Development And Application Of Monoclonal Antibody And Test Kit Against Clenbuterol

Clenbuterol (CL) is a beta2 -adrenergic agonist. It can exhibit metabolic effects to promote muscle growth and decrease fat deposition. However, CL can remains in tissue of treated animal and cause symptoms of acute poisoning in people. No country has permitted CL as a growth-promoting agent in farm animals now.For detection of residues of CL, there are two main methods: enzyme immunoassay and confirmatory methods. But the confirmatory methods are not suitable for screening a large number of samples because these methods are time-consumption, costly and highly skilled. Enzyme-linked immunosorbent assay (ELISA) as a rapid, special and sensitive biological method is gradually applied in drug residues assay. This research synthesized the antigen and developed the antibodies, established the ELISA method for detecting CL and developed the Clenbuterol ELISA test kit.Three Hybridoma cell lines producing monoclonal antibody against Clenbuterol were established by fusions between SP2/0 myeloma cells and spleen cells from Balb/c mice immunized with protein linked Clenbuterol. Monoclonal antibody (mAb) CL-3C3 could react with only Clenbuterol but not with other structurally related compounds, such as Salbuterol, Adrenaline, Noradrenalin.However mAb CL-4F3, CL-2F12 could react with not only Clenbuterol but also with Salbuterol. This result showed that mAb CL-3C3 is specificity against Clenbuterol.An indirect competitive enzyme-linked immunosorbent assay (ciELISA) was established successifully the Clenbuterol (CL) linking to Ovalbumin by Diazotization as coating antigen, called CL-OVA, in which Clenbuterol, a kind of haptens, was competitive. The method showed that the optimum concentrations of CL-OVA, monoclonal antibody against CL and rabbit anti-mouse IgG were 0.72 g/mL, 1:1000, 1:2000 and 1:30000, respectively. The sensitive range to detect CL varied from Ing/mL to 100ng/mL. The minimum detectable was 0.1ng/mL. The Kit showed high sensitivitywith an IC50 of 0.43 ng/mL when measuring CL and only 4.3% cross-reactivity with Salbutamol, but on cross-reactivity with other 3 -adrenergic receptor agonists. Fine precision and accuracy (87%~105%) was achieved when detecting CL spiked in pig urine within the Kit's shelf life. The variation coefficients of intra-assay and inter-assay were 2.16% and 7.59%, respectively. The established method is vital to rapidly detect the residues of CL in food of animal product.