| Clenbuterol (CL), one of the P -adrenergic agonists, has a powerful effect of promoting animal growth with increased accretion of skeletal muscle mass and decreased accretion of body fat. Thus, it is called a repartitioning agent, and also called "leanness enhancer ". For this purpose, CL was introduced as a growth-promoting agent to improve feed conversion efficiency and enhance lean meat to fat ration. However, accumulative residue of CL in food producing animals can cause symptoms of acute poisoning in people. Illegal use of CL has resulted hi severe cases of food poisoning at home and abroad, and gives highly rise to concern regarding consumer safety. So far, no country has authorized the use of CL for repartitioning purposes in food animals. And, of rules of law and directives have published by European United, FDA of the USA and China government, which underlines the necessity of recognizing the CL toxicosis and methods and control systems.In this study, CL instant detection competitive enzyme-linked immunosorbent assay ELISA Kit (CL-Kit) and test strips of immuno-chromatagraphy were developed by using CL monoclonal antibodies which were produced by conventional cell fusion technology. Meantime, the elimination of CL toxicosis was carried out. CL-conjugated antigen (BSA-CL or OVA-CL) were produced by coupling diazotized CL with Bovine Sera Albumin (BSA) or Ovalbumin (OVA), respectively. The spleen cells of Balb/c mouse immunized by BSA-CL were fused with NSO plasmacytoma cells using PEG4000, and selected cultured with HAT and HT medium. Four hybridoma cell lines of C4G1, C2H6, C2D6 and C4C9 were screened for specificity to CL with OVA-CL using an indirect ELISA, and cloned by limited dilution method for three times, whichsecret high affinity monoclonal antibodies against CL with indirect ELISA titers of 5x10-5, 3x10-4, 6x10-4 and 2x10-5 in astices, respectively. Also, the four mAb have isotypes of IgG1/K, IgGi1K, IgG2a/K IgG2a/, respectively, and affinity constant is 1.68x108L/mol in C4G1, 1.3x108L/mol in C2H6.CL mAb have specificity integrated with CL conjugated antigen that determined by WestGold protein blotting assay.On the basis of competitive ELISA, CL-kit was developed with C-4G1 and C-2H6. The CL-Kit showed good sensitivity with an IC50 of 7.94ng/mL toward CL, 1000ng/mL toward salbuterol and more than 6400ng/mL towards Norepinephrine, Epinephine, Isoprenaline and Ractopamine. The CL-Kit generally had 0.79% cross-reactivity towards Salbuterol, and showed little or no cross-reactivity towards Norepinephrine, Epinephine, Isoprenaline, Ractopamine, antibiotics and multi-vitamin added in animal feed. CL-Kit was performed using an indirect assay format with standard CL competitor concentrations ranging from 0.5 to 128ng/mL. Compounds that produced sigmoidal like curves were fitted to the four parameter logistic equation with linear detection of 0.5-128ng/mL (R2=0.996) and variation coefficient of 2.3%-3.6%. The CL-Kit also showed fine precision and accuracy of assay in shelf life. As expected, the recoveries of CL spiked in pig urine were 91%-105%.The inter-assay coefficient variation was below 20%. The intra-assay CV% was well below 10%.Test strips of CL instant detection were designed by CL mAb immuno-chromatographic assay. The limit of detection values for CL-conjugated antigen and urine specimens were determined to be 1.9ng/mL and 6~24ng/mL, respectively. In addition, CL mAb colloidal gold Dot Immunobinding Assay showed sensitivity with 1.25pg/mL and 2.5pg/mL of LOD and LOQ values for CL-conjugated antigen.The sensitivity, specificity, simplicity and reliability of CL-Kit were confirmed by the procedure utilizing GC-MS and HPLC for the identification and quantification of CL in pig urine and feed.The pharmacokinetics of CL were investigated to determine the elimination of residues in pigs that were dosed with CL added feed for 21 days with 5mg/kg for growth promoting treatment. The CL concentrations in urine at withdrawal time 12 hours were 181.15ng/mL, and come down less to Ing/...
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