抗人免疫球蛋白G多克隆荧光抗体的制备及在肾活检中的应用

Preparation of fluorescent polyclonal antibody against human IgG and its application in the diagnosis of nephrosis

目的:制备抗人免疫球蛋白G(IgG)多克隆荧光抗体,并用于肾病的病理诊断。方法:以免疫新西兰兔制备多克隆抗体,用亲和层析的方法纯化抗体,并用ELISA法双向琼脂扩散,W esten-b lot和免疫组化等方法对多克隆抗体进行测定。纯化的抗体标记荧光素,用免疫组化的方法鉴定并用于临床肾病的辅助诊断。结果:获得的抗体琼脂双扩滴度为1∶32。ELISA法测定效价为1∶128 000,W esten-b lot证实抗体特异性识别人IgG。纯化后抗体效价仍有1∶56 000,灰度分析证实纯化后的抗体纯度达80%。间接法免疫组化检测抗体滴度为1∶800。标记荧光后效价无明显下降,免疫组化鉴定在临床病例中该荧光抗体能特异性识别人IgG。结论:得到纯化后的抗人IgG抗体有良好的特异性和亲和性,抗体的结合力较高,标记荧光后得到的多克隆荧光抗体对具有IgG阳性的肾组织反应特异性强,与商品化的抗人IgG抗体比较,效价及特异性均无显著差异,可用于肾病的临床诊断。

Objective：To prepare polyclonal antibody against human immunoglobulin G （human IgG） and to use this antibody in the clinical diagnosis of nephrosis, Methods：New Zealand rabbit was immu- nized subcutaneously with human IgG, Enzyme linked immunosorbent assay （ELISA） was applied to reveal the titer of the prepared polyclonal antiserum against human IgG, Antiserum was purified with affinity chromatography, and the purified antibody was confirmed for＇its specificity by Western blot and immunohistochemical staining. The purified antbodies which have been indentified were fused with FITC （fluorescein isothiocyanate ） and then analyzed by immunohistochemical staining. Finally the fused antibody was useed in the clinical diagnosis of nephrosis. Results ： The titer of the obtained antiserum was up to 1：128 000, double agar diffusion test showed the titer of the antibody was 1 ： 32, By the method of affinity chromatography, we obtained purified antibody with the purity of 85%. ELISA, double agar diffusion and immunohistochemical staining tests showed that the specificity and titer of the antibody were not decreased sharply. The purified antibody fused with FITC also kept the specificity of the primitive antibody. When the FITC fused antibody was tried in nephrosis patients, it detected human IgG effectively. Conclusion：The polyelonal antibody can specifically recognize human IgG. This purified antibody fused with FITC can be used in the diagnosis of nephrosis.