Preparation Of Monoclonal Antibody Against Salbutamol And Establishment Of Immunoassay Method For Salbutamol

Salbutamol is one of the selectiveβ2 receptor agonists, with the function of promoting muscle (lean) growth and reducing fat accumulation in animals. It is often illegally used as growth promoter since it can improve the feed conversion rate, increase lean meat yield. Salbutamol residues in food can cause serious impact on people's health, such as palpitations, headache, dizziness and breathing difficulties. As the detection technology of salbutamol residues is outdated, some criminal use salbutamol in the farm instead of clenbuterol as a growth promoter to seek economic benefits.There are many methods for salbutamol detection at present, such as gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and enzyme-linked immunosorbent assay (ELISA). In these methods, GC-MS, HPLC and LC-MS are accurate and reliable, but they are not suitable for production, grass-roots units and on-site inspection with the defects of cumbersome pre-treatment and high technical requirements. ELISA is a detection method on the basic of antigen-antibody reaction and enzyme-based chemical, with the advantages of sensitive, simple, and large amount of test sample. This method doesn't require complex and expensive instrument or equipment, and the professional requirement of the operator is relatively low.In this research, Succinic acid derivatives of Sal were acquired by alcoholysis reaction between Sal and succinic anhydride, and then the derivatives were coupled to BSA and ovalbumin, synthesizing complete antigen Sal-HS-BSA and Sal-HS-OVA. The UV scanning and SDS-PAGE are used to confirm that the Sal has connected to the carrier protein. The coupling ratios of BSA and ovalbumin are 7.7:1 and 8.8:1, respectively. The BALB/C mice were immunized with Sal-HS-BSA as immunogen. The mice serum antibody titer is 1: 103-104 detected by indirect ELISA.Select the cells of the mice with strong immune (antibody titer is 1:2.56×10⁴, IC50 is 270 ng/mL at least) for cell fusion with the cell-fusion rate of 100%. There are 1² heterokaryon cells in every pore. Six hybridoma cell lines were acquired through positive cells cloning. In these hybridoma cells, two lines, which named 2D9 and 1C4 respectively, with the characteristic of sensitive and high titer, were obtained by detecting the IC50. The two hybridoma cells, 2D9 and 1C4, were injected into the Balb/c adult mice for collecting of ascites. The protein content was 3.6 mg/mL, and with the purity of 78%⁸6% after purification. The antibody titers are 1:2.56×10⁴ and 1:4.096×10⁶ in the cell culture supernatant and ascites of 2D9 detected by indirect ELISA, respectively. The antibody titers are 1:1.28×10⁴ and 1:2.048×10⁵ in the cell culture supernatant and ascites of 1C4 detected by indirect ELISA, respectively. The secreted antibodies of 2D9 and 1C4 are both IgG1 with the identification of antibody subtypes.Blocking-ELISA was used to detect the sensitivity and specificity of Sal monoclonal antibody. The IC₅0 value was about 1.14 ng/mL, indicated that Sal monoclonal antibody has highly sensitive for Sal. The cross reaction of Sal monoclonal antibody with the carrier protein,Sal, clenbuterol hydrochloride, ractopamine, isoproterenol and norepinephrine was detected. The result of the CR showed that Sal monoclonal antibody has no cross reaction to ractopamine and other adrenal hormones, while 26.09% to clenbuterol hydrochloride. It is indicated that the Sal monoclonal antibody has highly specific.In this research, a high-titer, sensitive and specific Sal monoclonal antibody was acquired. The method of immunological detection was initially established on the basis of monoclonal antibody 2D9. The conditions of ELISA experiment were optimized.The standard curve of detecting Sal was established by indirect competitive ELISA,The equation was y=-32.595x+48.1 (R²=0.9841),the sensitivity and detection limit were 1.14 ng/mL and 0.15 ng/mL respectively. This will provide the foundation for the research of salbutamol residue immunoassay.