牛奶及奶粉中三聚氰胺残留酶联免疫检测方法的研究

Development of an Enzyme Linked Immunosorbent Assay for Detection of Melamine Residues in Milk and Milk Powder

通过4,6-二氨基-2-氯-1,3,5-三嗪与对氨基苯丁酸反应获得三聚氰胺半抗原,再以活性酯法与载体蛋白偶联制备三聚氰胺免疫原或包被原。免疫BALB/c小鼠,利用杂交瘤技术制备出针对三聚氰胺的特异性单克隆抗体。采用间接竞争酶联免疫吸附法(ciELISA)建立检测三聚氰胺的标准曲线,线性范围是17.4～345.5ng/mL,50%抑制浓度(IC50)61.3ng/mL。牛奶和奶粉加标回收率在68.1%～91.0%,变异系数在2.4%～14.5%。结果表明,该方法可以满足牛奶和奶粉中三聚氰胺残留分析要求。

4-aminobenzenebtitanoic acid was reacted with 2-chloro-4,6-diamino-1,3,5-triazine to get hapten of melamine. An immunogen or coating antigen was prepared by coupling hapten to carrier protein with the activated ester method. BALB/c mice were immunized and the specific monoclonal antibody against melamine was generated with hybridoma technique. In a competitive indirect enzyme-linked immunosorbent assay （ciELISA）, the linear range of calibration curve to detect melamine was 17.4 ng/mL- 345.5 ng/mL with an 50% binding inhibition（IC50） value of 61.3 ng/mL. Recoveries ranged from 68. 1 to 91. 0% for fortified milk and milk powder at various levels with coefficients of variation between 2, 4%-14.5%. The results indicated that the method for the detection of melamine residues in milk and milk powder was effective.