Polyclonal Antibody-Based Noncompetitive Immunoassay for Small Analytes Developed with Short Peptide Loops Isolated from Phage Libraries
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- 作者：A. Gonzá ; lez-Techera ; H. J. Kim ; S. J. Gee ; J. A. Last ; B. D. Hammock ; G. Gonzá ; lez-Sapienza
- 刊名：Analytical Chemistry
- 出版年：2007
- 出版时间：December 1, 2007
- 年：2007
- 卷：79
- 期：23
- 页码：9191 - 9196
- 全文大小：160K
- 年卷期：v.79,no.23(December 1, 2007)
- ISSN：1520-6882
文摘
To date, there are a few technologies for the developmentof noncompetitive immunoassays for small molecules, themost common of which relies on the use of anti-immunocomplex antibodies. This approach is laborious, casespecific, and relies upon monoclonal antibody technologyfor its implementation. We recently demonstrated that,in the case of monoclonal antibody-based immunoassays,short peptide loops isolated from phage display librariescan be used as substitutes of the anti-immunocomplexantibodies for noncompetitive immunodetection of smallmolecules. The aim of this work was to demonstrate thatsuch phage ligands can be isolated even when the selectorantibodies are polyclonal in nature. Using phenoxybenzoicacid (PBA), a major pyrethroid metabolite, as a modelsystem, we isolated the CFNGKDWLYC peptide afterpanning a cyclic peptide library on the PBA/anti-PBAimmunocomplex. The sensitivity of the noncompetitiveenzyme-linked immunosorbent assay (ELISA) setup withthis peptide was 5-fold (heterologous) or 400-fold (homologous) higher than that of the competitive assay setupwith the same antibody. Phage anti-inmunocomplex assay(PHAIA) was also easily adapted into a rapid and highlysensitive dipstick assay. The method not only provides apositive readout but also constitutes a major shortcut inthe development of sensitive polyclonal-based assays,avoiding the need of synthesizing heterologous competinghaptens.