抗体检测胶体金免疫层析技术中不同拦截模式的初步比较研究

Preliminary Comparative Study of Different Interception Modes in Colloidal Gold Immunochromatography for Antibody Detection

胶体金免疫层析技术是以胶体金作为示踪标志物,应用于抗原抗体反应中的一种新型快速检测技术,目前国内外对于抗体检测胶体金免疫层析技术中不同拦截模式的比较研究较少。本研究将牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)的gD基因进行了原核表达,以纯化的IBRV gD-pAb作为检测靶标,将胶体金标记的g D蛋白、金黄色葡萄球菌蛋白A(staphylococcal protein A,SPA)、链球菌蛋白G(streptococcal protein G,SPG)、羊抗兔IgG分别包被于金垫,将未标记的上述4种蛋白分别包被于NC膜的检测线处,将金垫和NC膜进行组合,制备出7种抗体拦截模式的胶体金免疫层析试纸,通过比较其灵敏度、显色效果及批内重复性来评价不同拦截模式的实际性能。结果显示,标记抗原-SPG、标记抗原-SPA、标记抗原-二抗拦截模式(间接法)的灵敏度约为标记抗原-抗原拦截模式(双抗原夹心法)的200倍;间接法中标记抗原-SPG、标记抗原-SPA与标记SPG-抗原、标记二抗-抗原拦截模式的灵敏度相近,而标记抗原-二抗拦截模式及标记SPA-抗原拦截模式由于IgG结合蛋白的特性而导致试纸的灵敏度降低、显色效果及批内重复性变差;7种抗体拦截模式试纸在检测副结核分枝杆菌(Mycobacterium avium subsp.paratuberculosis,MAP)、赤羽病病毒(akabane virus,AKAV)、牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)、牛支原体(Mycoplasma bovis)、小反刍兽疫病毒(peste des petits ruminants virus,PPRV)、布氏杆菌(Brucella)、口蹄疫病毒(foot-and-mouth disease virus,FMDV)的标准阳性血清时均为阴性反应;IgG结合蛋白的实际性能排序为SPG>SPA>二抗。本研究为其他疫病抗体检测的胶体金免疫层析试纸的制备提供参考。

Colloidal gold immunochromatography is a new rapid detection technique using colloidal gold as a tracer marker applied to antigen-antibody reactions,and there are few comparative studies on different interception modes in colloidal gold immunochromatographic techniques for antibody detection at home and abroad.In this study,thegD gene of infectious bovine rhinotracheitis virus(IBRV)was expressed in prokaryote,and the purified IBRV gD-pAb was used as the target of the detection.The colloidal gold-labeled gD protein,staphylococcal protein A(SPA),streptococcal protein G(SPG),goat anti rabbit IgG were coated on the gold pad,while the unlabeled above four proteins were respectively coated at the detection line of NC membranes.Colloidal gold immunochromatographic test strips with seven antibody interception modes were prepared by combining gold pads and NC membranes,to evaluate the practical performance of different interception modes by comparing their sensitivity,color development effect and intra-batch reproducibility.The results showed that the sensitivity of labeled antigen-SPG,labeled antigen-SPA and labeled secondary antibody interception mode(indirect method)was about 200 times higher than that of labeled antigen-antigen interception mode(double antigen sandwich method).The sensitivity of labeled antigen⁃SPG,labeled antigen⁃SPA and labeled SPG⁃antigen,labeled secondary antibody⁃antigen interception mode in the indirect method was similar,while the sensitivity of labeled antigen⁃secondary antibody interception mode and labeled SPA⁃antigen interception mode was reduced with poor color rendering and intra⁃batch reproducibility due to the properties of IgG⁃binding protein.Seven antibody interception mode test strips were negative in detecting standard positive serum forMycobacterium avium subsp.paratuberculosis(MAP),akabane virus(AKAV),bovine viral diarrhea virus(BVDV),Mycoplasma bovis,peste des petits ruminants virus(PPRV),Brucella,and foot⁃and⁃mouth disease virus(FMDV).The actual performance of IgG⁃binding proteins was ranked as SPG>SPA>secondary antibody.This study provides reference for the preparation of colloidal gold immunochromatographic test strips for antibody detection of other diseases.