title: Characterization of a unique human single-chain antibody isolated by phage-display selection on membrane-bound mosquito midgut antigens.
abstract: The insect midgut is the primary site for food digestion, as well as for vector-borne pathogen infection into the invertebrate host. Accordingly, antigens of this critical insect organ are targets for anti-vector vaccines, insecticidal toxins, and transmission-blocking vaccines. We used midgut proteins of the African malaria vector mosquito Anopheles gambiae to select single-chain human antibody fragments (scFv) from a high-diversity, phage-displayed library. Using a phage-display selection method on western-blotted antigens, we selected an unusual truncated scFv clone, consisting of a heavy-chain only, which binds to An. gambiae midgut tissue. This clone binds a spectrum of mosquito antigens from the midgut and other mosquito tissues, as well as various mammalian glycoproteins, but binding was reduced when these glycoproteins were enzymatically deglycosylated. We also observed that this clone preferentially binds the lumenal midgut surface. Furthermore, antigen binding by our selected scFv was limited by competition with increasing concentrations of certain soluble carbohydrates, most dramatically by galactose and N-acetyl glucosamine. Our results show that the cognate epitope of this scFv is a carbohydrate moiety. This paper describes a phage-display selection of antibody fragments on mosquito midgut tissue and it also describes a method for phage-display selection on membrane-immobilized heterogeneous antigens. These selection methods resulted in the isolation of a novel, truncated, carbohydrate-binding human antibody fragment from a naive phage-display library.
authors.affiliation: Interdisciplinary Program of Molecular and Cellular Biology, Tulane University, New Orleans, LA, USA. bfoy@tulane.edu
authors.forename: Brian D
authors.initials: BD
authors.lastname: Foy
authors.forename: Gerry F
authors.initials: GF
authors.lastname: Killeen
authors.forename: Ross H
authors.initials: RH
authors.lastname: Frohn
authors.forename: Daniel
authors.initials: D
authors.lastname: Impoinvil
authors.forename: Andrew
authors.initials: A
authors.lastname: Williams
authors.forename: John C
authors.initials: JC
authors.lastname: Beier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000852:Anopheles; D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000302:isolation & purification*; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000302:isolation & purification*; D019476:Insect Proteins / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library
chemical_list: D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019476:Insect Proteins; D019151:Peptide Library; C055966:immunoglobulin Fv; D004247:DNA