[Preparation and characterization of monoclonal antibodies against EGFP]

To prepare monoclonal antibodies (mAbs) with high specificity and affinity against enhanced green fluorescent protein (EGFP), we used purified EGFP emulsified with Freund{L-End} 's adjuvant to immunize Balb/c mice. Mouse splenocytes were then fused with SP2/0 myeloma cells. Hybridoma cell lines stably secreting antibodies were obtained through indirect ELISA screening and subcloning with the limited dilution method. The epitope specificity, reactivity, and affinity of the antibodies were systematically analyzed via an additivity assay, isotype identification, Western blotting, and surface plasmon resonance (SPR). The results demonstrated the successful screening of two hybridoma cell lines, 1F11 and 3C11, which stably secreted antibodies specifically targeting EGFP, with isotypes of IgG2a and IgG2b, respectively. The additivity assay showed an additivity index (AI)>40%, indicating recognition of distinct antigenic epitopes. Antibody titers reached 1:512 000. Western blotting confirmed that the prepared antibodies could specifically recognize EGFP with a size of about 27 kDa. SPR analysis revealed affinity constants of 3.806×10^-10 mol/L and 2.631×10^-10 mol/L, respectively, both within the high-affinity range. These findings indicate that the prepared mAbs possess excellent specificity, reactivity, and high affinity, providing a specific and important biological tool for the immunological detection of EGFP-tagged proteins and the in vivo monitoring of vaccine strains.