Preliminary Preparation Of Enrofloxacin Colloidal Gold Test Strip

Experiment One,ovalbumin(OVA)and bovine serum albumin(BSA)were used as protein carriers to synthesize small molecule drugs EFLX complete antigens by active ester method,The conjugates formed in this way is EFLX-BSA and EFLX-OVA,The serum titer was detected by use of EFLX-BSA immunogen and EFLX-OVA coating antigen by ELISA,the specificity and detection limit(LD)was detected by Ci-ELISA.The results showed that the immunogen and coating antigen were successfully synthesized,The serum titer detection result is the first group was 25600,second and third groups were more than 51200,second were the highest of all;The result of Ci-ELISA indicated that This method was sensitive and had a linear detection range from 7.39 ng/mL to 403.43 ng/mL(R2=0.9939),with IC50 and LOD values of 54.60 ng/mL and 1.17 ng/mL.Experiment Two,to preparation and identification of monoclonal antibodies(mAb)against Enrofloxacin(EFLX)Immunogen is EFLX-BSA,which was synthesized using the activated ester method,the hybridoma cell strain secreting anti-EFLX mAb was obtained by hybridoma technology,The massive mAb were induced from in vivo method,the titer,affinity,sensitivity,specificity and subtype of the mAb were characterized.The hybridoma cell lines of 7C5 were screened for specificity to EFLX,the isotypes of the mAb were IgGl,The indirect ELISA titer of the mAb were 1.035×10-6 in supernatant,The result had a linear detection range from 2.05 ng/mL to 98.38 ng/mL(R2=0.9927),with IC50 and LOD values of 14.21 ng/mL and 0.56 ng/mL,the mAb had no cross reaction to other antibiotics.Experiment Three,the author purified the mouse ascites using HiTrapTM Protein A HP,and tested the optimum pH and the lowest amount of stability on enrofloxacin monoclonal antibody with colloidal gold-labeled,after that,the author labeled it with colloidal gold,tested the feasibility of enrofloxacin colloidal gold test strip by hand spotting method,and the dilution ratio with phosphate buffered saline(PBS).The results showed that T and C lines could be seen clearly when positive samples were detected;when the negative samples were detected,C line is clearly visible,T line is not visible.The results showed that enrofloxacin colloidal gold test strip was preliminarily developed in this experiment.In summary,this study successfully synthesized enrofloxacin artificial antigens,detected the status of the mice immunized,and prepared enrofloxacin monoclonal antibodies by cell engineering technology,and then have a preliminary research of enrofloxacin colloidal gold rapid diagnostic test strips.