Optimization of peptide arrays for studying antibodies to hepatitis C virus continuous epitopes

Abstract

Accurate and in-depth mapping of antibody responses is of great value in vaccine and antibody research. Using hepatitis C virus (HCV) as a model, we developed an affordable and high-throughput microarray-based assay for mapping antibody specificities to continuous antibody epitopes of HCV at high resolution. Important parameters in the chemistry for conjugating peptides/antigens to the array surface, the array layout, fluorophore choice and the methods for data analysis were investigated. Microscopic glass slide pre-coated with N-Hydroxysuccinimide (NHS)-ester (Slide H) was the preferred surface for conjugation of aminooxy-tagged peptides. This combination provides a simple chemical means to orient the peptides to the conjugation surface via an orthogonal covalent linkage at the N- or C-terminus of each peptide. The addition of polyvinyl alcohol to printing buffer gave uniform spot morphology, improved sensitivity and specificity of binding signals. Libraries of overlapping peptides covering the HCV E1 and E2 glycoprotein polypeptides (15-mer, 10 amino acids overlap) of 6 major HCV genotypes and the entire polypeptide sequence of the prototypic strain H77 were synthesized and printed in quadruplets in the assays. The utility of the peptide arrays were confirmed using HCV monoclonal antibodies (mAbs) specific to known continuous epitopes and immune sera of rabbits immunized with HCV antigens. The methods developed here can be easily adapted to studying antibody responses to antigens relevant in vaccine and autoimmune research.

Prediction of B Cell Epitopes Among Hantavirus Strains Causing Hemorragic Fever With Renal Syndrome

“…The peptides may also be useful for quantum dot nanotechnological tools. Similar approaches have been taken for other infectious agent successfully [Tripp et al, ; Ruwona et al, ].…”

Screening Complex Biological Samples with Peptide Microarrays: The Favorable Impact of Probe Orientation via Chemoselective Immobilization Strategies on Clickable Polymeric Coatings

“…Click reactions’ suitability to immobilize bioligands on microarray surfaces is now well-established, yet, to the best of our knowledge, literature is still lacking a rigorous and critic comparative evaluation of click-reaction performances in terms of binding efficiency and influence of the immobilization chemistry on the diagnostic accuracy in a serological assay. Similarly, the comparison between random versus oriented immobilization under a quantitative perspective is still to be reported, the optimization of chemoselective immobilization is often restricted to one type of bioconjugation strategy, − and other parameters, such as the effect of spacing peptide ligands from microarray surface by means of suitable spacers, have been so far only intuitively discussed and mostly for proteases assays …”

Peptide Arrays of Three Collections of Human Sera from Patients Infected with Mosquito-Borne Viruses

“…A subset of the previously-predicted diagnostic peptides [21], representing multiple mosquito-borne virus species and subtypes, were synthesized at the Center for Protein and Nucleic Acid Research at The Scripps Research Institute (TSRI) [23,24]. This selected collection of peptides consisted of 15-mers with sequences that represented the consensus amino acid sequence among strains belonging to each of our six target taxa including: CHIKV, DENV1, DENV2, DENV3, WNV, and ZIKV.…”

“…Immediately prior to interrogating the arrays, slides were blocked for 1 h with ethanolamine buffer to quench any unreacted NHS-ester on the slide. All slides were used within 2 months of printing and were stored at -20°C [23].…”

“…This algorithm has been used previously to identify residues within 15-mer linear peptide regions that have high predicted specificity and sensitivity values and that could therefore be useful for detecting antibodies against a variety of mosquito-borne Flavivirus species [22]. Quantifying the reactivity of this set of peptides using high-throughput custom peptide arrays enabled the efficient and simultaneous testing of all peptides against each serum sample with higher efficiency than what is possible with manual ELISA-based technology [23].…”