Monoclonal Antibody Production Targeting Recombinant Goat CD4 and CD25

Expression vector PET32a and PGX-6P-1 were preserved in our laboratory. Recombinant expression and affinity column purification of the extracellular region of goat CD4 and CD25 genes were conducted in E. coli (Gao et al. 2019 (link); Yang et al. 2019 (link)). Two recombinant strains were successfully constructed using expression vectors CD4-PGX-6P-1 and CD25-PET32a, which were named as “rCD4” and “rCD25.” Six-week-old female BALB/c mice were immunized four times with purified recombinant proteins, and the procedures for immunization and cell fusion using PEG1500 (Sigma-Aldrich) were performed as previously described (Wagner et al. 2006 (link)). Hybridoma lines producing mAbs that only recognized the recombinant proteins but not the tag protein were screened by ELISA. Positive hybridoma cells that had been cloned three times were used to make ascites (Li et al. 2019b (link); Liu et al. 2022 (link); Ma et al. 2019 (link)). The ascites of mice were purified by protein G agarose (Beyotime, Shanghai, China), labeled with a conjugation kit (Bioss, Beijing, China).