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目的:制备具有高特异性的三聚氰胺多克隆抗体,建立三聚氰胺快速检测方法。方法:通过抗原偶联嫁接技术,将不具备免疫原性的三聚氰胺与特定蛋白连接,制备免疫抗原(MB1st、MB2st、KM和ML2)。对家兔进行皮下多点注射,初次免疫后取抗体血清,用SDS-PAGE检测血清中IgG的产生情况。采用酶联免疫吸附法测定各抗体血清的效价,最终以亲和层析法纯化血清,获得高特异性的抗体。将纯化后的抗体包被于酶标板上,建立三聚氰胺ELISA方法,并验证其有效性。结果:通过SDS-PAGE证明免疫前、后血清中IgG显著增长。进一步使用间接ELISA法测得各兔血清效价。MB1st免疫的效果最佳,其抗体血清对三聚氰胺的竞争率由纯化前的36.97%上升至纯化后的75.57%。MB2st免疫的抗体血清对三聚氰胺的竞争率由纯化前的30.80%上升至纯化后92.43%。KM免疫的抗体血清效价较低,其对三聚氰胺的竞争率由纯化前的11.95%上升至纯化后的96.09%。ML2免疫效价低,且其抗体血清在纯化前、后均对三聚氰胺没有显著的竞争性。由此建立ELISA方法,并初步验证了该方法的有效性。结论:本试验中制备的三聚氰胺抗体血清,经分离、纯化后得到对三聚氰胺具有较高竞争性的多克隆抗体,为建立酶联免疫方法,以检测食品中三聚氰胺含量提供了理论基础。
Objective: To prepare polyclonal antibodies with high specificity and to establish a rapid detection method of melamine. Methods: The immunogenic antigens (MB1st, MB2st, KM and ML2) were prepared by antigen-linked grafting technique by linking non-immunogenic melamine to specific proteins. The rabbits were injected subcutaneously in multiple subcutaneous injection. After the first immunization, the serum of the antibody was taken, and the production of IgG in serum was detected by SDS-PAGE. The titer of each antibody serum was determined by enzyme-linked immunosorbent assay. Finally, the serum was purified by affinity chromatography to obtain highly specific antibodies. The purified antibody was coated on a microtiter plate to establish a melamine ELISA method and to verify its effectiveness. Results: Serum IgG was significantly increased by SDS-PAGE before and after immunization. The serum titer of each rabbit was further measured by indirect ELISA. MB1st immunized the best, the competitive rate of antibody serum to melamine increased from 36.97% before purification to 75.57% after purification. The competitive rate of MB2st antibody serum to melamine increased from 30.80% before purification to 92.43% after purification. The antibody titer of KM immunized was lower, and the competition rate of melamine increased from 11.95% before purification to 96.09% after purification. ML2 immune titer low, and its serum before and after the purification of melamine are not significantly competitive. The establishment of ELISA method, and preliminary validation of the effectiveness of the method. CONCLUSION: The serum of melamine antibody prepared in this study was isolated and purified to obtain polyclonal antibody with high competitive melamine. This study provided a theoretical basis for the detection of melamine in foodstuff by establishing ELISA method.