Development Of ELISA Kit For Detecting Ractopamine Residues In Meat

Objectives: To produce the ascitictype monoclonal antibodies(MAbs) againstRactopamine(RAC) based on the positive hybridoma cell strain. To optimize andestablish an Enzyme-Linked Immunosorbent Assay(ELISA) for detecting ractopamineand develop the ELISA(ciELISA) kit for detecting ractopamine residue in meat.Methods: Ractopamine complete antigen was synthesized by using the active estermethod. The hybridoma cell lines which screening high titer and high affinity antibodieswere selected by cell cloning，and were used to produce ascitictype MAbs. Based onthese monoclonal antibodies produced，Ractopamine ELISA conditions includingmicrowell-plate，coating buffer，coating condition，blocking buffer，blocking time，reaction buffer，reaction time and antibody stabilizer were optimized. Then the indirectcompetitive ELISA for detecting ractopamine was established. Recoveries of ractopaminespiked in meat were determined by ciELISA. Accuracy and precision of the ELISA wereevaluated through testing the coefficient of variation of intra-assay and inter-assay.Results: The complete antigen of ractopamine were synthesized successfully. Themonoclonal antibodies titer was up to105，with low cross reactivity with other β-agonist.The optimum conditions of ELISA were given as follows：JCH microwell-plate wascoated with the antigen in CBS(pH9.6，0.05M).The plate was incubated at4℃for16hours.5%skimmed milk powder in PBS-T blocking the plate at37℃for1hours couldreduce the non-specific adsorption. Primary antibody and second antibody were diluted inPBS-T and incubated at37℃for1hours，TMB was used to develop the colour for10min.Under optimized conditions， the sensitivity of ELISA was1.05ng/ml(IC50)， thedetection limits (LOD）was0.97ng/ml(IC10)， the linear concentrations ranged wasfrom0.25to8ng/mL，the rate of recoveries were72.38%-85.69%，the intra-assay andinter-assay coefficients of variation were less than15%，the detection limit was0.47ng/g in swine muscle. The reactivities of the monoclonal antibodies could be remained at2-8℃for6months by using the optimized stabilizer. The accuracy and precision ofELISA kit met the requirements of the standard issued by Ministry of Agriculture of thePeople’s Republic of China.Conclusions: The high affinity and high specific McAbs against ractopamine wereprepared. A rapid，sensitive and specific indirect competitive ELISA for ractopamine detection was optimized and established. A sensitive，specific，accurate，stable andconvenient indirect competitive ELISA kit for detecting ractopamine in meat wasdeveloped.