The Study On The Preparation Of McAb To Anti-Melemine And Establishment Of Ic-ELISA For Detetion Of Melamine

Melamine are important azacyclo- organizing chemical materials. Melamine is added to Food, animal feeds and milk by unlawful venders. It is as pseudo-protein to increase the protein level. Recent research show that melamine is little toxic lonely, but animals maybe cause the damages of reproductive system and urinary system, bladder calculus and kidney stones, even bladder carcinoma with intaking melamine for long-term. So it is requisite to establish a fast, easy and accurate detection method that can avoid melamine's harmful factors to the healthy of human through food chain.First, using Diazo synthesis method produced immunizing antigen and detecting antigen with carrier BSA and OVA. Then analyzing the conjugate by SDS-PAGE, BCA and ultraviolet spectrophotometer. The average antigenic content of melamine-BSA is 1917.10μg.mL-1, and the average antigenic content of melamine-OVA is 3546.68μg.mL-1. The couple ratio of melamine-BSA is 17.8, and the couple ratio of melamine-OVA is 26.Second, immunizing female strong and healthy, lively Balb/c mice (six-week) with immunizing antigen melamine-BSA by peritoneal cavity path. Utilizing indirect ELISA to detect mice serum antibody titer by coating melamine-OVA with density 5μg.mL"'. After five immunization, the spleen cells of the immune mice were fused with mouse SP2/0 myeloma cells to produce hybridoma by PEG3350 that titer of blood serum is more than 1:16000. One hybridoma cell line that stably secretes monoclonal antibody against melamine was selected, by called 3H6, heavy chain antibody isotype is IgG2b and light chain antibody isotype isκ. Chromosome average number is 93 pairs, the titer of culture medium supernate is 1:1600; the titer of purified ascites is 1:105; the affinity constant is 2.28×107L.mL-1 and non-cross-reactivity with carrier protein.Finally, by ic-ELISA optimal conditions, develop the best concentration of coating antigen, antigen coating condition, blocking condition, monoclonal antibody, competitive style, competitive time, second antibody marked with HRP, second antibody condition and substrate active time being of 1.0μg·mL-1,4℃overnight,4% milk 37℃2h,1:1000, intra-plate style,37℃2h,1:70000,37℃45min and 15min respectively. The equation of linear regression is y=-15.12x+107.3, coefficient correlation is R2=0.997, The linear range is 1ng～1000ng.mL-1, The limit of detection is 2ng.mL-1, the sensitivity is 6.236～20.985μg.mL-1, IC50 of melamine is 0.527μg.mL-1. The cross-reactiv ratio of the monoclonal antibody clone was 29.88%,13.36%and 0% against tricyanic acid, tricyanic acid monoamide and tricyanic acid diamide. The average value of Intra-assay is 3.05%, the average value of Inter-assay is 7.4%. The recovery of pets food, vegetable casein, forage, milk and eggs is 73.8%～84.32%,67.3%～88.27%,70.3%～85.9%,83.47%～89.76% and 76.2%～87.29%. The detection method developed by the optimal conditions, was highly specific and sensitive for detecting melamine.