Abstract:

【Objective】 The aim of the study is to prepare high sensitivity and specificity monoclonal antibody (mAb) against Gentamicin (GM) and identifiy its immunological characteristics. The monoclonal antibody against GM would lay a foundation for development of GM ELISSA kit and strip. 【Method】 The GM was conjugated to carrier protein BSA and OVA by EDC to obtain the artificial immunogen and reactionogen and SDS-PAGE was used to identify these artificial antigens. Balb/c mice were immunized with BSA-GM. The titer of polyclonal antibody (pAb) was detected by indirect ELISA and blocking ELISA after three times immunization. The high titer, sensitivity and specificity mouse was selected for cell fusing. The hybridoma lines that secrete GM mAb were established by using monoclonal antibody hybridoma technology and the immunological characteristics such as titer, sensitivity and specificity of the mAb were characterized. A rapid test ELISA Kit was established and identified by blocking ELISA and has been used for the GM standard samples in milk detection. 【Result】 SDS-PAGE results showed that GM artificial antigen was synthesized successfully. Three BALB/c mice indirect ELISA titer against GM were above 10-3 and the IC50 of No. 2 mice was 17.28 ng•mL-1. Four hybridoma cell lines, 5E2-D7, 1A3-A9, 5E2-A12, and 2A6-B5, were screened for specificity to GM after cell fusing, the indirect ELISA titer of the mAb were 1﹕1600, 1﹕800, 1﹕800, and 1﹕800 in supernatant, 1﹕1.02×107, 1﹕5.12×106, 1﹕2.56×106, and 1﹕2.56×106 in ascites, the mAb of 5E2-D7 showed good sensitivity with an IC50 of 0.30 ng•mL-1 to GM, no cross-reactivity to other compounds. The recovery of GM spiked in milk was 96.0% and the coefficient variation was below 15%. 【Conclusion】 The high-titer, sensitivity and specificity GM mAb has been prepared and laid a foundation for GM residue detection.

Key words: gentamicin')">gentamicin, monoclonal antibody, hybridoma, preliminary application