Development And Application Research Of The Monoclonal Antibody Against Melamine

Melamine, a industrial chemicals, in which nitrogen content as high as 66.6 % ,was illegally added to milk and animal feedstuffs, which brought bad effect on human and animal's health. However actual methods (such as HPLC, GC-MS and so on) were time consuming and needed expensive instruments. Thus development of a rapid and convenient detection method for local Mela analysis is extremely desirable. In this study artificial antigen was synthesized and a hybridoma cell strain secreting anti-Mela monoclonal antibody (McAb) was developed using the cell fusion technique. Then the Mela-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.Mela was activated by the multi-anhydride method and the activated Mela was conjugated to a carrier protein (BSA or OVA) by the mixed-anhydride method.The spleen cells from BALB/c mice immunized with Mela-BSA were fused with SP2/0 myeloma cells and a fused cell line,which secreted stable McAb against Mela was screened with a indirect ELISA .The hybridoma were cloned and subcloned by limited dilution method . The titer of McAb in astices was 1:204800 . McAb has specificity integrated with Mela conjugated antigen that determined by gold-immuno chromatography assay (GICA) which showed good sensitivity with an IC50 of 4.15μg/mL for detection of Mela and had not cross reactivity with Cyanuric acid and antibiotics . The cell line for McAb secretion was still stable after repeated passages and frozen-recoveries.Colloidal gold, of which the diameter is about 20nm, was obtained by reducing the gold chloride with sodium citrate and labeled with anti-Mela McAb as detection reagent. The optimum pH for labelling was about 9.0 and the amount of mAb was 20μg/mL. The colloidal gold-labeled mAb was stabilized by 1% PEG30000. The glass fibre membrane Millipore FB-08 and nitrocellulose (NC) Sartorius CN-140 were used as chromatographic materials, The detection sensitivity of Mela-kit was 1μg/mL and the validity of kit at 25℃was lasted more than eighteen months.It is biggest advantage that Mela residues can be instantly detected by the kits sensitively, specifically, simply and reliably without any other equipment in 5 min. The method of producing the kit for Mela detection could be used as a reference in the development of kits for the detection of other animal drugs.