Development Of An Colloidal Gold Immunochromatographic Strip For Detection Of Ractopamine Based On Monoclonal Antibody

Ractopamine (RAC) and Clenbuterol (CL) areβ₂-agonists and prohibited drug. RAC has a wide range of physiological effects to human and is often used for bronchial asthma, bronchospasm and obstetric disease treatment. When used in the animal's diet, RAC is a good nutrient repartitioning agents which can significantly improve lean percentage of swine carcasses and feed efficiency. Illegally adding RAC in the animal's diet has been seriously increasing since CL and other drugs have been prohibited as feed additive in most countries around the world. Meanwhile, its abuses and residues in the animal food threat to people's health and life safety sincerely. RAC detection methods are lagging behind than otherβ₂-agonists' detection methods such as CL, and cann't satisfy instant-detected requirements.Thus,to explore and establish a rapidly , highly efficient, and sensitive economic method for RAC residue detection is emergency.In this study artificial antigen was synthesized and a hybridoma cell strain secreting anti-RAC monoclonal antibody (mAb) was developed using the cell fusion technique. Then the RAC-Gold Immunochromatographic Assay was successfully developed which was highly sensitable, specific and stable that can be well used in conventional testing with a highly commercial importance.1. Synthesis of artificial antigen and preparation of anti-RAC mAb:RAC was activated by the multi-anhydride method and the activated RAC was conjugated to a carrier protein (BSA or OVA) by the mixed-anhydride method.The spleen cells from BALB/c mice immunized with RAC-BSA were fused with SP2/0 myeloma cells and a fused cell line, which secreted stable mAb against RAC with a indirect ELISA titer of 1:409600 in astices was selected and cloned by limited dilution method. The mAb has specificity integrated with RAC conjugated antigen that determined by gold-immuno chromatography assay (GICA). The mAb showed good sensitivity with an IC50 of 2.75 ng·ml^-1 to RAC. The cross reactivity with clenbuterol hydrochloride, epinephrine, norepinephrine, isoprenaline, isoxsuprine, salbutamol and dopamine was less than 0.03%, while with dobutamine was 9.8%. The mAb secretion of the cell line was still stable after repeated passages and frozen-recoveries.2. Preparation of Colloidal gold and strips for RAC detection:Colloidal gold, of which the diameter is about 18nm, was obtained by reducing the gold chloride with sodium citrate and labeled with anti-RAC mAb as detection reagent. The optimum pH for labelling was about 8.2, and the amount of mAb was 7.2μg·ml^-1. The colloidal gold-labeled mAb was stabilized by 1% PEG20000. The glass fibre membrane SB-06 and nitrocellulose (NC) Sartorius CN^-140 were used as chromatographic materials, on which the antigen was coated at 1mg·ml^-1 (1μl·cm^-1). The sheep anti-mouse IgG was coated at 1mg·ml^-1 (1μl·cm^-1) and the colloidal gold-labeled mAb was diluted 3 times and coated with 1 ml / pad (50mm×60mm) . The detection sensitivity of RAC-Strip was 5 ng·ml^-1 and the validity of strip at 25℃was lasted more than twelve months.The biggest advantage of this study is that RAC residues can be instantly detected by the strips sensitively, specifically, simply and reliably without any other equipment in 10 min and that all needed reagents were included in the strip. The method of producing the strip for RAC detection could be used as a reference in the development of strips for the detection of other veterinary drugs.