title: Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response.
abstract: Single-domain antibodies (sdAbs) found in camelids lack a light chain, and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. Although this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low-affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC(50) 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, K(D) 0.98-1.37 nM (SPR), which allowed development of a competitive assay for TCC with an IC(50) = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC(50) attained with other antihapten VHHs reported thus far. Despite the modest overall antihapten sdAbs response in llamas, a small subpopulation of high-affinity VHHs is generated that can be isolated by careful design of the selection process.
authors.affiliation: Cátedra de Inmunología, Facultad de Química, Instituto de Higiene, Universidad de la República, Montevideo, Uruguay.
authors.forename: Sofia
authors.initials: S
authors.lastname: Tabares-da Rosa
authors.forename: Martin
authors.initials: M
authors.lastname: Rossotti
authors.forename: Carmen
authors.initials: C
authors.lastname: Carleiza
authors.forename: Federico
authors.initials: F
authors.lastname: Carrión
authors.forename: Otto
authors.initials: O
authors.lastname: Pritsch
authors.forename: Ki Chang
authors.initials: KC
authors.lastname: Ahn
authors.forename: Jerold A
authors.initials: JA
authors.lastname: Last
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
authors.forename: Gualberto
authors.initials: G
authors.lastname: González-Sapienza
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D001665:Binding Sites; D002161:Camelids, New World / Q000276:immunology; D002224:Carbanilides / Q000276:immunology; D006241:Haptens / Q000276:immunology*; D008297:Male; D019151:Peptide Library; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D000906:Antibodies; D002224:Carbanilides; D006241:Haptens; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C009540:triclocarban