Study On The Immunoassay For Multi-residue Detection Of β-agonists Based On Dual-antigen

β-receptor agonists are divided into two classes: the aniline type and the phenol type.The clenbuterol,which belongs to the aniline type β-receptor agonists;and ractopamine,belongs to the phenol type β-receptor agonists.As an animal feed additive,it can significantly improve the feed conversion rate and lean lean conversion rate.But its residue in the animal body,through the food chain into the human body,thereby endangering human health.They are therefore banned worldwide.In this paper,using enzyme-linked immunosorbent assay(ELISA)and gold immunochromatography assay(CGIA)was established for β-receptor agonist multi residue detection technology.The results obtained are as follows:1.To the starting material of the p-hydroxybenzaldehyde and the nitrocarbol,and combined with Raney nickel reduction method,de novo synthesis of ractopamine amino derivatives,namely RAC-NH2.Using RAC-NH2 as the starting material,like clenbuterol,RAC-HSA,RAC-BSA,CLE-HSA and CLE-BSA can be produced by the diazotized reaction.On the basis of this,the RAC-NH2 and CLE are mixed in a certain molar ratio,and the reaction is carried out.Using UV spectrophotometer and SDS-PAGE electrophoresis as the test methods.Respectively,the Immunogen CLE-HSA-RAC and the coating antigen CLE-BSA-RAC were synthesized.2.Using the synthesized CLE-HSA-RAC as immunogen,the mice were immunized with the conventional immunization method,Comprehensive valence and sensitivity of two aspects,mice of CLE-RAC-2 were selected to carry on the shock immunity,and the cell fusion test was carried out.After three rounds of sub clones,two 3A2 and 4D8 strains were obtained to secrete anti CLE-RAC cell lines,4D8,the cell strain secreting monoclonal antibody,had the best sensitivity to CLE-RAC,IC50 was 0.325± 0.021 ng/m L and the titer was 64000.Therefore,4D8 cell line was injected into mice to induce ascites.3.An indirect competitive ELISA method for the detection of CLE and RAC was established by using CLE-BSA-RAC artificial antigen and CLE-RAC monoclonal antibody(4D8).This method detects CLE and RAC IC50 value is 0.77 ng/mL,the minimum detection limit of IC10 is 0.14 ng/mL.The cross reaction of clenbuterol and ractopamine and mabuterol rate is very high(CR>96%).At the same time,cimbuterol,bromide,mapenterol,cimetrol,bambuterol and clenproperol also specificity(17%<CR<85%).It implements multi residue detection.The rest of the several receptor agonists did not cross react with(CR<0.1%).In the actual samples,the minimum detection limit of pig urine,pork,pork liver,and feed are 0.302,0.603,0.804 and 12.42 ng/mL.The lowest limit of quantification were 0.518,0.858,1.152 and 20.583 ng/mL.The recovery rate was 78%-118.00%,and the recovery rate was 84.00%-114.00%.The coefficient of variation within the plate is less than 14.04% and the coefficient of variation of the plate is less than 7.69%.LC-MS/MS method is used to verify this method is reliable.4.CLE-RAC colloidal gold labeled antibody was prepared by sodium citrate reduction method,and colloidal gold test strips were successfully prepared.Naked eye detection limits of RAC and CLE and other 9 kinds of β-receptor agonists can reach the level of 5 ng/mL,consistent with icELISA,It implements a multi residue detection,and detection time is short,can be detected in the field.In this paper,we used the technology of hybrid tumor cells to produce CLE-RAC monoclonal antibody,and established the icELISA method and gold standard test strip detection technology,which provided a reliable multi residue detection method for CLE,RAC and other β-receptor agonists.