Generation and characterization of monoclonal antibodies against chicken interleukin 4.

Xiaoyu GUAN; Zhichao XU; Yongqiang WANG; Xiaoqi LI; Hong CAO; Shijun ZHENG

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Generation and characterization of monoclonal antibodies against chicken interleukin 4.

Author: Xiaoyu GUAN ¹ ; Zhichao XU ¹ ; Yongqiang WANG ¹ ; Xiaoqi LI ¹ ; Hong CAO ¹ ; Shijun ZHENG ¹
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1. State Key Laboratory of Agrobiotechnology, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
Publication Type:Journal Article
Keywords: chicken interleukin 4; monoclonal antibody; prokaryotic expression
From: Chinese Journal of Biotechnology 2017;33(1):44-54
CountryChina
Language:Chinese
Abstract: To develop monoclonal antibodies (McAbs) against chicken interleukin 4 (chIL-4), we subcloned the mature chIL-4 gene into prokaryotic expression vectors pET-28a and pGEX-6P-1, then expressed and purified the recombinant proteins. We immunized BALB/c mice with the purified His-chIL-4 protein and fused the murine splenocytes with SP2/0 after 4 times of immunization. We used the GST-chIL-4 protein as a coating antigen to establish an indirect ELISA to screen positive clones. After screening and 3 rounds of cloning process, we obtained 3 hybridomas that stably secreted McAbs against chIL-4, and named 1G11-3B, 2E5-3D, and 1G11-5H. The isotypes of these McAbs were all IgG1 and the dissociation constant (Kd) of these McAbs were 1.79×10⁻⁹, 1.61×10⁻⁹, and 2.36×10⁻⁹, respectively. These McAbs specifically bound to chIL-4 expressed by either prokaryotic or eukaryotic system as determined by Western blotting and indirect immunofluorescence assay. The binding domains of chIL-4 recognized by 1G11-3B, 2E5-3D, and 1G11-5H were located between aa 1-40, 80-112, and 40-80, respectively, as determined by Western blotting. These McAbs would help to detect chIL-4 and to elucidate the biological roles of chIL-4 in immune responses.