title: Generation of recombinant guinea pig antibody fragments to the human GABAC receptor.
abstract: To generate monoclonal antibodies to the human ρ1 GABA(C) receptor, a ligand-gated chloride ion channel that is activated by the neurotransmitter γ-aminobutyric acid (GABA), we recovered the immunoglobulin variable heavy chain (V(H)) and light chain (V(L)) regions of a guinea pig immunized with a 14-mer peptide segment of the N-terminal extracellular domain of the ρ1 subunit. Oligonucleotide primers were designed and used to amplify the V(H) and V(L) regions of guinea pig RNA by the reverse transcriptase polymerase chain reaction. The amplified and cloned V(H) and V(L) regions were transferred together into a phagemid vector, yielding a library of 5×10(6) members, which displayed chimeric fragments of antigen binding (Fabs) with guinea pig variable and human constant regions fused to protein III of M13 bacteriophage. Through affinity selection of this phage-display library with the biotinylated 14-mer peptide segment of GABA(C), we isolated four different antibody fragments that bound specifically to the immunogenic peptide. Phage particles displaying two of these antibodies, but not negative controls, bound selectively to the surface of neuroblastoma cells expressing the ρ1 GABA(C) receptor. Such antibody fragments will be useful in future studies involving targeting of specific neural tissues that express the GABA(C) receptor.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, 60607, USA.
authors.forename: Adnan
authors.initials: A
authors.lastname: Memic
authors.forename: Veronica V
authors.initials: VV
authors.lastname: Volgina
authors.forename: Hélène A
authors.initials: HA
authors.lastname: Gussin
authors.forename: David R
authors.initials: DR
authors.lastname: Pepperberg
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D006168:Guinea Pigs; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D019151:Peptide Library; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D018079:Receptors, GABA / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D012333:RNA, Messenger; D018079:Receptors, GABA; D011994:Recombinant Proteins