三聚氰胺多克隆抗体的制备、分离纯化及ELISA方法的测试研究

The Preparation and Purification of Specific Polyclonal Antibody Against Melamine and Detection by ELISA

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 the highly specific anti-melamine polyclonal antibody,and establish fast detection method for melamine.Method：The melamine has been coupled with a specific protein in order to prepare the immune antigen.There were MB1st,MB2st,KM and ML2.Four rabbits were separately immunized with these antigens by way of subcutaneous multi-point injection.The antiserum after the primary immunization was tooken,and it had been verified to produce IgG by the experiments,such as SDS-PAGE.The titer changes of rabbits＇ antiserums had been detected by enzyme-linked immunosorbent assay（ELISA）.Eventually,the highly specific anti-melamine polyclonal antibodies were acquired from antiserums through the purification with affinity chromatography.In order to establish ELISA method for melamine,the antibodies after purification were wrapped in elisa plate.Then the effectiveness of elisa plate for melamine should be determined.Result：The generation of IgG was verified with SDS-PAGE.Then the titrations of serum could be determined by methods of indirect ELISA.The result of antigen MB1st was the best.The competitive rate of melamine was only 36.97% in the original antiserum,and then the rate was up to 75.57% by purification.The competitive rate of melamine of serum to MB2st rise from 30.80% to 92.43% after the purification with affinity chromatography.The titer of atiserum to KM was low.The competitive rate of melamine in the original antiserum was just only 11.95%;however,the rate had been greatly improved by the purification and could reach to 96.09% at last.Comparatively speaking,the immunization of antigen ML2 showed an unfavorable result.The titer of antiserum was low.Either purified or not,the antiserum had an unremarkable specificity to melamine.Finally the effectiveness of ELISA method for melamine had been verified.Conclusion：The results showed that the antiserums could be prepared by using the polyclone antibodies preparation technique,and then could be purified to obtain the highly specific anti-melamine polyclonal antibodies by means of affinity chromatography.The anti-melamine polyclonal antibodies could build an advantageous experimental foundation for the detection of melamine in food by ELISA.