Development Of Enzyme-Linked Immunosorbent Assay For The Determination Of Melamine

An enzyme-linked immunosorbent assay (ELISA) for the determination of melamine pollution in foods was developed.Firstly the biguanide, ethyl chloroacetate and sodium methoxide were used to generate the melamine analogues 6-(chloromethyl)-1,3,5-triazine-2,4-diamine, then the hapten was synthetized with 6-(chloromethyl)-1,3,5-triazine-2,4-diamine and 3-Mercaptopropionic acd. The hapten was conjugated to OVA to get immunogen via the active ester method, and conjugated to BSA and HRP to obtain coating antigen and enzyme tracer respectively. Two male rabbits were immunized. The antiserum titer was about 200000. Antibody purification be completed by protein A-sepharose 4B immunoaffinity chromatography.For the indirect competive ELISA, the plate was coated with coating antigen at 0.005 μg per well, the blocking solution is 0.5% skim milk powder, and the antibody dilution was 1:4000. The assay sensitivity was 45.45±2.62 ng/mL and the limit of detection was 2.72±0.18 ng/mL. For the direct competive ELISA, the antibody was coated with 1.0 μg per well, while the dilution ratio of enzyme tracer was 1:800.The sensitivity of this method was 7.8±1.3 ng/mL,while the limit of detection was 0.66 ±0.18 ng/mL. The antibody showed low cross-reactivity with other tested compounds except for cyromazine(426%).The recoveries of melamine from spiked milk and milk powder samples were studied, the recovery in milk was between 79.3%-93.6%, while in milk powder was between 94.1%-113.2% the and the coefficients of variation (CV) were less than 20%. The linear regression equation between data obtained using the ELISA and LC-MS/MS was y= 0.8266x+20.791 with a correlation coefficient (R2) of 0.968. It showed that the two methods had a good correlation.We established a simple, rapid, sensitive and specific enzyme-linked immunosorbent assay, it can be applied to detect melamine in dairy.