Development of anti-aflatoxin B1 nanobodies from a novel mutagenesis-derived synthetic library for traditional Chinese medicine and foods safety testing

Yu-Ching Lee; Gar-Hwa Lai; Tsai-Yu Lin; Tien-Sheng Tseng; Tsung-Hsun Tsai; Wang-Chuan Chen; Cheng-Chung Lee; Keng-Chang Tsai

doi:10.1186/s13036-023-00350-y

Development of anti-aflatoxin B1 nanobodies from a novel mutagenesis-derived synthetic library for traditional Chinese medicine and foods safety testing

J Biol Eng. 2023 Apr 24;17(1):30. doi: 10.1186/s13036-023-00350-y.

Authors

Yu-Ching Lee^#^{1

2

3

4}, Gar-Hwa Lai^#⁵, Tsai-Yu Lin², Tien-Sheng Tseng⁶, Tsung-Hsun Tsai⁷, Wang-Chuan Chen^{8

9}, Cheng-Chung Lee¹⁰, Keng-Chang Tsai^{11

12}

Affiliations

¹ TMU Research Center of Cancer Translational Medicine, Taipei Medical University, Taipei, Taiwan.
² Ph.D. Program for Cancer Molecular Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
³ Ph.D. Program in Drug Discovery and Development Industry, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.
⁴ Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
⁵ Department of Orthopedics, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
⁶ Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan.
⁷ Department of Psychiatry, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan.
⁸ The School of Chinese Medicine for Post Baccalaureate, I-Shou University, Kaohsiung, Taiwan.
⁹ Department of Chinese Medicine, E-Da Hospital, Kaohsiung, Taiwan.
¹⁰ The Ph.D. Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
¹¹ Ph.D. Program in Medical Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan. tkc@nricm.edu.tw.
¹² National Research Institute of Chinese Medicine, Ministry of Health and Welfare, No. 155-1, Sec. 2, Linong St., Beitou District, Taipei, 11221, Taiwan. tkc@nricm.edu.tw.

^# Contributed equally.

Abstract

Background: The main commercially available methods for detecting small molecules of mycotoxins in traditional Chinese medicine (TCM) and functional foods are enzyme-linked immunosorbent assay and mass spectrometry. Regarding the development of diagnostic antibody reagents, effective methods for the rapid preparation of specific monoclonal antibodies are inadequate.

Methods: In this study, a novel synthetic phage-displayed nanobody Golden Glove (SynaGG) library with a glove-like cavity configuration was established using phage display technology in synthetic biology. We applied this unique SynaGG library on the small molecule aflatoxin B1 (AFB1), which has strong hepatotoxicity, to isolate specific nanobodies with high affinity for AFB1.

Result: These nanobodies exhibit no cross-reactivity with the hapten methotrexate, which is recognized by the original antibody template. By binding to AFB1, two nanobodies can neutralize AFB1-induced hepatocyte growth inhibition. Using molecular docking, we found that the unique non-hypervariable complementarity-determining region 4 (CDR4) loop region of the nanobody was involved in the interaction with AFB1. Specifically, the CDR4's positively charged amino acid arginine directed the binding interaction between the nanobody and AFB1. We then rationally optimized the interaction between AFB1 and the nanobody by mutating serine at position 2 into valine. The binding affinity of the nanobody to AFB1 was effectively improved, and this result supported the use of molecular structure simulation for antibody optimization.

Conclusion: In summary, this study revealed that the novel SynaGG library, which was constructed through computer-aided design, can be used to isolate nanobodies that specifically bind to small molecules. The results of this study could facilitate the development of nanobody materials to detect small molecules for the rapid screening of TCM materials and foods in the future.

Keywords: Aflatoxin B1; Glove-like cavity; Nanobody; Nonhypervariable complementarity-determining region 4 loop; Phage display technology.

Abstract

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