Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response

Sofia Tabares-Da Rosa, Martin Rossotti, Carmen Carleiza, Federico Carrión, Otto Pritsch, Ki Chang Ahn, Jerold A Last, Bruce D. Hammock, Gualberto González-Sapienza

Research output: Contribution to journalArticle

40 Scopus citations

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 (IC50 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, KD 0.98-1.37 nM (SPR), which allowed development of a competitive assay for TCC with an IC50 = 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 IC50 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.

Original languageEnglish (US)
Pages (from-to)7213-7220
Number of pages8
JournalAnalytical Chemistry
Volume83
Issue number18
DOIs
StatePublished - Sep 15 2011

ASJC Scopus subject areas

  • Analytical Chemistry

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    Tabares-Da Rosa, S., Rossotti, M., Carleiza, C., Carrión, F., Pritsch, O., Ahn, K. C., Last, J. A., Hammock, B. D., & González-Sapienza, G. (2011). Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response. Analytical Chemistry, 83(18), 7213-7220. https://doi.org/10.1021/ac201824z