1. Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response
- Author
-
Martín A. Rossotti, Otto Pritsch, Federico Carrión, Bruce D. Hammock, Carmen Carleiza, Ki Chang Ahn, Sofía Tabares-da Rosa, Gualberto González-Sapienza, Catedra de Inmunología, Universidad de la República [Montevideo] (UCUR)-Facultad de Quimica-Instituto de Higiene [Montevideo], Universidad de la República (UDELAR)-Universidad de la República (UDELAR), Zoo Parque Lecocq, Intendencia Municipal de Montevideo, Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP), Departamento de inmunobiologia, Facultad de Medicina- Universidad de la República [Montevideo] (UCUR), Department of Entomology, School of Medicine-University of California, Pulmonary/Critical Care Medicine, and This work was supported in part by NIH Fogarty International Center Grant TW05718, NIEHS Superfund Basic Research program, P42 ES04699, and the NIEHS Center, P30 ES05707. B.D.H. is a George and Judy Marcus Senior Fellow of the American Asthma Foundation.
- Subjects
Male ,MESH: Carbanilides ,Immunoglobulin light chain ,01 natural sciences ,Antibodies ,Article ,Analytical Chemistry ,law.invention ,MESH: Recombinant Proteins ,03 medical and health sciences ,Antigen ,Peptide Library ,law ,Animals ,MESH: Animals ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Binding site ,Peptide library ,030304 developmental biology ,0303 health sciences ,MESH: Single-Chain Antibodies ,Binding Sites ,Chemistry ,MESH: Antibodies ,010401 analytical chemistry ,Surface Plasmon Resonance ,Molecular biology ,Recombinant Proteins ,MESH: Male ,0104 chemical sciences ,MESH: Surface Plasmon Resonance ,Single-domain antibody ,MESH: Binding Sites ,MESH: Haptens ,Recombinant DNA ,MESH: Camelids, New World ,MESH: Peptide Library ,Camelids, New World ,Haptens ,Hapten ,Carbanilides ,Single-Chain Antibodies - Abstract
International audience; 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.
- Published
- 2011
- Full Text
- View/download PDF