Generation And Identification Of Monoclonal Antibody For Anti-Salbutamol And Establishing Immunoassay Method Of Salbutamol

Salbutamol(Sal), one of the synthesized β2-adrenergic agonists, used for asthma and other human respiratory system disease in the early time. It has been approved that salbutamol has a powerful effect promoting animal growth. It can increases accretion of skeletal muscle mass and decrease accretion of body fat. For this purpose, Sal has been illegally used at home and abroad as a growth-promoting agent to improve feed conversion efficiency and enhance lean meat to fat ration. As an kind of growth-promoting agent the dosage of salbutamol far more exceeds the normal curing dosage, the accumulative residue of sal in food producing animals can cause poisoning symptoms such as dizzy, quickening heartbeat, muscle dindling, palpitation and so on in human. The government of our country has proclaimed β 2.adrenergic agonists is prohibited as additive of animals feed. FDA and WHO proposed maximal residue of sal in urine did not exceed 1.2ng/ml, 0.6ng/g in tissue. The government of HONGKONG has legislated prohibiting β 2.adrenergic agonists such as sal as feed additive. As a member of WTO the residue of β- 2.adrenergic agonists such as sal will badly impacts the export of animal products of our country. It is very important how to establish a kind of credible, sensitive and quick assaying method.Sal conjugated antigen (Sal-KLH Sal-OVA) were procuced by coupling Sal with Keyhole limpet haemocyanin (KLH) or Ovalbumin (OVA) through EDC. Sal-KLH was used as immune antigen Sal-OVA as coating antigen. Anti-Sal monoclonal antibodies were produced by conventional cell fusion technology. The spleen cells of immunizedBlab/c mouses were fused with SP2/0 plasmacytoma cells using PEG4000, and selected with HAT and HT medium. Three hybridoma cell lines of 2A6 3B3 2H7 were screened for specificity to Sal with Sal-OVA using an indirect ELISA, and cloned by dilution method for three times, which secrete high affinity monoclonal antibodies anti-Sal with indirect ELISA titers of 1: 8.0X104 1: 3.2 X105, 1: 5.0 X 104.On the basis of prohibitive ELISA, Anti-Sal monoclonal antibodies showed good sensitivity with an IC50 of 40.5ng/ml toward Sal, 40.5ng/ml toward CL, more than 3906ng/ml towards Norepinephrine, Epinephine, Isoprenaline and Ractopamine.With Anti-Sal monoclonal antibodies we established method immunoassay of Sal based on prohibitive ELISA. We also optimized the condition so as to provide gist for immunoassay kit.