Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response
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 fo...
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Published in | Analytical chemistry (Washington) Vol. 83; no. 18; pp. 7213 - 7220 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
15.09.2011
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Subjects | |
Online Access | Get full text |
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Summary: | 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, K D 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. |
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Bibliography: | PMCID: PMC3193053 |
ISSN: | 0003-2700 1520-6882 |
DOI: | 10.1021/ac201824z |