title: Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.
abstract: The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: Sujatha P
authors.initials: SP
authors.lastname: Koduvayur
authors.affiliation: Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: Hélène A
authors.initials: HA
authors.lastname: Gussin
authors.affiliation: Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, Illinois, United States of America ; Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: Rajni
authors.initials: R
authors.lastname: Parthasarathy
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: Zengping
authors.initials: Z
authors.lastname: Hao
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
authors.affiliation: Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, Illinois, United States of America ; Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: David R
authors.initials: DR
authors.lastname: Pepperberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D051381:Rats; D011963:Receptors, GABA-A / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D010455:Peptides; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies