Production Of RabMab Of Ractopamine And Establishing Of ELISA Detecting Method

Ractopamine is a kind of phenethanolamine P -adrenergic agonist with N-alkylphenyl substituents. It has a lot of physiological effects. It can be used as clinical medicine for bronchitis and obstetrical diseases. When the dosage is 5-10 times higher than its clinical dosage, it can also regulate growth rate and repartition nutrition. Accumulation of P -adrenergic agonist in animals is dangerous for consumers, especially for those persons who have heart disease, diabetes, hypertension, sthenic thyroxin, glaucoma and prostate hypertrophy, and even cause death. At present, usage of P -adrenergic agonists as growth accelerant are strictly prohibited in China. But some poisoning events which caused by illegal usage of ractopamine are still happening. Effective detection methods are needed in order to prohibit illegal usage of ractopamine and protect consumer's safety. To date, there's no standard detection method for detecting ractopamine in China. It is lagged behind the detection method of other -adrenergic agonists such as clenbuterol. So to develop a sensitive, effective, specific and rapid analytic method for ractopamine detection has theoretical and practical significances.At present, there are no reports on RabMab for P -Adrenergic agonists. In our study, we synthesized the immunogen of ractopamine, immunized rabbits and obtained immunized spleen of rabbit. Hybridoma technology was used to produce a rabbit monoclonal antibody against the ractopamine. We established ELISA rapid detection process and ELISA kit for ractopamine. The results were shown as below:1. Immunogens were synthesized by mixed anhydride. Ractopamine-glutarate-BSA was used as the immunogen and ractopamine-glutarate-OVA was used as the coating antigen. The final compounds were identified by UV spectrophotometer, infrared spectrometer and LabChip kit of Agilent 2100 Bioanalyzer. The immunogens was synthesized successfully. The molecular weight of Ractopamine-glutarate-BSA was 76.4 kDa, the coupling ratio was 28:1. Bradford protein assay was used to detect the final concentrations of the protein. The protein concentration of ractopamine-glutarate-BSA and ractopamine-glutarate-OVA were 3.62 mg/mL and 1.156 mg/mL.2. WHB dark eye rabbits were immunized by Ractopamine-glutarate-BSA. The subcutaneous, lymphous and venous injection methods were used. Anti-sera with high titers were obtained after seven times immunization. Working concentrations of No.5 and No.6 rabbit anti-sera were 1:32X 10⁴ and 1:16 X 10⁴. Anti-sera were purified by saturated ammonium sulphate.3. After immunization, we obtained immunized rabbit spleen. Splenocytes from rabbit were fused with 240E cells at presence of 50% PEG6000. Fusion cells were plated in a total of 40 96-well culture plates. The fused cells were maintained in HAT selection medium. Indirect ELISA, competitive indirectELISA, and IgG quantitative assay were used for screening clones. 116 positive clones were selected by primary screening. A3-2-4, A3-2-6, B5-4-4 and B5-4-5 clones were selected after the second subclone. These hybridomas could secrete specific antibodies continuously and stably after freezing and anabiosis again and again. RabMab was produced by extended cultivating hybridomas in vitro.4. The screening process of ELISA was optimized. The best reaction conditions were: 96 ELISA plate coated by ractopamine-glutarate-OVA was incubated at 4°C for 12h, the primary antibody was incubated at 37°C for 60min, the second antibody was incubated at 37°C for 100 min, and color was developed and incubated at 37°C for 15min. TMB and H₂O₂ affect markedly the sensitivity of ELISA process. The best TMB and H₂O₂ concentration were 0.25g/L TMB and 0.02% H₂O₂.5. The indirect ELISA titer and working concentration of supernatant A3-2-4, A3-2-6, B5-4-4 and B5-4-5 were 1:1600, 1:3200, 1:50 and 1:100, respectively. The competitive inhibition curves of clone A3-2-4, A3-2-6, B5-4-4 and B5-4-5 were typical S-shaped. IC₅₀ of these supernatants were less than 20 ng/ml. It showed the supernatants had good affinities.6. The standard curve of A3-2-6 was established by inhibition indirect ELISA. The linear range of the inhibition rate vs log ractopamine competition curve generated in PBST was 0.1¹00ng/ml. And the IC₁₀ and IC₅₀ value of A3-2-6 were 0.2227ng/ml and 4.0907 ng/ml.7. Inhibition indirect ELISA was used to detect the specificity of supernatant A3-2-6. All cross-reactivity rates except dobutamine were less than 0.005% which means compounds that have similar structure to ractopamine had no cross-reactivity except dobutamine. Cross-reactivity rate of dobutamine was 32.23%. Clenbuterol, isoxsuprine, epinephrine, norepinephrine, isoproterenol, dopamine and salbutamol had no cross-reactivity with A3-2-6. Common veterinary medicine such as ephedrine hydrochloride, ampicillin sodium, penicillin sodium, oxacillin sodium, amoxicillin sodium, streptomycin sulfate and kanamycin sulfate had no cross-reactivity with A3-2-6. This showed that the RabMab to ractopamine hydrochloride possessed a high specificity.8. Veracity experiments of indirect ELISA assay showed recovery rates of urine samples with different concentration were high. The recovery rates were between 94.76% and 110.22%. Coefficients of variation were well below 10% except the sample added 5ng/mL ractopamine. Precision experiments of indirect ELISA assay showed the Coefficients of variation of between days assay were well below 10%. These results showed the ELISA assay had good accuracy. An indirect ELLSA kit was produced in this study.