Bioconjugate Chem. 2002, 13, 713−720
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Peptide Arrays for Highly Sensitive and Specific Antibody-Binding Fluorescence Assays Oleg Melnyk,*,† Xavier Duburcq,*,‡,§ Christophe Olivier,§ Florence Urbe`s,§ Claude Auriault,‡ and He´le`ne Gras-Masse† UMR CNRS 8525, UMR CNRS 8527, and Sedac-Therapeutics, Biological Institute of Lille, 1 rue du Pr Calmette, 59021 Lille, France. Received November 27, 2001; Revised Manuscript Received March 6, 2002
We report a novel generation of peptide arrays fabricated by site-specific ligation of glyoxylyl peptides onto glass slides covered by a semicarbazide sol-gel layer. These arrays allowed the highly sensitive and specific detection of antibodies in very small blood samples from infected individuals using three model peptidic epitopes (HCV Core and NS4, EBV Capsid) in an immunofluorescence assay. Comparison with standard enzyme-linked immunosorbent assays (ELISAs) demonstrated a large gain in sensitivity and specificity. These unique properties, combined with the possibility to immobilize glycoproteins such as antibodies, offer the possibility to perform sandwich immunofluorescent assays in a highly parallel format.
INTRODUCTION
EXPERIMENTAL PROCEDURES
Today, the miniaturization of biological assays is at the origin of crucial biological research and medical advances. In this respect, the design of miniaturized devices for the simultaneous highly sensitive and specific detection of antibodies from complex biological samples should represent a breakthrough. Over the past decade, the trends toward miniaturization of high-throughput screening systems have generated a great deal of interest in the scientific community. Microfabricated analytical devices have found applications in the field of gene expression profiling (1) or large-scale functional analysis of peptides (2-4) or proteins (5-7). Owing to the possibility to implement simultaneously a large variety of assays based on antibody or antigen detection, microarrays may revolutionize the diagnostic of infections. So far, applications of protein arrays to the detection of antibodies have displayed high background levels, low signal intensities, and cross-reactivities (8-12). As a result, the design of miniaturized devices for the highly sensitive and specific detection of antibodies from complex biological samples has not been achieved to date (13). Here we report that a novel generation of peptide arrays, assembled by site-specific semicarbazone ligation of medium-sized peptides onto glass slides, allows the highly sensitive and specific detection of antibodies in very small blood samples from infected individuals. Three model peptidic epitopes (HCV Core and NS4, EBV Capsid) were used for antibody detection studies based on an immunofluorescent assay. The peptide arrays were analyzed with a fluorescence array scanner. A collection of referenced sera permitted demonstration of a large gain in sensitivity and specificity for our arrays as compared to the standard ELISA tests.
Peptide Synthesis. The peptides were synthesized in an automated peptide synthesizer (model Pioneer, Perseptive Biosystems Inc., MA) using the Fmoc/tert-butyl strategy (14). Peptides 3 and 5-7 were synthesized on a poly(ethylene glycol) polyacrylamide copolymer (amino PEGA resin (15), Novabiochem, Meudon, France) derivatized with an isopropylidene tartrate linker as described in refs 27-29. The peptides were purified by preparative RPHPLC (C18-VYDAC, buffer A: deionized water containing 0.05% TFA; buffer B: acetonitrile/deionized water: 4/1 by vol. containing 0.05% TFA). Peptide 3, yield 21%, MALDI-TOF [M + H]+ calcd 3587.0 found 3587.0. Peptide 5, yield 21%, MALDI-TOF [M + H]+ calcd 3733.1, found 3732.7. Peptide 6, yield 25%, MALDI-TOF [M + H]+ calcd 2755.4, found 2755.7. Peptide 7, yield 34%, MALDI-TOF [M + H]+ calcd 2606.3, found 2606.5. For peptide 8 (0.1 mmol scale), peptide elongation was followed by acylation with diglycolic anhydride/DIEA: 4 equiv/4 equiv in DMF during 30 min. The peptidyl resin was washed with DMF (4 × 2 min) and then treated successively with 10 equiv of 4,7,10-trioxa-1,13-tridecanediamine (TTD), 3 equiv of DIEA, and 2 equiv of PyBOP (Novabiochem, Meudon, France) in DMF. After 30 min of reaction, the beads were washed with DMF (4 × 2 min) and acylated with Fmoc-L-Ser(OtBu)-OH/ HBTU/HOBt/DIEA: 10 equiv/10 equiv/10 equiv/30 equiv in DMF during 45 min. The resin was washed with DMF (4 × 2 min) and deprotected with DMF/piperidine: 4/1 by vol. The resin was washed with DMF (4 × 2 min) and CH2Cl2 (4 × 2 min) and dried. Deprotection and cleavage from the solid support was performed with TFA/H2O/TIS: 95/2.5/2.5 by vol (2 h). The peptide was precipitated in cold ether, redissolved in aqueous acetic acid, and lyophilized. Periodic oxidation was performed in a pH 7.0 0.1 M phosphate buffer with 2 equiv of NaIO4 (5 min). The peptide was purified by RP-HPLC on a C18 Nucleosil 320 × 12 mm column (100 Å, 5 µm), detection at 220 nm, 50 °C, 3 mL/min, 0-20% B in 20 min. Peptide 8, yield 25%, MALDI-TOF [M + H]+ calcd 2722.4, found 2722.5.
* To whom correspondence should be addressed. O.M. and X.D. contributed equally to this work. Oleg Melnyk: Tel: 33(0)3 20 87 12 15, fax: 33(0)3 20 87 12 33, e-mail: oleg.melnyk@ pasteur-lille.fr. † UMR CNRS 8525. ‡ UMR CNRS 8527. § Sedac-Therapeutics.
10.1021/bc015584o CCC: $22.00 © 2002 American Chemical Society Published on Web 06/04/2002
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For peptide 4 (0.1 mmol scale), peptide elongation was followed by acylation with Fmoc-L-Lys(Mtt)-OH. Removal of the Mtt group (16) with 1% TFA in CH2Cl2 (17) was followed by acylation with Fmoc-L-Ser(OtBu)-OH as described for peptide 8. The peptide was deprotected, cleaved with TFA/phenol/H2O (10 mL/0.5 mL/0.75 g) (2 h), and precipitated in cold diethyl ether. The crude peptide was then oxidized with periodate as described for peptide 8. Peptide 4 was purified by RP-HPLC on a C3 Zorbax 250 × 15 mm column, detection at 225 nm, 60 °C, 4 mL/min, 0-80% B in 60 min. Peptide 4, yield 18%, MALDI-TOF [M + H]+ calcd 3544.0, found 3543.8. Amino acid composition of all the peptides was determined on a Beckman amino acid analyzer (model 7300, ninhydrin detection) following hydrolysis of aliquots in 6 N HCl/phenol (10/1 by vol) (48 h at 110 °C). Semicarbazide Slides. Full details of semicarbazide glass slide optimization, characterization, and preparation will be reported elsewhere. Rapidly, glass slides (Esco, 114 slides at a time) cleaned with a H2SO4/H2O2 (1/1 by vol) solution were silanized with 3-aminopropyltrimethoxysilane. The amino slides were treated with triphosgen/diisopropylethylamine in 1,2-dichloroethane and Fmoc-NHNH2 in DMF, respectively, to install the semicarbazide functions. Finally, removal of the Fmoc groups was performed with piperidine/DBU in dimethylformamide. Four slides were used for the quality control. Three slides were immersed 1 h at 37 °C in a 10-4 M solution of Rho-Lys-Arg-NH(CH2)3NH-CO-CHO (synthesized according to ref 27-28, where Rho is (5)-6carboxytetramethylrhodamine). One slide was treated with the control peptide Rho-Lys-Arg-NH2. The fluorescence intensity of the slides was quantified using an Affymetrix 418 Array Scanner (MWG). Typically, the mean fluorescence intensity of semicarbazide slides treated with the glyoxylyl-rhodaminated probe was found to be 36260 (SD 3940) at L35, PMT50 scanner sensitivity (control slide: mean fluorescence 3450, SD 486). Both fluorescent probes were found to adsorb similarly on untreated slides (mean fluorescence for the glyoxylyl probe: 7790, SD 670, mean fluorescence for the amide probe: 5270, SD 930, L35, PMT50). Human Sera. Sera were collected from the clinical laboratory of the Centre Hospitalier Re´gional (Lille, France). The reactivity of HCV-referenced sera was determined with the AxSYM HCV version 3.0 Abbott ELISA kit (30 HCV-negative and 100 HCV-positive individuals). The reactivity of EBV-referenced sera (36 HCV-positive and 15 HCV-negative individuals) was determined using an immunofluorescence assay (antiVCA IFA, Immunoconcept). ELISAs. Microtiter plate wells (Carbo-bind, Costar, Corning Inc., NY) were coated 60 min at 37 °C with the peptide (5 µg/mL in 0.1 M acetate buffer pH 5.5). The wells were washed and then saturated with 2.5% milk in PBS (0.01 M phosphate buffer supplemented with 1.8% NaCl pH 7.4) at 37 °C. After four washings with PBS 0.05% Tween 20 (PBS-T, Sigma-Aldrich, Saint Quentin Fallavier, France), human sera diluted 1/100 in PBS plus 0.5% Tween 20 plus 2.5% milk were incubated in coated wells for 120 min at 37 °C. After four washings, the peroxidase-conjugated goat antibodies to human IgG-A-M (Diagnostic Pasteur, Marnes la Coquette, France), diluted 1/10000 in PBS plus 0.5% Tween 20 plus 2.5% milk, were incubated for 60 min at 37 °C. Bound conjugated antibodies were detected using o-phenylenediamine dihydrochloride substrate (Sigma) plus H2O2 in 0.05 M citrate buffer pH 5.5, for 30 min in the dark at room temperature. The reaction was blocked by addition of 2 N H2-
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SO4. The absorbance was recorded against a blank at 492 nm through a multichannel automatic plate-reader (MR 5000, Dynatech, Grafton, Ohio). Mean at 492 nm + 3 standard deviation (SD) of HCV-sera were used as cutoff values in the ELISAs. The ELISAs were performed at least twice to assess their reproducibility. Peptide Arrays. A manual arrayer (Microarray Printer XMM 47832, Xenopore, Hawthorne, NJ) equipped with 32 pins was used to print the slides. Similar results were obtained using an Affymetrix 417 arrayer. The printed slides were incubated overnight at 37 °C in a humid chamber and stored until use at room temperature for a period ranging from days to several months. The slides were washed and incubated during 120 min at 37 °C under microscope slide with 50 µL of human sera diluted 1/50 in PBS plus 0.5% Tween 20 plus 2.5% milk. After four washings, 50 µL of the rhodamine(TRITC)-conjugated goat antibodies to human IgG-A-M (Jackson ImmunoResearch Laboratories, Baltimore, MD), diluted 1/100 in PBS plus 0.5% Tween 20 plus 2.5% milk, were incubated for 60 min at 37 °C under cover glass. The slides were then washed, dried, and scanned with an Affymetrix 418 Array Scanner (MWG). The data where analyzed using the ScanAlyse software (Stanford University). Immobilization of Antibodies on Semicarbazide Slides. The antibodies (donkey IgG anti-goat IgG F(c), Rockland, Gilbertsville, PA, or goat IgG anti-human Ig G-A-M, Jackson ImmunoResearch Laboratories, Baltimore, MD) were oxidized as described in ref 32. Following oxidation, the antibodies (600 µg) were dialyzed (nanosep tubes, Pall Gelman Laboratory, Ann Arbor, MI) against acetate buffer pH 5.5 0.1 mM (six washings), solubilized in 40 µL of acetate buffer pH 5.5 0.1 mM and placed in a 384-well plate. Ten slides were printed using a manual Xenopore XMM 47832 printer and incubated one night at 37 °C in a humid chamber. The slides were washed and incubated under cover glass with 50 µL of rhodamine conjugated goat IgG F(c) fragment (Rockland, Gilbertsville) diluted 1/100 in PBS plus 0.5% Tween 20 plus 2.5% milk (60 min at 37 °C). The slides were washed, dried in air, and analyzed with an Affymetrix 418 Array Scanner. The data were processed with the ScanAlyse software. RESULTS AND DISCUSSION
Glass Slide Design and Preparation. The design of miniaturized devices for the highly sensitive and specific detection of antibodies from complex biological samples represents a challenge which, besides the selection of the probes, relies mainly upon the surface properties of the array. We first hypothesized that the optimal surface for antibody detection would be uncharged at neutral pH and highly hydrated. Indeed, neutral hydrophilic polymers have been found to reduce protein adsorption on different surfaces (18, 19). Second, the surface should be chemically inert toward immunoglobulins but should allow the covalent site-specific and dense immobilization of synthetic polypeptides so as to favor the antigen accessibility and to reach high signalto-noise ratios. Glass slides covered by a semicarbazide sol-gel layer were found to meet all these criteria, and in conjunction with the well developed chemistry of glyoxylyl peptides (20) were used to elaborate a novel generation of peptide arrays assembled by semicarbazone site-specific ligation (Scheme 1). The optimization, characterization, and preparation of semicarbazide glass slides will be reported in a paper devoted to the fabrication of oligodeoxynucleotide mi-
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Bioconjugate Chem., Vol. 13, No. 4, 2002 715
Scheme 1. Semicarbazone Assembled Peptide Arrays
Scheme 2. Slides
Preparation of Semicarbazide Glass
croarrays using a similar immobilization strategy (21, 22). Very rapidly as shown in Scheme 2, microscope glass slides were cleaned with H2SO4/H2O2 (piranha solution) and silanized with 3-aminopropyltrimethoxysilane. The amino slides were then treated successively with triphosgen/DIEA and Fmoc-NHNH2 (23) in DMF to install the semicarbazide groups. Finally, removal of the Fmoc groups was performed with piperidine/DBU in dimethylformamide. The density and homogeneity of the semicarbazide sol-gel layer was evaluated by immersing the slides in a solution of a glyoxylyl dipeptide labeled with (5)-6-carboxytetramethylrhodamine (Rho-Lys-Arg-NH(CH2)3NH-CO-CHO, peptide 1) followed by quantification of the fluorescence using the Cy3 channel of a DNA microarray scanner. Immobilization through site-specific semicarbazone formation was demonstrated by several control experiments using either underivatized dipeptide 2 Rho-Lys-Arg-NH2 on semicarbazide slides or glyoxylyl peptide 1 on slides lacking the semicarbazide functionality (see Experimental Section). Synthesis of Model Glyoxylyl Peptide Antigens. The three model sequences selected for our study are derived from immunodominant and conserved regions of hepatitis C virus (HCV, core p21 15-45 (24), NS4 19251947 (25)) and Epstein-Barr virus (EBV, VCAp18 153176 (26)). The corresponding glyoxylyl peptides are shown in Figure 1. For the HCV core p21 antigen, the glyoxylyl group was placed either at the N-terminus at the extremity of a Lys side-chain (peptide 4) or at the C-terminus. In this later case, two different spacers were used to document the influence of the distance between the peptide and the glass surface upon the detection of antibodies (peptides 3 and 5). N- or C-glyoxylyl derivatives were also synthesized for the EBV VCAp18 peptide (peptides 7 and 8). Only one derivative was evaluated for the HCV NS4 peptide (peptide 6).
Figure 1. Model glyoxylyl peptide antigens used in this study.
Peptides 3, 5-7 were synthesized on the solid phase using an isopropylidene tartrate-based linker and the Fmoc/tBu strategy as described elsewhere (27, 28, 29). Peptides 4 and 8 were obtained from the corresponding seryl precursors by treatment with NaIO4 according to Geoghegan et al. (30). For H-Lys(Ser)-HCV core p21 1545, the peptide elongation step using the Fmoc/tBu strategy was followed by coupling with Boc-L-Lys(Fmoc)OH. The Fmoc group was removed with piperidine and the -amino group was acylated with Fmoc-L-Ser(OtBu)OH. The peptidyl resin was deprotected and cleaved from the solid support as usual with concentrated trifluoroacetic acid. For the seryl precursor of peptide 8, the peptidyl resin was treated with diglycolic (DG) anhydride to introduce a carboxylic acid function on the N-terminus.
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Figure 2. Detection of antibodies to peptides 5 (HCV p21) and 6 (HCV NS4) expressed in arbitrary units (AU). AU ) signal value/ cutoff. (Cutoff ) 1 AU ) mean of the background + 3 SD). (a) Scanning image of HCV+ (NS4+P21+, NS4+P21-, NS4-P21+) and HCV- sera at a low (L35, PMT50) or high (L50, PMT70) scanner sensitivity. (b) Detection of antibodies to peptide 5 by ELISA for 15 sera. (c) Detection of antibodies to peptide 6 by ELISA for 16 sera. (d) Peptide 5 array fluorescence for the same sera as b. (e) Peptide 6 array fluorescence for the same sera as c.
In situ activation of the carboxylic acid group with PyBOP in the presence of 4,7,10-trioxa-1,13-tridecanediamine (TTD) was followed by acylation with Fmoc-L-Ser(OtBu)-OH. Deprotection and cleavage from the support afforded H-Ser-TTD-DG-EBV VCAp18 peptide. Peptide Array Spotting. The glyoxylyl peptides were printed using a manual arrayer from Xenopore, which led to a spot diameter of about 550 µm. Similar results were obtained using an Affymetrix 417 arrayer (spot diameter of 160 µm) showing that the data did not depend on the printing technology. As for rhodaminated probe 1, the presence of a glyoxylyl group on the HCV p21 peptide is essential for an efficient immobilization on semicarbazide slides. The glyoxylyl antigens were printed at a concentration of 10-4 M in a pH 5.5 acetate buffer. HCV p21 and NS4 peptides were also printed as a 1/1 mixture using the same experimental conditions. The slides were incubated overnight in a humid chamber at 37 °C and then directly used for antibody detection. Importantly, no further chemical treatment for the
capping of the remaining semicarbazide groups was found to be necessary before the incubation with the sera. Detection of Antibodies by an Immunofluorescent Assay. Comparison with Enzyme-Linked Immunosorbent Assays. The peptide arrays were utilized for the detection of antibodies as shown in Scheme 1. Incubation with diluted human sera was followed by detection of immobilized antibodies with a rhodaminelabeled anti-human Ig-G-A-M antibodies. These two steps were performed under a cover glass and required minutes quantities of sera (1µL) and labeled antiboby (1 µg). The slides were imaged using the Cy-3 channel of a DNA microarray scanner. A referenced collection of 130 sera of HCV infected (100) or not (30) individuals was analyzed using the peptide array technology and by ELISA (AxSYM HCV version 3.0 Abbott, or ELISA with p21 or NS4 antigens coated on carbo-bind plates). The background noise of the arrays, defined as the fluorescence signal between the printed spots, was found to be very low for all the tested
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Table 1. Comparison between ELISAs and Peptide Arrays for HCV Antibodies Detection ELISAs entry
Nb of sera
1 2 3 4 5 6 7 8
73 14 1 1 5 2 4 30
reference + + + + + + + -
testa
peptide array
Carbobindc + + + + -
p21
Western-blotb
NS4
p21
NS4
p21+NS4
core
NS4
NS3
+ + + -
+ + + -
+ + + -
+ + + + -
nd nd + nd
nd nd + + nd
nd nd + + nd
remarks
false ELISAs false ELISAs false ELISAs false ELISAs false ELISAs
positive positive positive negative negative
a AxSYM HCV version 3.0, Abbott. b CHIRON RIBA HCV 3.0 SIA, Ortho-Clinical Diagnostics. c Carbo-bind plates, Costar, Corning Incorporated.
Figure 3. Scanning image of HCV+ (NS4+P21+, NS4+P21-, NS4-P21+), HCV-, and EBV VCAp18+ or EBV VCA p18- sera at a L35, PMT50 scanner sensitivity (peptides 5, 6, and 8).
sera and corresponded to 0.1% of the highest signal observed. Interestingly, the fluorescence obtained with the sera of noninfected individuals was always found to be below the mean of the background + 3 SD, even at a high scanner sensitivity (Figure 2A). Analogously, no fluorescence was observed for HCV+p21- or HCV+NS4- sera with p21 or NS4 peptides, respectively. Thus, to the contrary of ELISAs, no nonspecific binding could be detected with peptide arrays. Consequently, the cutoff value of positivity was defined as the mean of the background + 3 SD. The data collected in Table 1 show that peptide arrays and ELISAs were in agreement for 117 sera (entries 1, 2, and 8). For the 13 sera, where discrepancies were observed between the different tests (entries 3-7, Table 1), an additional analysis was performed using a western-blot assay (CHIRON RIBA HCV 3.0 SIA, Ortho-Clinical Diagnostics), which is the most sensitive and specific test for HCV antibodies detection. The western-blot assay confirmed the peptide array results, and that these sera were false positive or false negative by ELISA. Thus, at least for the selected model epitopes, peptide arrays displayed 100% of sensitivity and specificity. In addition,
the arrays were found to be much more sensitive than ELISAs, since comparison of ELISA and peptide array data indicated an average gain in sensitivity of about 8. Figure 2b-e detail the fluorescence and absorbance values for HCV+p21+ or HCV+NS4+ sera just above the cutoff value in ELISA, expressed in arbitrary units (AU) as defined in the legend of Figure 2. The consequence of this gain in sensitivity is that false negative sera or sera just above the ELISA cutoff were detected without ambiguity using the peptide arrays. The same sensitivity and specificity levels were reached with the VCAp18 EBV peptides 7 or 8. Indeed, 36 EBVpositive sera were found positive with the peptide array technology, whereas the fluorescence values associated with the 15 EBV-negative sera were always below the cutoff value of positivity (as for HCV sera, background + 3SD). Interestingly, when peptides 5, 6, and 8 were printed on the same slide, no cross-reaction was observed between a particular peptide and antibodies specific for the two others (Figure 3). These data illustrate the high specificity of this peptide array technology, an essential characteristic to permit the application of the array format for the simultaneous detection of different antibodies.
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Figure 4. Influence of the spacer and of the glyoxylyl group position upon the detection of antibodies directed against HCV core p21 15-45 antigen, HCV+ sera, L35, PMT 50.
Figure 5. Influence of the glyoxylyl group position upon the detection of antibodies directed against EBV VCAp18 antigen, data expressed in arbitrary units (AU), HBV+ sera, L35, PMT50.
Influence of the Spacer and of the Glyoxylyl Group Position. Peptides 3-5 permitted us to evaluate both the influence of the spacer separating the peptide from the glass surface and of the site of immobilization for the HCV p21 antigen. The data presented in Figure 4 show that no significant difference could be observed between these three peptides. For the site-specific immobilization through the C-terminus, the size of the spacer seemed to have little or no effect on antigen recognition. To the contrary of HCV p21, detection of antibodies directed against EBV VCAp18 antigen was found to be highly dependent upon the site of attachment to the glass
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surface (Figure 5). Indeed, peptide 8 permitted to improve significantly the detection of antibodies in the cases where peptide 7 displayed low ratios. Nevertheless, both peptides displayed the same levels of sensibility and specificity for the serodetection of EBV infections, at least for the tested sera. Quality Control, Reproducibility, Stability upon Storage. An important aspect of antibody detection is the reproducibility of the assay. To document the spot to spot reproducibility, peptide 5 was spotted 600 times on the same slide. The array was incubated with an HCV p21 + serum and treated as described before (Figure 6a). The standard deviation (SD) of the fluorescence for 600 spots was found to be 5.1%. Figure 6b details the fine structure of a spot and shows that the fluorescence inside the spot was uniform, an essential characteristic for the automation of fluorescence measurements. Another important aspect of the peptide array methodology is the possibility to reach high slide to slide reproducibility levels. To check this point, 12 slides were spotted with peptide 5 (600 spots) and treated with the same HCV+ serum. The SD between the mean fluorescence of the spots and of the background for the different slides was found to be only 4.5%. Finally, the peptide arrays were also tested for their stability upon storage. For peptides 5-7, no significant variation in fluorescence intensity of the spots and of the background was observed following up to 6 months of aging at 37 °C in a humid chamber (Figure 7). This feature can be attributed to the high chemical stability of the semicarbazone linkage and of the sol-gel layer. Site-Specific Immobilization of Antibodies. To broaden the utility of this methodology, we have also shown that semicarbazide slides allow the immobilization of glycoproteins and thus can be used for the preparation of protein arrays or mixed peptide-proteins arrays. Indeed, periodic oxidation is a well established and mild method for generating aldehyde groups on glycoproteins (31, 32). The reaction of these aldehyde groups with semicarbazide functionalized slides leads to the covalent immobilization of the proteins. For example, oxidized donkey IgG anti goat IgG Fc and goat IgG anti human
Figure 6. (a) Spot to spot reproducibility, peptide 5, Affymetrix 418 Array Scanner, scanner sensitivity L35, PMT50; (b) fine structure of a spot, same slide as in a, array WoRx microarray scanner, Applied Precision, Issaquah, Washington.
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Bioconjugate Chem., Vol. 13, No. 4, 2002 719 LITERATURE CITED
Figure 7. Stability of peptide arrays upon accelerated aging, peptides 5-7, HCV+ and EBV+ referenced sera, data expressed in arbitrary units (AU), L35, PMT50.
Figure 8. Site-specific immobilization of oxidized antibodies, scanner sensitivity L35, PMT50.
IgG-A-M were immobilized on semicarbazide slides and incubated with goat IgG Fc fragment labeled with rhodamine. Figure 8 shows that the fluorescence associated with donkey antibodies was found to be high, whereas fluorescence associated with the control goat antibodies corresponded to the background. Importantly, the slides were printed, washed, and incubated using the procedures employed for peptide arrays. Since these conditions retained the binding capacity and the specificity of immobilized antibodies, the simultaneous detection of antibodies and of circulating antigens on the same slide seems conceivable. CONCLUSION
At the origin, microarrays were developed to utilize the huge amount of information provided by genome projects, but they have clear potential for many other fields where multiple tests have to be performed using very small biological samples. We have demonstrated that arrays, assembled by semicarbazone site-specific ligation of synthetic polypeptides to glass slides, displayed very high sensitivities and specificities for antibodies directed toward several model peptidic epitopes. These unique properties combined with the possibility to immobilize glycoproteins should find many applications such as sandwich immunofluorescent assays, detection of circulating antigens, epitope mapping, and high throughput screenings. ACKNOWLEDGMENT
We thank CNRS, Universite´ de Lille 2, Institut Pasteur de Lille and Sedac-Therapeutics for financial support, Re´mi Desmet, Annick Blanpain, and Eric Diesis for technical assistance, Fre´de´ric Malingue for data studies, David Hot and Ludovic Huot for fluorescence measurements, Thomas Heim for AFM studies, and Steven Brooks for proofreading the manuscript. We thank Pr Yves Lemoine for giving us an access to the Affymetrix 418 array scanner and Affymetrix 417 arrayer.
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