Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassayAnalytica Chimica Acta

About

Authors
Yu-Lou Qiu, Qing-Hua He, Yang Xu, Arun K. Bhunia, Zhui Tu, Bo Chen, Yuan-Yuan Liu
Year
2015
DOI
10.1016/j.aca.2015.06.033
Subject
Analytical Chemistry / Spectroscopy / Biochemistry / Environmental Chemistry

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Text

Accepted Manuscript

Deoxynivalenol-mimic nanobody isolated from a na?ve phage display nanobody library and its application in immunoassay

Yu-Lou Qiu, Qing-Hua He, Yang Xu, Arun K. Bhunia, Zhui Tu, Bo Chen, Yuan-Yuan.

Liu

PII: S0003-2670(15)00834-X

DOI: 10.1016/j.aca.2015.06.033

Reference: ACA 233998

To appear in: Analytica Chimica Acta

Received Date: 26 March 2015

Revised Date: 19 June 2015

Accepted Date: 25 June 2015

Please cite this article as: Y.-L. Qiu, Q.-H. He, Y. Xu, A.K. Bhunia, Z. Tu, B. Chen, Y.-Y. Liu,

Deoxynivalenol-mimic nanobody isolated from a na?ve phage display nanobody library and its application in immunoassay, Analytica Chimica Acta (2015), doi: 10.1016/j.aca.2015.06.033.

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Graphical Abstract

Anti-DON

ScFv

Anti-DON

ScFv

Nanobody

N-28

DON

Mimicking

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ACCEPTED MANUSCRIPT 1

Deoxynivalenol-mimic nanobody isolated from a na?ve phage display nanobody library and its application in immunoassay

Yu-Lou Qiu a, Qing-Hua He a,*, Yang Xu a,**, Arun K. Bhunia b, Zhui Tu a, Bo Chen a,

Yuan-Yuan, Liu a a State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute,

Nanchang University, No. 235 Nanjing East Road, Nanchang 330047, China b Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University,

West Lafayette, Indiana, USA * Corresponding author at: State key laboratory of Food Science and Technology, Sino-German

Joint Research Institute, Nanchang University, NO. 235 Nanjing East Road, Nanchang 330047,

China. Tel.:+86-791-8830 5177; E-mail Address: heqinghua@ncu.edu.cn (Q.-H. He). ** Corresponding author at: State key laboratory of Food Science and Technology, Sino-German

Joint Research Institute, Nanchang University, NO. 235 Nanjing East Road, Nanchang 330047,

China. Tel.:+86-791-8833 3708; E-mail Address: xuyang@ncu.edu.cn (Y. Xu).

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ABSTRACT

In this study, using mycotoxin deoxynivalenol (DON) as a model hapten, we developed a nanobody-based environmental friendly immunoassay for sensitive detection of DON. Two nanobodies (N-28 and N-31) which bind to anti-DON monoclonal antibody (MAb) were isolated from a naive phage display library. These nanobodies are clonable, thermally stable and mycotoxin-free products and can be served as coating antigen mimetics in heterologous immunoassay. The half inhibition concentration (IC50) of the immunoassay developed with N-28 and N-31 was 8.77? 0.41 ng mL-1 and 19.97?0.84 ng mL-1, respectively, which were 18- and 8-fold more sensitive than the conventional coating antigen (DON-BSA) based immunoassay. In order to better understand the molecular mechanism of antigen mimicry by nanobody, the 3D structure of ?nanobody (N-28) - anti-DON MAb? complex was presented and verified by molecular modeling and alanine-scanning mutagenesis. The results showed that hydrogen bond and hydrophobic interaction formed between Thr 102 Ser 106 of N-28 and CDR H3 residues of anti-DON antibody may contribute to their binding. This novel concept of enhancing sensitivity of immunoassay for DON based on nanobody may provide potential applications in a general method for immunoassay of various food chemical contaminants.

Keywords: Nanobody, immunoassay, deoxynivalenol, mimetics

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ACCEPTED MANUSCRIPT 3 1. Introduction

The presence of chemical contaminants (e.g. mycotoxins, pesticides, and veterinary drugs) in foods is a serious concern since it poses a threat to the health of humans and animals. Therefore, it is necessary to develop rapid analytical and detection techniques for continued surveillance of foods and feedstuffs [1, 2]. To date, a number of analytical techniques have been developed for chemical contaminant analysis, including chromatography [3], mass spectrometry [4], surface enhanced

Raman scattering [5], NMR spectroscopy [6] and immunoassays [7]. Among these, immunoassays have been found widespread application as rapid screening techniques because of specificity, sensitivity, low cost, and suitability for high throughput screening.

At present, improving the sensitivity of immunoassays has been the major focus by employing a wide variety of approaches: development of natural or artificial ligands such as antibody fragments, aptamers and peptides with high affinity [8-10]; the application of signal amplification strategies, such as avidin-biotin system [11], immunogold-silver [12], immune-PCR [13], and novel immunoassay-based sensor platforms [14]. Furthermore, for development of sensitive and reliable immunoassay for small molecules, the quality and selectivity of hapten-protein conjugates are critical. For detection of hapten-protein conjugates, the heterologous immunoassay system (i.e., the immunizing hapten and the coating hapten differ in their molecular structures) presents a good alternative to homologous assays in achieving higher sensitivity in competitive immunoassay format [15-17]. Although heterology format is

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ACCEPTED MANUSCRIPT 4 very useful, the procedures of chemical synthesis of hapten-protein conjugates are over elaborative and complicated. Especially, in regard to the synthesis of toxin-carrier conjugates, which are usually chemosynthesized by ?trial and error? approach, involving handling of the toxin which pose a threat to the environment and human health, and the lot-to-lot variation, affecting immunoassay results [18, 19].

In recent years, researchers have found that peptides [20-22], peptide-protein conjugates [19] and anti-idiotypic antibodies [23] can replace the conventional hapten-protein conjugates of small molecules. In addition, the use of nanobodies as antigen mimetics in immunoassay for mycotoxin detection was reported. Wang et al. constructed a phage displayed anti-idiotypic nanobody library from an Alpaca immunized with anti-aflatoxin B1 (AFB1) MAb. Three anti-idiotypic nanobodies were isolated from this library and applied to immunoassay as a coating antigen for detection of AFB1 with a detection value of IC50 was 0.16 ng mL-1 [24]. Compared to peptide-based mimetics of hapten-protein conjugates, the nanobody-based mimetics have the advantages of high solubility, thermal and chemical stability, thus suitable for detection application in harsh conditions.