Preparation Of Monoclonal Antibodies Against Ractopamine And The Establishment Of An Indirect Competition ELISA Method

Ractopamine(RAC) is a kind of β2-adrenergic receptor agonist which is used to treat bronchial asthma, bronchospasm and obstetric diseases clinically. Just like clenbuterol it can alter the metabolism way of nutrient, promote protein synthesis, inhibit the synthesis and accumulation of fat, and ultimately improve the quality of the carcass and promot the growth rate of animals. But high dosage of ractopamine has certain toxic side effects to animals, also it will produce harmful effects to human health after human consumption of animal food. EU has already explicity prohibited the use of all β2-adrenergic receptor agonist in animal food, including ractopamine. RAC is allowed to be used as feed additive for food animals in USA, but there are strict rules of the dosage and the operating procedures. In China, it is banned, but the phenomenon of the illegal use in swine production is still serious.Many physical and chemical methods to detect residue of RAC have been established at home and abroad, such as HPLC, GC-MS. Although these methods are accurate, sensitive and effective, they are not suitable for large batch samples’screening because of their expensive equipments, high technical request, complex sample preparation procedures and long time consuming. Enzyme-linked immunosorbent assay (ELISA) is now the most common method which is fast, sensitive, convenient and can screen a large number of samples at one time. Especially the detect results of ELISA have high consistent with the results of HPLC and GC-MS. Therefore ELISA is often used as a screening method.For exploring a method that can detect residue of RAC, we prepared anti-ractopamine monoclonal antibody, using monoclonal antibody and ELISA technique. Also we establish the CiELISA method for detecting RAC. The addition and reclamation test of samples proved that the established CiELISA method for detecting the residue of RAC was feasible.1. Synthesis and identification of RAC artificial antigens.RAC was linked with BSA and HSA, using the methods of MA and EDC. Then the artificial antigens RAC-BSA and RAC-HSA were prepared, and the synthesized antigens were demonstrated to be successful by UV-scanning and immunological methods. We selected RAC-BSA as immunogen and RAC-HSA as coating antigen after comparing experiments.2. The preparation of the Monoclonal Antibodies against RAC. Mice (BALB/c) were immunized by RAC-BSA, in the way of routine method. The titer of blood serum collected after the fifth immunization reached1:32,000. Then a hybridoma cell line (5D7) which could excrete anti-ractopamine monoclonal antibody steadily was gained by hybridoma’s technique. Monoclonal antibody against ractopamine was prepared by inside body induced ascites method. The protein concentration of McAb we got was21.3mg/mL, the titer of the McAb was1:128,000.3. The establishment of ELISA method. The CiELISA’s method was founded base on the McAb, RAC standard curve was prepared with the linear equation:Y=-0.4095x+0.7134(R2=0.991), IC504.75ng/mL, LOD under0.5ng/mL, LOQ0.92ng/mL. The results of specificity test showed that the McAb was100percent inhibition rate anti ractopamine while the cross-reactivity with other β2-adrenergic receptor agonists.4. The addition and reclamation test of RAC. The CiELISA’s method was applied in the addition and reclamation test of the liver sample of swine. The intervention disappeared when the extraction of liver sample was diluted4times. The standard curve equation was Y=-0.3972x+0.7237(R2=0.9892) ranging from0-50ng/mL and IC50was5.31ng/g, LOD was0.41ng/g. Adding1,2,5,10,20and50ng/mL RAC to the liver of swine, the recovery ratio was between80%and100%. The intraassay coefficient of variation was between3.28%and9.37%, and the interassay coefficient of variation was between4.75%and10.27%.