构建检测隐孢子虫特异性抗体的多重微粒子免疫检测法

Development of multiplex microbead immunoassay for detection of specific antibodies against Cryptosporidium parvum

目的建立一种检测微小隐孢子虫特异性抗体的微粒子免疫检测法(MIA)。方法以纯化的重组蛋白CP23、SA35和SA40为检测抗原,以牛血清白蛋白(BSA)为内参,偶联微粒子,并进行蛋白偶联效率验证。比较单一MIA法和多重MIA法的一致性,以及多重MIA法检测的板内和板间差异。结果纯化蛋白和BSA成功偶联到相应的微粒子上,建立了多重MIA法检测血清隐孢子虫抗体的最佳检测条件,且证实多重检测并不降低检测的敏感性和特异性。结论多重MIA法可以快速检测隐孢子虫感染后人体产生的多种特异性抗体,敏感性、特异性均较高,可成为人群大规模血清流行病学调查的有力工具之一。

Objective To develop a multiplex microbead immunoassay for detection of specific antibodies against Cryptosporidium parvum using recombinant proteins CP23,SA35 and SA40.Methods By using purified recombinant proteins CP23,SA35 and SA40 as detected antigens,and bovine serum albumin（BSA） as internal control,the four proteins aforementioned were coupled with micro beads and MIA was developed.Then,the efficiency of the coupled proteins was tested,the difference between the single MIA method and the multiple MIA method was compared,and the difference between plates was also compared.Results The purified proteins and BSA were coupled with microbeads successfully,and the MIA method was developed.Finally,the sensitivity and specificity of MIA method were confirmed.Conclusions The multiplicate MIA method could be used to detect multiple antibodies after Cryptosporidium parvum infection,and the specificity and sensitivity of MIA are very high.The multiplicate MIA method can be one of the tools used in epidemiological survey.