title: 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 and 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 was 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.
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, United States.
authors.forename: Tinashe B
authors.initials: TB
authors.lastname: Ruwona
authors.affiliation: Microarray Core Facility, The Scripps Research Institute, La Jolla, CA, United States.
authors.forename: Ryan
authors.initials: R
authors.lastname: Mcbride
authors.affiliation: Center for Protein and Nucleic Acids Research, The Scripps Research Institute, La Jolla, CA, United States.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Chappel
authors.affiliation: Microarray Core Facility, The Scripps Research Institute, La Jolla, CA, United States.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Head
authors.affiliation: Center for Protein and Nucleic Acids Research, The Scripps Research Institute, La Jolla, CA, United States.
authors.forename: Phillip
authors.initials: P
authors.lastname: Ordoukhanian
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, United States.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Burton
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, United States. Electronic address: mlaw@scripps.edu.
authors.forename: Mansun
authors.initials: M
authors.lastname: Law
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000276:immunology*; D015415:Biomarkers / Q000097:blood; D002021:Buffers; D000939:Epitopes; D004952:Esters / Q000737:chemistry; D016174:Hepacivirus / Q000276:immunology*; D018937:Hepatitis C Antibodies / Q000097:blood*; D057166:High-Throughput Screening Assays; D019151:Peptide Library; D011142:Polyvinyl Alcohol / Q000737:chemistry; D040081:Protein Array Analysis; D011817:Rabbits; D015203:Reproducibility of Results; D013388:Succinimides / Q000737:chemistry; D014759:Viral Envelope Proteins / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D015415:Biomarkers; D002021:Buffers; C087035:E1 protein, Hepatitis C virus; D000939:Epitopes; D004952:Esters; D018937:Hepatitis C Antibodies; D019151:Peptide Library; D013388:Succinimides; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus; D011142:Polyvinyl Alcohol; C001426:N-hydroxysuccinimide
keywords: E1E2 glycoprotein; HCV; Peptide array