Kock W C
Department of Pediatrics, Medical College of Virginia/Virginia Commonwealth University, Richmond 23298, USA.
J Virol Methods. 1995 Sep;55(1):67-82. doi: 10.1016/0166-0934(95)00046-w.
To establish a renewable source of parvovirus B19 antigens for diagnostic tests, gene sequences for the viral capsid proteins, VP1 and VP2, were cloned into baculovirus expression vectors and the recombinant viruses used to infect Sf9 insect cells. Cell lysates examined by immunoblotting demonstrated reactive proteins corresponding to the expected sizes of native VP1 (83 kDa) and VP2 (58 kDa). The VP2 protein was produced efficiently in quantity and self-assembled into empty capsids as shown by density equilibration in a CsCl step gradient. The VP2 protein was purified and used as an antigen in antibody-capture enzyme immunoassays for the detection of B19 IgG and IgM antibodies. Compared to a standard antibody-capture EIA based on whole viral antigen, the VP2-EIA gave a sensitivity of 100% and specificity of 97% in detection of B19 IgM in 138 patients suspected of B19 infection. No IgM-positive specimens were missed. IgG detection yielded a sensitivity of 100% and specificity of 96% in the same population. Recombinant VP2 capsid proteins expressed in baculovirus-infected insect cells can substitute for serum-derived B19 virus in standard antibody-capture EIA for the detection of B19 IgG and IgM with comparable results.
为建立用于诊断测试的细小病毒B19抗原的可再生来源，将病毒衣壳蛋白VP1和VP2的基因序列克隆到杆状病毒表达载体中，并使用重组病毒感染Sf9昆虫细胞。通过免疫印迹检测的细胞裂解物显示出与天然VP1（83 kDa）和VP2（58 kDa）预期大小相对应的反应性蛋白。VP2蛋白大量高效产生，并通过在CsCl阶梯梯度中的密度平衡显示自组装成空衣壳。VP2蛋白被纯化并用作抗体捕获酶免疫测定中的抗原，用于检测B19 IgG和IgM抗体。与基于全病毒抗原的标准抗体捕获酶免疫测定相比，VP2-酶免疫测定在检测138例疑似B19感染患者的B19 IgM时，灵敏度为100%，特异性为97%。没有漏检IgM阳性标本。在同一人群中，IgG检测的灵敏度为100%，特异性为96%。在杆状病毒感染的昆虫细胞中表达的重组VP2衣壳蛋白可以替代血清来源的B19病毒，用于标准抗体捕获酶免疫测定中检测B19 IgG和IgM，结果相当。
