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Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library

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Abstract

Compared with peptide-based mimotope, anti-idiotypic antibodies (AIds) are considered as promising biosynthetic surrogate antigen because these antibodies display stable protein conformation. Nevertheless, conventional AIds are generated by immunizing animals with heterologous idiotypic antibody in vivo; isolated AIds commonly exhibit a higher affinity to primary antibodies than target analytes because AIds undergo an affinity-matured process during immune responses, resulting in low sensitivity in competitive immunoassay. In the present study, an anti-citrinin monoclonal antibody (anti-CIT McAb) was designed as primary antibody; one β-type AI alpaca heavy chain single domain antibody (β-AI VHH) was selected as a citrinin (CIT) surrogate from a naive phage-displayed VHH library. The affinity constant (K D) of obtained β-AI VHH to anti-CIT McAb (160 nM) is 2.35 times lower than that of CIT and ovalbumin conjugates (CIT-OVA) to anti-CIT McAb (68 nM). The developed VHH-based enzyme-linked immunosorbent assay (V-ELISA) can be used to perform dynamic linear detection of CIT in 10 % (v/v) methanol/PBS from 5.0 to 300.0 ng/mL, with a median inhibitory concentration (IC50) of 44.6 ng/mL (n = 3); this result was twice as good as that of indirect competitive ELISA (ic-ELISA, IC50 = 96.2 ng/mL) with CIT-OVA as a coating antigen. Moreover, the precision of V-ELISA was evaluated by analyzing average recoveries and coefficient of variations of CIT-spiked cereal sample; the reliability of V-ELISA was also validated with a conventional ic-ELISA. In summary, the proposed strategy has a great potential for panning other β-AI VHH toward small organic molecules from a naive VHH library.

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Acknowledgments

This work was financially supported by grants from National Basic Research Program of China (grant 2013CB127804), the National Natural Science Foundation of China (grants NSFC-31360386, NSFC-31201360, and NSFC-31171696), the Program of State Key Laboratory of Food Science and Technology (project no. SKLF-ZZA-201302).

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Authors and Affiliations

  1. State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University, Number 235 Nanjing East Road, Nanchang, 330047, Jiangxi, China

    Yang Xu, Liang Xiong, Yanping Li, Yonghua Xiong, Zhui Tu, Jinheng Fu & Bo Chen

  2. Department of Preventive Medicine, Gannan Medical College, Number 1 Yixueyuan Road, Ganzhou, 341000, Jiangxi, China

    Liang Xiong

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  1. Yang Xu
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  2. Liang Xiong
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  3. Yanping Li
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  4. Yonghua Xiong
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  5. Zhui Tu
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Correspondence to Yanping Li.

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Xu, Y., Xiong, L., Li, Y. et al. Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library. Anal Bioanal Chem 407, 5333–5341 (2015). https://doi.org/10.1007/s00216-015-8693-3

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  • DOI: https://doi.org/10.1007/s00216-015-8693-3

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