interest. Finally, we recommend to include a spot with a control peptide on the chip and to perform periodical injections of a control sample. The reduction of signal measured on this control spot can be modelled using a polynomial curve (Fig. 10A), which can be used to determine a correction factor for the others injections.

Fig. 9. Effect of glycerol and protease inhibitors on SPR signal loss. C131 and Ova75 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-C131 and anti-Ova75 serums (1/50) were performed, followed by HCl-Glycine regeneration and signal loss after 76 injections was quantified. + Gly: presence of glycerol 0.1% in the running buffer and regeneration solution. + Inh: addition of protease inhibitors in the serum samples.

upon the subsequent injections. Thus we suggest performing 10 – 12 blank injections/regeneration cycles before sample analysis to limit the conformational change between the first and the last sample of interest. Finally, we recommend to include a spot with a control peptide on the chip and to perform periodical injections of a control sample. The reduction of signal measured on this control spot can be modelled using a polynomial curve (Fig. 10A), which can be used to determine a correction factor for the

Fig. 9. Effect of glycerol and protease inhibitors on SPR signal loss. C131 and Ova75 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-C131 and anti-Ova75 serums (1/50) were performed, followed by HCl-Glycine regeneration and signal loss after 76 injections was quantified. + Gly: presence of glycerol 0.1% in the running

buffer and regeneration solution. + Inh: addition of protease inhibitors in the serum

others injections.

samples.

Fig. 10. SPR signal correction: 14 peptides were immobilized in triplicate on gold chips surface via pyrrole electropolymerization. Saturation of the chip surface was ensured using NIS and glycerol was added to running solutions. Successive injections of serums from HDV infected or healthy donors (1/50) were performed, followed by HCl-Glycine regeneration. Rabbit anti-C131 serum was periodically injected. A) Remaining signal obtained on C131 spot upon anti-C131 serum injections. B) Standard deviation of the SPR signal obtained before and after signal correction, for three independent sets of injections.

Stability of Peptide in Microarrays: A Challenge for High-Throughput Screening 213

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To validate this correction procedure, we realised the following experiments: 14 peptides (11 from HDV, 1 from HEL, 1 from Ova and the control peptide C131) were grafted in triplicates on 2 chips. A set of 17 serums (14 from HDV infected patients and 3 from healthy donors) was injected twice on the first chip and once on the second chip. Serums were in a different random order within each set. Rabbit anti-C131 serum was periodically injected (at least every 12 injections) to establish a control curve for signal decrease. The sum of the SPR signals obtained for each peptide was calculated for each injection set, before and after application of the correction, as well as the standard deviation (Fig. 10B). Indeed, as signal loss depends on the peptide/ligand pair, this correction is not optimal, but nevertheless improves the results.
