Preparation Of Monoclonal Antibody Against Benzodiazepine And Establishment Of Indirect Competitive ELISA Assay

Benzodiazepines have been illegally added to animal feed as sedatives and anti-stress drugs in recent years to accelerate animal fattening.Residues in animal foods endanger human health.The Ministry of Agriculture prohibits the addition of animal feed.Benzodiazepines such as nitrazepam,diazepam and oxazepam.At present,there are few studies on the rapid detection technology of benzodiazepine residues in China.In this study,monoclonal antibodies were prepared using diazepam complete antigen,and an indirect competitive ELISA assay for benzodiazepines was initially established using this monoclonal antibody.Obtaining high sensitivity and high titer monoclonal antibodies is one of the prerequisites for the establishment of ELISA assay methods.Comparing the adsorption rates of different types of carbon nanotubes(carboxy multi-wall short carbon nanotubes,carboxyl single-wall long carbon nanotubes,multi-wall short carbon nanotubes,single-wall long carbon nanotubes)to BSA,the results show that single-wall long carbon The adsorption rate of nanotubes at the concentration of 5 mg/m L can reach 94.8%,which is better than the other three.BALB/c mice were immunized with Freund's adjuvant and different concentrations of single-walled long carbon nanotubes,and the antibody titer in serum was determined.The results showed that compared to Freund's adjuvant,the same potency range(1:8000~1:16000)was achieved,and 2.5 mg/m L single-walled long carbon nanotubes required only half the dose of diazepam-bovine serum albumin.(DZP-BSA),and can also reduce hair loss and stress death in mouse skin,but the antibody prepared by the single-wall long carbon nanotube immunization group has low affinity for the diazepam hapten,and the antibody level is weak,which is not suitable.Establish an ELISA assay.The immune spleen cells of the Freund's adjuvant group were fused with myeloma cells(1:5).After subcloning three times,a cell line secreting benzodiazepine monoclonal antibodywas obtained,which was named 4G6A5E9C7.Cloned antibodies in 30 benzodiazepines against diazepam,metronidazole,temazepam,flunitrazepam,2-hydroxyethylfluoxetine,tetrahydrocitabine,alprazolam,pu There is a significant cross-reaction of the drug of Racine.The indirect competitive ELISA assay established with this monoclonal antibody has a sensitivity of 1.67 ng/m L.For the detection of pig urine samples,the detection limit of this method is 0.08 ng/m L,the intra-assay recovery rate(79.36%~95.61%)and the inter-assay recovery rate(75.85%~103.78%)are all in the range of(70%~120%).The intra-assay coefficient of variation(4.78%~6.79%)and the inter-assay coefficient of variation(6.05%~9.38%)are within 15%,which meets the basic requirements of ELISA detection,laying the foundation for the development of benzodiazepine ELISA rapid test kit.basis.