Contribution of CDR3 in characterization of camelid VHH and its aflatoxin B1-binding interaction
The single variable domain of heavy chain (VHH) antibodies, owing to their unique properties, has gained attention in immunoassays. However, compared with macromolecule antigens, in the case of small molecule haptens, such as aflatoxin B1 (AFB1), the role of the longer complementarity determining re...
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Elsevier
2025-03-01
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| Series: | Food Chemistry Advances |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2772753X25000012 |
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| author | Hina Mukhtar Lan Ma Qian Pang Xiaohong Wang Jia Wang |
| author_facet | Hina Mukhtar Lan Ma Qian Pang Xiaohong Wang Jia Wang |
| author_sort | Hina Mukhtar |
| collection | DOAJ |
| description | The single variable domain of heavy chain (VHH) antibodies, owing to their unique properties, has gained attention in immunoassays. However, compared with macromolecule antigens, in the case of small molecule haptens, such as aflatoxin B1 (AFB1), the role of the longer complementarity determining region 3 (CDR3) of VHH is still indistinct. In this study, anti-AFB1 VHH was selected from a naïve VHH library, and its CDR3 roles in VHH characteristics were further explored. Anti-AFB1 VHH-2 was selected with 6.33 × 10–5 M of KD value. Two VHH chimeras (C3-Nb28 and C3-VHH2) were constructed by interchanging the CDR3 of VHH-2 with Nb28, a previously reported anti-AFB1 VHH from immunized alpaca, and vice versa. Refolded C3-Nb28 was obtained from the inclusion body, and C3-VHH2 was purified as a soluble protein for prokaryotic expression. Computational analysis showed that the binding of VHH-2 to AFB1 is mediated by two hydrogen bonds (Thr106 and Tyr115) and two hydrophobic interactions (Trp109 and Thr114) in CDR3. In contrast, no binding pockets were available in the CDR3 in either VHH chimera, which was further confirmed by VHH ELISA. Studying the CDR3 role of VHH can elucidate its effects and affinity for small molecular weight molecules and broaden its applications. |
| format | Article |
| id | doaj-art-c2c03e183b4e40ab9ff6b2a0eef37056 |
| institution | DOAJ |
| issn | 2772-753X |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
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| series | Food Chemistry Advances |
| spelling | doaj-art-c2c03e183b4e40ab9ff6b2a0eef370562025-08-20T02:55:44ZengElsevierFood Chemistry Advances2772-753X2025-03-01610088510.1016/j.focha.2025.100885Contribution of CDR3 in characterization of camelid VHH and its aflatoxin B1-binding interactionHina Mukhtar0Lan Ma1Qian Pang2Xiaohong Wang3Jia Wang4College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR ChinaCollege of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR ChinaCollege of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR ChinaCollege of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China; Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR ChinaCollege of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China; Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China; Corresponding author.The single variable domain of heavy chain (VHH) antibodies, owing to their unique properties, has gained attention in immunoassays. However, compared with macromolecule antigens, in the case of small molecule haptens, such as aflatoxin B1 (AFB1), the role of the longer complementarity determining region 3 (CDR3) of VHH is still indistinct. In this study, anti-AFB1 VHH was selected from a naïve VHH library, and its CDR3 roles in VHH characteristics were further explored. Anti-AFB1 VHH-2 was selected with 6.33 × 10–5 M of KD value. Two VHH chimeras (C3-Nb28 and C3-VHH2) were constructed by interchanging the CDR3 of VHH-2 with Nb28, a previously reported anti-AFB1 VHH from immunized alpaca, and vice versa. Refolded C3-Nb28 was obtained from the inclusion body, and C3-VHH2 was purified as a soluble protein for prokaryotic expression. Computational analysis showed that the binding of VHH-2 to AFB1 is mediated by two hydrogen bonds (Thr106 and Tyr115) and two hydrophobic interactions (Trp109 and Thr114) in CDR3. In contrast, no binding pockets were available in the CDR3 in either VHH chimera, which was further confirmed by VHH ELISA. Studying the CDR3 role of VHH can elucidate its effects and affinity for small molecular weight molecules and broaden its applications.http://www.sciencedirect.com/science/article/pii/S2772753X25000012VHHComplementarity determining regionAflatoxin B1VHH chimeraAntigen binding |
| spellingShingle | Hina Mukhtar Lan Ma Qian Pang Xiaohong Wang Jia Wang Contribution of CDR3 in characterization of camelid VHH and its aflatoxin B1-binding interaction Food Chemistry Advances VHH Complementarity determining region Aflatoxin B1 VHH chimera Antigen binding |
| title | Contribution of CDR3 in characterization of camelid VHH and its aflatoxin B1-binding interaction |
| title_full | Contribution of CDR3 in characterization of camelid VHH and its aflatoxin B1-binding interaction |
| title_fullStr | Contribution of CDR3 in characterization of camelid VHH and its aflatoxin B1-binding interaction |
| title_full_unstemmed | Contribution of CDR3 in characterization of camelid VHH and its aflatoxin B1-binding interaction |
| title_short | Contribution of CDR3 in characterization of camelid VHH and its aflatoxin B1-binding interaction |
| title_sort | contribution of cdr3 in characterization of camelid vhh and its aflatoxin b1 binding interaction |
| topic | VHH Complementarity determining region Aflatoxin B1 VHH chimera Antigen binding |
| url | http://www.sciencedirect.com/science/article/pii/S2772753X25000012 |
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