title: Selection and expression of recombinant single domain antibodies from a hyper-immunized library against the hapten azoxystrobin.
abstract: Three V(H)Hs against the model hapten, azoxystrobin (MW 403), were isolated from a hyper-immunized phage-displayed V(H)H library. This library was constructed by isolating the V(H)H-coding genes from the lymphocytes collected from a Llama glama that was immunized with azoxystrobin conjugated to bovine serum albumin (BSA). Six rounds of panning were performed against azoxystrobin conjugated to either ovalbumin (OVA) or rabbit serum albumin (RSA) to enrich clones containing V(H)Hs specific to the hapten. After screening 95 clones, three V(H)Hs (A27, A72, and A85) with different amino acid sequences were identified, expressed in soluble format in Escherichia coli HB2151, and purified using nickel-immobilized metal affinity chromatography. Competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) showed that A27 and A85 were specific to azoxystrobin while A72 was not. The IC(50) values of A27 and A85 V(H)Hs were 7.2 and 2.0μM, respectively. To our knowledge A85 is one of the highest affinity V(H)Hs that has yet been isolated against a hydrophobic hapten such as azoxystrobin.
authors.affiliation: School of Environmental Sciences, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Shokouh
authors.initials: S
authors.lastname: Makvandi-Nejad
authors.forename: Ted
authors.initials: T
authors.lastname: Fjällman
authors.forename: Mehdi
authors.initials: M
authors.lastname: Arbabi-Ghahroudi
authors.forename: C Roger
authors.initials: CR
authors.lastname: MacKenzie
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Hall
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity / Q000276:immunology; D002161:Camelids, New World / Q000235:genetics / Q000276:immunology; D002417:Cattle; D006241:Haptens / Q000276:immunology; D007114:Immunization; D015151:Immunoblotting; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008297:Male; D008689:Methacrylates / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019151:Peptide Library; D011743:Pyrimidines / Q000737:chemistry / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000073739:Strobilurins
chemical_list: D000906:Antibodies; D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D008689:Methacrylates; D019151:Peptide Library; D011743:Pyrimidines; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D000073739:Strobilurins; D012710:Serum Albumin, Bovine; C087670:azoxystrobin