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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 establish a microparticle immunoassay (MIA) for the detection of Cryptosporidium parvum-specific antibodies. Methods The purified recombinant proteins CP23, SA35 and SA40 were used as detection antigen, and bovine serum albumin (BSA) was used as an internal standard to couple the microparticles. The protein coupling efficiency was also verified. The agreement between single MIA and multiple MIA was compared, and the intra- and inter-plate differences detected by multiplex MIA were compared. Results The purified protein and BSA were successfully coupled to the corresponding microparticles, and the optimal detection conditions for the detection of serum Cryptosporidium parvum antibodies by multiplex MIA method were established. It was confirmed that multiple detection did not reduce the sensitivity and specificity of detection. Conclusion The multiplex MIA method can rapidly detect a variety of specific antibodies produced by Cryptosporidium infection in humans. It has high sensitivity and specificity and can be used as a powerful tool for large-scale serological epidemiological investigation.