title: Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury.
abstract: Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago (UIC), 845 W. Taylor St, 3240 SES-MC 066, Chicago, IL 60607-7060, United States of America.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kierny
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago (UIC), 845 W. Taylor St, 3240 SES-MC 066, Chicago, IL 60607-7060, United States of America.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Cunningham
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago (UIC), 845 W. Taylor St, 3240 SES-MC 066, Chicago, IL 60607-7060, United States of America.
authors.forename: Rachida A
authors.initials: RA
authors.lastname: Bouhenni
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago (UIC), 845 W. Taylor St, 3240 SES-MC 066, Chicago, IL 60607-7060, United States of America.
authors.forename: Deepak P
authors.initials: DP
authors.lastname: Edward
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago (UIC), 845 W. Taylor St, 3240 SES-MC 066, Chicago, IL 60607-7060, United States of America.
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D015374:Biosensing Techniques; D015153:Blotting, Western; D002645:Chickens; D003460:Crystallization; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D007834:Lasers; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D017785:Photons; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012164:Retinal Diseases / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000096:biosynthesis*; D012995:Solubility; D014020:Tissue Extracts; D014771:Virion / Q000276:immunology
chemical_list: D015415:Biomarkers; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D014020:Tissue Extracts; D000409:Alanine