树鼩IgG纯化鉴定及其多克隆抗体制备和检测

Purification of Tree Shrews(Tupaia) Immunoglobulin G and Preparation of Anti- IgG Polyclonal Antibody

目的:比较两种抗体纯化方法在分离纯化树鼩IgG抗体的应用,制备抗IgG的多克隆抗体及检测。方法:采用两种商品化IgG抗体纯化试剂盒分离树鼩血清IgG抗体,采用SDS-PAGE和蛋白定量测定提纯IgG。以树鼩IgG作为抗原,与等量弗氏完全佐剂(第一次)、弗氏不完全佐剂(第二次)混合皮下注射免疫兔,对分离血清进行多克隆抗体纯化及Western Blot检测及定量分析。结果:两种方法均能有效分离纯化树鼩IgG,在经过Montage PROSEP-A试剂纯化后的IgG在纯度和含量方面均优于Protein A/G Matrix试剂。通过纯化后的树鼩IgG免疫兔制备的抗IgG抗体能有效识别树鼩IgG。结论:纯化的树鼩IgG具有良好免疫原性,由此制备的抗体具有高度特异性。研究结果为利用树鼩作为实验动物提供了必要的实验基础。

Objective： To isolate and purify tree shrew Ig G antibody, and perform the preparation and detection of anti-Ig G polyclonal antibody. Methods： The two commercial Ig G antibody purification kits were used to separate tree shrew serum Ig G, determination of purified Ig G by SDS-PAGE and protein quantification were also performed. The rabbits were immune injected with tree shrew Ig G as antigen, combined with the equivalence Freund＇s complete adjuvant as prime, incomplete Freund＇s adjuvant as boost. Western Blot was performed to detect and analyze the purification and preparation of polyclonal antibody. Results： The two purification methods could effectively separate and purify the tree shrew Ig G, the purity and content of Ig G after purification of Montage PROSEP-A reagent were better than using Protein A/G Matrix kit. The anti-Ig G antibodies after purification against tree shrew Ig G immunization with rabbit could effectively identify the tree shrew Ig G. Conclusions： the purification of tree shrew Ig G had good immunogenicity, thus prepared with a highly specific antibody. The results supported for usage of tree shrews as an experimental animal to providing experimental basis.