Development and application of a camelid single-domain antibody recognizing a linear B-cell epitope in glutamate dehydrogenase of Clostridium difficile
10.3760/cma.j.cn112309-20241009-00342
- VernacularTitle:识别艰难梭菌谷氨酸脱氢酶线性B细胞抗原表位的驼源单域抗体的研制与应用
- Author:
Huaqian ZHAI
1
;
Zhezhou LI
;
Mengting CAI
;
Kai ZHANG
;
Lijun SHEN
;
Yongneng LUO
;
Dazhi JIN
;
Hui HU
Author Information
1. 杭州医学院检验医学院，杭州 310053
- Publication Type:Journal Article
- Keywords:
Clostridium difficile;
Glutamate dehydrogenase;
Epitope;
Camelid;
Single-domain antibody
- From:
Chinese Journal of Microbiology and Immunology
2025;45(8):629-635
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To develop a camelid single-domain antibody (SdAb) recognizing linear B-cell epitopes in glutamate dehydrogenase of Clostridium difficile(CD-GDH), and to apply it in Western blot and ELISA. Methods:Purified recombinant CD-GDH was used as bait to screen phage-displayed camelid SdAb library and obtain positive clones. Then those clones were confirmed by Western blot, and their variable domain of heavy chain of heavy chain antibody(VHH) nucleotide sequence were determined. The VHH sequence was synthesized after codon optimization and cloned into the expression vector pET28a. The SdAb was then expressed and purified, and its ability to detect CD-GDH protein in multiple assays was further explored.Results:Six positive clones were obtained, among which clone GA4 was chosen for recombinant expression in Escherichia coli and further purification. The purified GA4 binded well with CD-GDH with a Kd value of 3 nmol/L. In Western blot and ELISA, GA4 was proven to be able to selectively detect both recombinant and endogenous CD-GDH. Conclusions:A camelid SdAb targeting a linear B-cell epitope in CD-GDH is successfully developed, which provides a very useful tool for detecting CD-GDH.
