Clinically Related Protein–Peptide Interactions Monitored in Real Time on Novel Peptide Chips by Surface Plasmon Resonance Imaging

materials and reagents

Prisms bearing a gold surface were prepared as described previously (17) and provided by Genoptics (Orsay, France). Activated pyrrole was obtained according to the protocol described by Livache et al. (22). One peptide derived from ovalbumin (Ova 273–288) and 11 peptides from hepatitis C virus (HCV) structural proteins (core, C 1–21, C 20–40, and C 131–150; envelope 1, E1 311–330; envelope 2, E2 528–546) and nonstructural proteins (NS2, 958–977; NS3, NS3 1248–1265; NS4, NS4/1 1688–1708; NS4/2 1921–1940; and NS5, NS5/1 2294–2313 and NS5/2 2944–2959) were synthesized with a maleimide-modified NH₂ terminus by Neosystem (Strasbourg, France). Full sequences can be found on the ExPASy server (http://ca.expasy.org/) under the accession number P26664 (accessed June 2005). Peroxidase-conjugated goat anti-human IgG was purchased from Jackson ImmunoResearch Laboratories, bovine serum albumin (BSA) was from Sigma, and succinimidyl acetyl thiopropionate was from Pierce. The SPR imager was from Genoptics. Rabbit immune sera against the C 20–40 and C 131–150 peptides were prepared by Neosystem. Human sera from healthy donors (n = 5) and HCV-infected patients (n = 10) were kindly provided by the Etablissement Français du Sang (Grenoble, France) and by the Département d’Hépato-Gastroentérologie (Centre Hospitalo Universitaire, Grenoble, France), respectively. Sera were stored at −20 °C.

preparation of pyrrole–peptide conjugates

Peptides (2 mmol/L) were conjugated with SH-activated pyrrole at a 1:10 molecular ratio in phosphate-buffered saline (PBS; 137 mmol/L NaCl, 3 mmol/L KCl, 1.5 mmol/L KH₂PO₄, 8 mmol/L Na₂HPO₄, pH 7.2) for 2 h at room temperature. At the end of the process, ∼20% of the input peptides were conjugated to pyrrole, as estimated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Because it is still difficult to separate peptide–pyrrole conjugates from the free peptide, this reaction mixture was used for electrochemical polymerization without further purification.

polymerization of pyrrole–peptide conjugates on a gold layer

Briefly, the electrochemical polymerization (electrospotting) process was carried out in sodium phosphate buffer (50 mmol/L; pH 6.8) containing 20 mmol/L free pyrrole (from a 1 mol/L solution in acetonitrile), 0.2 mmol/L pyrrolated peptide, and 100 mL/L glycerol. The chip was a prism (Genoptics) coated with a 50-nm-thick layer of gold. For electrochemical polymerization, an electrical pulse (2.4 V for 200 ms) was generated between the working electrode (gold layer) and the counter electrode (located in the tip filled with the pyrrole/pyrrole conjugate mixture) (17). The distance of the counter electrode from the gold surface was 4 mm, and the tip was rinsed with water between each electrochemical polymerization. Successive addressing of the tip filled with different peptides was carried out by an x/y/z robot from Microcontrole. After all peptide spots were synthesized, the slides were rinsed with PBS and stored at 4 °C.

antibody binding assay

All reactions were carried out in PBS at room temperature in a 10-μL Teflon cell connected to a peristaltic pump. The flow rate of solutions within the cell was 35 or 47 μL/min. The chip was saturated with PBS containing 10 g/L BSA, after which serum was injected and the sensor surface was rinsed with PBS to remove unbound molecules. The chip was then regenerated with 0.1 mol/L glycine (pH 2) for 1 min and washed with PBS before a new injection. Competition assays were carried out in 2 steps: in the first step, diluted immune serum (1:200) was injected into the Teflon cell and the chip was washed with PBS for 5 min; in the second step, relevant (competition) or irrelevant (control) free peptide (8, 15, and 24 μmol/L) diluted in PBS was injected.

SPRi interaction monitoring

The optical setup has been detailed elsewhere (17). Briefly, light was shone on the reverse side of the chip, which was constructed of a high optical index glass prism coated with thin layers of chromium and gold (5 and 45 nm, respectively). Changes in light reflectivity attributable to antibodies interacting with the immobilized peptides were recorded by a 12-bit charge-coupled device (CCD) camera. Sequential images were recorded at 1.5-s intervals, and binding kinetics were monitored by dedicated software (Genoptics).

preparation of bsa–peptide conjugates for elisa control

BSA was activated by use of succinimidyl acetyl thiopropionate according to the manufacturer’s recommendations and was further purified by gel filtration through Sephadex G50-fine columns (Amersham Biosciences). Maleimide-modified peptides were mixed with SH-activated BSA (10:1 molar ratio) in PBS for 2 h at room temperature and stored at −20 °C.

elisa

Plates were coated with BSA–peptide conjugates (equivalent to 0.5 mmol/L BSA). The ELISA was performed as described by Villiers et al. (23), and bound antibodies were revealed with peroxidase-conjugated goat anti-human IgG. Results represent the absorbance measured for a 1:1000 serum dilution.

peptide chip preparation and SPRi analysis

To prepare the peptide chips, we conjugated peptides N-terminally modified with maleimide to thiol-activated pyrrole and immobilized the conjugate on a gold surface, as described in the Materials and Methods. A spacer was inserted between the pyrrole moiety and the peptide to favor epitope accessibility to the antibody (Fig. 1A⇓ ). The electrochemically directed polymerization of pyrrole and peptide–pyrrole conjugates, initially developed for DNA arrays (17), scatters a polypyrrole film on the surface of the working electrode. Different peptides were immobilized on spatially defined areas of the gold surface of the chip through successive polymerizations with different peptides (Fig. 1A⇓ ). The resulting chip hosted 40 different spots on a 0.5-cm² area. The typical diameter of a spot ranged between 300 and 500 μm (internal diameter of the tip used for the electrochemical polymerization). The electrochemical charge needed for electrocopolymerization for all spots was recorded to ensure the reproducibility of the process. For SPRi measurements, the functionalized gold layer was deposited on a prism; a polarized light illuminated the whole surface, and the beam reflected by each point was analyzed individually, allowing simultaneous monitoring of each peptide spot (Fig. 1B⇓ ). A CCD camera converts the changes in reflectivity into gray-level variations that are directly related to the mass(es) of the compound(s) bound to the surface (see the movie in the Data Supplement that accompanies this article at http://www.clinchem.org/content/vol52/issue2/ ).

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Figure 1.

General scheme of the experimental setup.

Peptide chip preparation and SPRi device. (A), electrocopolymerization of pyrrole and pyrrole–peptide conjugates is carried out on a prism surface coated with a thin gold layer. M, maleimide. (B), the sensor chip is illuminated by a p-polarized LED beam, and the reflected light is recorded by a CCD camera. As shown in the sensorgrams (C), the CCD camera monitors the whole sensor chip surface, allowing simultaneous analysis of binding events on all spots (26 in this case). All curves show a typical profile with an association phase during sample injection, followed by a dissociation phase on washing. Regeneration of the chip allows for further sample measurements.

The example presented in Fig. 1C⇑ shows typical curves (sensorgrams) obtained when the reflectivity of each of the 26 spots analyzed simultaneously on this chip is plotted against time: injection of the ligand induces an initial increase in reflectivity; nonspecific interactions are then removed by washing, and the chip surface is regenerated with a glycine-based solution. Thus, the initial signal is recovered.

specificity of antibody binding to peptide chips and SPRi detection

Three peptides (C 20–40, C 131–150, and Ova 273–288) were spotted on the surface of a chip, and different rabbit immune sera were successively injected. The chip surface was regenerated after each assay. As shown in Fig. 2A⇓ , injection of an anti-C 20–40 immune serum led to an initial increase (association) and then to the stabilization (equilibrium) of the reflectivity signal corresponding to the spot bearing the C 20–40 peptide, whereas low signals were observed with the irrelevant C 131–150 and Ova 273–288 peptides as well as with the negative control (polypyrrole alone). The difference at the plateau (ΔR) between the sensorgrams generated by the positive assay and the control (irrelevant peptide) corresponds to the specific signal attributable to antibody capture. In contrast, injection of nonimmune serum under the same conditions led to a low and similar reflectivity from all of the spots, which corresponded to nonspecific binding (background value). Subsequent injection of the anti-C 131–150 immune serum confirmed the specificity of the method; only the C 131–150 spot emitted a strong reflectivity signal. Moreover, this third injection demonstrated that the peptide chip was still efficient after regeneration. Thus, consecutive injections (n = 4) of the same serum led to a reproducible signal (within 0.03%).

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Figure 2.

Specificity of antibody binding on peptide chips.

(A), direct assays. Rabbit anti-C 20–40 and anti-C 131–150 immune sera were successively injected on a peptide chip bearing peptides C 20–40 (green line), C 131–150 (red line), Ova 273–288 (gray line), or polypyrrole alone (blue line). Rabbit nonimmune serum was injected as control. All sera were used at the same dilution (1:100), and a regeneration step was performed between 2 successive injections. (B and C), competition assays. Rabbit anti-C 131–150 immune serum was injected first (1); after a 5-min wash, the C 20–40 peptide (B) or the C 131–150 peptide (C) was injected (2); the chip was then regenerated (3).

The specificity of the signal observed by SPRi was further assessed by competition assays: anti-C 131–150 antiserum was injected first and produced an increased reflectivity signal corresponding to the C 131–150 spot. Free C 20–40 (Fig. 2B⇑ ) or C 131–150 (Fig. 2C⇑ ) peptides, used as competitors, were subsequently injected. In the former case, the signal from all of the spots was slightly enhanced, likely because of an optical index shift, and then stabilized with time (Fig. 2B⇑ ). By contrast, injection of free C 131–150 peptide persistently and specifically decreased the reflectivity signal from the corresponding peptide spot (Fig. 2C⇑ ). This latter result indicates that the bound antibodies are displaced from the peptide chip by the relevant free peptide through selective competition and in a dose-dependent manner (see Fig. 1 in the online Data Supplement).

SPRi quantification of antibody binding on peptide chips

To evaluate the usefulness of this method for analyzing peptide–antibody interactions, we assessed whether the SPRi signal was indeed related to the amount of antibodies present in the input solution. Serial dilutions of anti-C 131–150 antiserum were successively injected into the Teflon cell, and the chip surface was regenerated after each assay. As shown in Fig. 3A⇓ , only background reflectivity was detected from the C 20–40 spot, whereas a dose-dependent signal was emitted from the relevant C 131–150 spot. More specifically, the measured ΔR correlated linearly with the dilution factor (Fig. 3B⇓ ), until a plateau was reached on saturation of the system. The value obtained at the plateau (8% of reflectivity) corresponded to a monolayer of antibody (24) equivalent to 1 pmol/mm².

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Figure 3.

SPRi analysis and quantification of antibodies bound to peptide chips.

(A), sensorgrams obtained when rabbit anti-C 131–150 immune sera serially diluted in PBS were successively injected on a peptide chip on which C 20–40 (gray line) or C 131–150 (black line) was immobilized, with a regeneration step between 2 successive injections. (B), ΔR plotted against the dilution factor of the immune serum in buffer ( ) and in nonimmune serum (▪).

One important application of this method is the detection of antibodies (or other molecules) present, even at low concentrations, in a complex medium such as serum. Because this technique was optimized by use of sera diluted in an appropriate buffer, we tested its efficacy in more stringent conditions. To that end, we diluted the anti-C 131–150 immune serum in whole nonimmune serum, mimicking a weak antibody concentration in a complex medium. Sera were successively injected on the chip under the same conditions used for the previous series of experiments (see Fig. 2 in the online Data Supplement). As shown in Fig. 3B⇑ , the curves featured similar profiles regardless of whether the screened antibodies were diluted in nonimmune serum or in buffer, confirming that the method could indeed be used for the analysis of a complex medium. The shift between the 2 curves was likely attributable to the blocking effect of the serum used to dilute the samples.

analysis of patient-derived sera by use of peptide chips and SPRi detection

Patients infected with HCV raise a broad antibody response that persists long after the disease has resolved. We used peptide chips and SPRi detection to screen sera from 10 patients who had recovered from HCV infection, as assessed by undetectable viral RNA, for anti-C 20–40 and anti-C 131–150 antibodies. As shown in Table 1⇓ , 3 sera induced a significant enhancement in reflectivity (ΔR >0.3) from the C 131–150 spot and 8 sera induced an increased reflectivity signal from the C 20–40 spot, revealing the presence of specific antibodies against these 2 peptides in patient-derived sera. On the other hand, the 5 sera from healthy donors did not produce a significant change in reflectivity.

To further demonstrate the relevance of our technique, we compared these results with those obtained by an ELISA. Correlation between the 2 methods was good (Fig. 4⇓ ): sera giving negative or weakly positive ELISA results also generated a weak SPR signal and sera generating high ELISA values also gave high SPRi signals. We confirmed this correlation by use of the two-tailed Spearman test (P = 0.008).

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Figure 4.

Correlation between graphs plotting anti-C 20–40 antibodies in sera from patients quantified by SPRi (ΔR, difference in reflectivity between positive and negative peptide spots) and by ELISA (A, absorbance).

The statistical analysis was performed with the two-tailed Spearman test.

Because microarrays are designed for assaying multiple analytes, we broadened our technique to a wider panel of epitopes. We implanted 11 HCV peptides on a chip. These epitopes were derived from structural (C 1–21, C 20–40, C 131–150, E1 311–330, and E2 528–546) and nonstructural (NS2 958–977, NS3 1248–1265, NS4/1 1688–1708, NS4/2 1921–1940, NS5/1 2294–2313, and NS5/2 2944–2959) proteins. Ova 273–288 was used as negative control. Eleven sera from HCV-positive patients and from healthy donors were successively injected on this chip, with a regeneration step between each new injection. The reflectivity changes from each spot (n = 12) were recorded simultaneously (Fig. 5A⇓ ), with each curve accounting for the reactivity of each serum against each peptide. As shown in Fig. 5B⇓ , the specific SPRi signal (ΔR) varied according to the patient and the peptide, revealing donor-related differences in the anti-HCV antibody responses (specificity, amount, and affinity). It is noteworthy that antibodies against some peptides were never (NS5/1) or rarely (E2) detected in the sera, suggesting that these epitopes induce only a weak humoral response.

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Figure 5.

SPRi analysis of sera from 10 HCV patients (P1–P10) and from 1 healthy donor (H1).

The sera were injected on a chip bearing 11 HCV peptides and Ova 273–288 as a control, with a regeneration step between 2 successive injections. (A), sensorgrams obtained after injection of the 11 sera (diluted 1:4 in PBS). (B), ΔR for each serum.