title: Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay.
abstract: Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed, and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC(50) of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC(50) = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structural analogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.
authors.affiliation: Department of Entomology and UCD Cancer Center, University of California, Davis, California 95616, United States.
authors.forename: Hee-Joo
authors.initials: HJ
authors.lastname: Kim
authors.forename: Mark R
authors.initials: MR
authors.lastname: McCoy
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Majkova
authors.forename: Julie E
authors.initials: JE
authors.lastname: Dechant
authors.forename: Shirley J
authors.initials: SJ
authors.lastname: Gee
authors.forename: Sofia
authors.initials: S
authors.lastname: Tabares-da Rosa
authors.forename: Gualberto G
authors.initials: GG
authors.lastname: González-Sapienza
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
mesh_terms: D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology / Q000302:isolation & purification* / Q000652:urine; D001565:Benzoates / Q000276:immunology*; D002161:Camelids, New World / Q000276:immunology*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens / Q000276:immunology*; D006801:Humans; D007118:Immunoassay; D007143:Immunoglobulin Heavy Chains / Q000276:immunology*; D008297:Male; D019151:Peptide Library; D011722:Pyrethrins / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology / Q000302:isolation & purification* / Q000652:urine
chemical_list: D000888:Antibodies, Anti-Idiotypic; D001565:Benzoates; D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D019151:Peptide Library; D011722:Pyrethrins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C017618:3-phenoxybenzoic acid