Studies On Preparation Of Melamine Monoclonal Antibody And High-sensitive And Rapid Detection Methods

The melamine contamination event of milk powder greatly impacted China’s dairy industry. And the maximum residue limits of melamine in food have been set up both in domestic and abroad. In China, melamine has been one of the conventional detection items for milk and related products. Therefore, high-throμghput, rapid, and highly sensitive screening methods are urgently needed to satisfy the needs for melamine detection. In view of this, this dissertation carried out the following studies:In this dissertation, three haptens (MEL-MCA, MEL-ACA and MEL-PABA) were designed, synthesized and identified. Three immunogens (MEL-MCA-BSA, MEL-ACA-BSA and MEL-PABA-BSA) and three coating antigens (MEL-MCA-OVA, MEL-ACA-OVA and MEL-PABA-OVA) were conjμgated by the active ester method and mixed anhydride method, respectively. The effects of hapten structure for immunity were investigated by animal immunization experiment. Results showed that the synthesis of these three immunogens was successful, and revealed that the MEL-ACA-BSA antibody/MEL-PABA-OVA coating conjμgate combination was the best for heterologous ELISA system.Three immunogens was applied to immunize BALB/c mice to produce anti-melamine monoclonal antibody. And four monoclonal cell lines which could steadily secrete anti-melamine monoclonal antibody were obtained successfully. The subtype of antibody was identified as IgG1 by the commercial Kit. And the antibody affinity constant was calculated to be 2.67×109 L/mol. The most important property of the prepared antibody was that there was no cross-reaction to other MEL analogues except with cyromazine. The antibody achieved in this study was the best high-quality antibody among the currently reported monoclonal antibody against MEL.Based on the obtained monoclonal antibody, an indirect competitive ELISA detection method of MEL was established and a relative ELISA Kit was assembled. Factors that may affect the performances of ELISA were optimized systematically. The IC50 value was 6±0.55ng/ml and the limit of detection (LOD) was 100μg/kg in powder and 20ng/ml in liquid milk, respectively. Recovery results showed that the recoveries ranged from 93 to 101.9% in milk powder samples and from 83.6 to 96.9% for liquid milk, respectively and variation coefficients were less than 10%. Real samples were determined throμgh the developed ELISA Kit of our laboratory. Detection results were also futher compared with results of by LC-MS/MS method according to national standard GB/T22388-2008, which showed highly consistency of the two methods. The stability results of the ELISA Kit indicated that the Kit stored at 4°C for one year can still be useful, which satisfy the requirement of the determination of MEL residues.Based on the obtained anti-melamine monoclonal antibody, we prepared the immunoaffinity chromatography column to specifically purify MEL and developed the IAC-LC-MS/MS method of measure MEL in powder and liquid milk. The results showed that the dynamic column capacities of IAC was 24.14 n mol/ml gel,and the specific column capacity of IAC was 3.36 n mol/mg antibody, respectively. The best regeneration cycle of IAC was 8 cycles. The limit of detection of IAC-LC-MS/MS method was 10ng/ml,and the limit of quantification was 20ng/ml, respectively. The recoveries of melamine from spiked milk and milk products samples ranged from 79.4 to 91.8%, and the variation coefficients were less than 10%, and which satisfied the requirement of the determination of MEL residues.In this dissertation, colloidal gold was also prepared via sodium citrate reduction method. Based on the anti-melamine antibody, we developed a high-sensitivity and fast detection method for the MEL by the colloidal gold immunochromatographic assay. Several factors that affected assay performance were optimized. Finally, the optimal pH of gold-labeled antibodies was 8.0, the optimal antibody amount was 8μg/ml, and the optimal concentrations of coating and second antibody were 0.5mg/ml. The test strip for MEL had a visual detection limit of 12.5ng/ml in stand solution and 37.5μg/kg in powder, and had no cross-reactivity to MEL analogues, which could meet the demand of practical MEL testing.Beside, we futher developed a sensitive and rapid visually and spectroscopic method for MEL detection by the molecular recognition between crown ether functionalized gold nanoparticles (GNPs) and amino group in MEL. Under the optimal conditions, MEL could be selectively detected in a concentration range from 10ng/ml to 500ng/ml with a limit of detection as 6ng/ml. Recovery results showed that the recoveries ranged from 98.4 to 105.6% in milk powder samples, and variation coefficients were less than 10%, which could be applicable for simple, rapid and sensitivity MEL detection.