Preparation And Characterization Of Monoclonal Antibody To Clenbuterol

Clenbuterol, a substituted phenylamino ethanol that has beta-2 adrenomimetic properties at verylow doses. It is used as a bronchodilator in asthma. CL can enhance animal's nervous excitation, assignnutrition again, reduce the fat to deposit and may also lead to the development of muscle protein,because of these, CL was used as an adjunct to animal feeds, however, it is difficult to discharge withsupersession, CL propably will keep higher residue amount in the body and bring serious danger to anyanimals. Though the policies and regulations of the country have already been forbidden strictly using itas the additive of feed, there is application in violation of rules and regulations. So, in order to guaranteemeat product quality and human health, it is necessary and significant to set up a method which is fast,simple and convenient to monitor.At present, the ELISA kits we used mainly relies on import, they are expensive and can't easy tospread. So, developing it with independent property right already becomes task of top priority. Todevelop ELISA kit, we have to produce monoclonal antibodies which have high specificities and goodtiters against clenbuterol firstly. Objective of this research was to produce and identify monoclonalantibodies against clenbuterol.In this study, CL-conjugated antigens (BSA-CL and OVA-CL) were produced by couplingdiazotized CL with Bovine Sera Albumin (BSA) and Ovalbumin (OVA), respectively. The BSA-CLemulsified in freund's complete adjuvant were chosen to immunize 6 to 8 weeks old female Balb/c micewith 96μg at the first time. Fourteen days later, the same antigen emulsified in freund's incompleteadjuvant were intraperitoneally immunized.OVA-CL was used in detection of ELISA. After four timesimmunization to the mouse, Splenocytes from the immunized mice were fused with Sp2/0 myelomacells. 365 hybridomas holes were observed, the fusing rate is 54%.291 McAbs against to CL from 365 hybridomas holes were chosen by indirect ELISA., the positiverate is 80%. Positive hybridomas which could secreted McAb were cloned three times by technique oflimiting dilutions, 18 hybridoma cell lines were established, three clones in the 18 lines named E9-C11,C11-E3,E7-G8 were choosed to identify.This reseacher indicated that ELISA titer of hybridoma supernatants of three clones were 1:1280 inE9-C11,1:1000 in C11-E3,1:800 in E7-G8, respectively, The ascites titers were 1×10^-6. The affinityconstant was 2.4×10^-8mol/L,2.83×10^-8mol/L,3.28×10^-8 mol/L, respectively, and all three McAbbelong to IgG₁ subclass. McAb of E9-C11 was indicated high sensitivity with an IC₅₀ of 6.1035μg/L to CL, higher than 781.25μg/L to Salbuterol, and showed lower than 0.78% cross activity towardsSalbuterol, no cross reactivity towards Epinephine, Norepinephrine, Isoprenaline, antibiotics andvitamin. Furthermore, protein blotting assay showed that these McAb have high specificity to CL andcan be used to detect CL residues...