Preparation and identification of monoclonal antibodies against cat allergen Fel d 1.
- Author:
Linying CAI
1
;
Zichen ZHANG
1
;
Zhuangli BI
1
;
Shiqiang ZHU
2
;
Miao ZHANG
2
;
Yiming FAN
2
;
Jingjie TANG
1
;
Aoxing TANG
2
;
Huiwen LIU
3
;
Yingying DING
3
;
Chen LI
3
;
Yingqi ZHU
2
;
Guijun WANG
4
;
Guangqing LIU
5
Author Information
1. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036; Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai 200241, China.
2. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai 200241, China.
3. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China.
4. College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China. *Corresponding authors, E-mail: wgj@ahau.edu.cn.
5. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, Shanghai 200241, China. *Corresponding authors, E-mail: liugq@shvri.ac.cn.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies, Monoclonal/biosynthesis*;
Mice, Inbred BALB C;
Cats;
Mice;
Allergens/genetics*;
Glycoproteins/genetics*;
Enzyme-Linked Immunosorbent Assay;
Hybridomas/immunology*;
Recombinant Proteins/genetics*;
Female;
Antibody Specificity
- From:
Chinese Journal of Cellular and Molecular Immunology
2025;41(4):348-354
- CountryChina
- Language:Chinese
-
Abstract:
Objective Currently, there is no commercially available quantitative detection kit for the main Felis domestic allergen (Fel d 1) in China. To establish a rapid detection method for Fel d 1, this study aims to prepare monoclonal antibodies against Fel d 1 protein. Methods The codon preference of Escherichia coli was utilized to optimize and synthesize the Fel d 1 gene. The prokaryotic expression plasmid pET-28a-Fel d 1 was constructed and used to express and purify the recombinant Fel d 1 protein. Subsequently, the recombinant protein was immunized into BALB/c mice and monoclonal antibodies (mAbs) were prepared by the hybridoma technique. An indirect ELISA was established using the recombinant Fel d 1 as the coating antigen, and hybridoma cell lines were screened for positive clones. The specificity and antigenic epitopes of the mAbs were confirmed by Western blot analysis. Finally, the selected hybridoma cells were injected into the peritoneal cavities of BALB/c mice for large-scale monoclonal antibody production. Results The recombinant plasmid pET-28a-Fel d 1 was successfully constructed, and soluble Fel d 1 protein was obtained after optimizing the expression conditions. Western blot and antibody titer assays confirmed the successful isolation of two hybridoma cell lines, 7D11 and 5H4, which stably secreted mAbs specific to Fel d 1. Antibody characterization revealed that the 5H4 mAb was of the IgG2a subtype and could recognize the amino acid region 105-163 of Fel d 1, while the 7D11 mAb was the IgG1 subtype and could recognize the amino acid region 1-59. Conclusion The high-purity recombinant Fel d 1 protein produced in this study provides a promising alternative for clinical immunotherapy of cat allergies. Furthermore, the monoclonal antibody prepared in this experiment lays a material foundation for the in-depth study of the biological function of Fel d 1 and the development of ELISA detection.
