title: Synthetic peptide vaccine development: measurement of polyclonal antibody affinity and cross-reactivity using a new peptide capture and release system for surface plasmon resonance spectroscopy.
abstract: A method has been developed for measurement of antibody affinity and cross-reactivity by surface plasmon resonance spectroscopy using the EK-coil heterodimeric coiled-coil peptide capture system. This system allows for reversible capture of synthetic peptide ligands on a biosensor chip surface, with the advantage that multiple antibody-antigen interactions can be analyzed using a single biosensor chip. This method has proven useful in the development of a synthetic peptide anti-Pseudomonas aeruginosa (PA) vaccine. Synthetic peptide ligands corresponding to the receptor binding domains of pilin from four strains of PA were conjugated to the E-coil strand of the heterodimeric coiled-coil domain and individually captured on the biosensor chip through dimerization with the immobilized K-coil strand. Polyclonal rabbit IgG raised against pilin epitopes was injected over the sensor chip surface for kinetic analysis of the antigen-antibody interaction. The kinetic rate constants, k(on) and k(off), and equilibrium association and dissociation constants, KA and KD, were calculated. Antibody affinities ranged from 1.14 x 10(-9) to 1.60 x 10(-5) M. The results suggest that the carrier protein and adjuvant used during immunization make a dramatic difference in antibody affinity and cross-reactivity. Antibodies raised against the PA strain K pilin epitope conjugated to keyhole limpet haemocyanin using Freund's adjuvant system were more broadly cross-reactive than antibodies raised against the same epitope conjugated to tetanus toxoid using Adjuvax adjuvant. The method described here is useful for detailed characterization of the interaction of polyclonal antibodies with a panel of synthetic peptide ligands with the objective of obtaining high affinity and cross-reactive antibodies in vaccine development.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, CO, 80262, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Cachia
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Kao
authors.forename: Robert S
authors.initials: RS
authors.lastname: Hodges
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000032:analysis / Q000276:immunology*; D000915:Antibody Affinity; D001428:Bacterial Vaccines / Q000737:chemistry / Q000276:immunology*; D003429:Cross Reactions; D036781:Fimbriae Proteins / Q000276:immunology; D007074:Immunoglobulin G / Q000032:analysis / Q000276:immunology; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology*; D017434:Protein Structure, Tertiary; D011552:Pseudomonas Infections / Q000517:prevention & control; D011550:Pseudomonas aeruginosa / Q000276:immunology*; D011817:Rabbits; D011971:Receptors, Immunologic / Q000276:immunology / Q000502:physiology; D020349:Surface Plasmon Resonance / Q000379:methods*; D022223:Vaccines, Subunit / Q000276:immunology; D014614:Vaccines, Synthetic / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D007074:Immunoglobulin G; D008024:Ligands; D010455:Peptides; D011971:Receptors, Immunologic; D022223:Vaccines, Subunit; D014614:Vaccines, Synthetic; C037872:pili, bacterial receptor; D036781:Fimbriae Proteins