title: Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol.
abstract: A single-domain variable heavy chain (V(H)H) antibody fragment specific to the mycotoxin 15-acetyldeoxynivalenol (15-AcDON) was obtained after immunization of a llama (Llama glama) with the protein conjugate 15-DON-BSA plus TiterMax Classic adjuvant. After confirmation of a polyclonal response to DON toxin in both conventional (cIgG) and heavy chain antibody (HCAb) fractions, a V(H)H library was constructed from amplified cDNA by nested PCR. V(H)H fragments with binding affinity for the mycotoxin were selected by panning of the phagemid library against microtiter plates coated with 15-DON-OVA. The dominant clone (NAT-267) was expressed in E. coli and was purified as a V(H)H monomer (mNAT-267) at a final concentration of 1.3 mg mL(-1). Isolated NAT-267 V(H)H DNA was fused to the homopentamerization domain of the B subunit of verotoxin to generate the pentabody format of single-domain antibody (sAb). The V(H)H pentamer (pNAT-267) was expressed in E. coli and was purified at a final concentration of 1.0 mg mL(-1). Surface plasmon resonance (SPR) analysis of soluble mNAT-267 binding kinetics to immobilized 15-DON-Horse Radish Peroxidase (HRP) indicated a dissociation constant (K(D)) of 5microM. Competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and fluorescence polarization assay (FPA) inhibition experiments with monomer and pentamer confirmed binding to 15-AcDON. Competitive inhibition FPAs with mNAT-267 and pNAT-267 determined IC(50) values of 1.24 and 0.50 microM, respectively, for 15-AcDON hapten. These values were similar to the IC(50) value of 1.42 microM for 15-AcDON given by polyclonal llama serum sampled 56 days after immunization. Competition formats for structurally related trichothecenes resulted in no cross-reactivity to: DON; 3-acetyldeoxynivalenol (3-AcDON); neosolaniol (NEO); diacetoxyscirpenol (DAS); and T-2 toxin. Our study confirmed that recombinant V(H)H fragments capable of binding low molecular weight haptens can be produced through the creation and panning of hyper-immunized single-domain (sdAb) libraries.
authors.affiliation: Department of Environmental Biology, University of Guelph, Guelph, Ontario, N1G 2W1 Canada.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Doyle
authors.forename: Mehdi
authors.initials: M
authors.lastname: Arbabi-Ghahroudi
authors.forename: Nathalie
authors.initials: N
authors.lastname: Gaudette
authors.forename: Gordon
authors.initials: G
authors.lastname: Furzer
authors.forename: Marc E
authors.initials: ME
authors.lastname: Savard
authors.forename: Steve
authors.initials: S
authors.lastname: Gleddie
authors.forename: Michael D
authors.initials: MD
authors.lastname: McLean
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; D000915:Antibody Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009183:Mycotoxins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D014255:Trichothecenes / Q000737:chemistry*
chemical_list: D007128:Immunoglobulin Fragments; D009183:Mycotoxins; D011994:Recombinant Proteins; D014255:Trichothecenes; C038972:acetyldeoxynivalenol