Yoshimatsu K, Arikawa J, Yoshida R, Li H, Yoo Y C, Kariwa H, Hashimoto N, Kakinuma M, Nobunaga T, Azuma I
Institute of Immunological Science, Hokkaido University, Sapporo, Japan.
Lab Anim Sci. 1995 Dec;45(6):641-6.
The recombinant nucleocapsid protein (rNP) of Hantaan virus was expressed by a baculovirus vector in silkworm hemolymph and was used as an antigen in western blotting (WB). The rNP is expressed in insoluble form in hemolymph; therefore simple washing of the insoluble fraction with phosphate-buffered saline by low-speed centrifugation allowed preparation of purified antigen for WB. The rNP had strain-specific and hantavirus-common epitopes similar to the authentic NP antigen of hantavirus and was stable after transfer to membrane. For detection of antibody in serially obtained sera from experimentally infected rats, WB enabled detection of IgM antibodies 3 days after infection, which was at least 2 days earlier than detection by the indirect immunofluorescent antibody test (IFA). Thus WB had a higher sensitivity than the IFA for detection of hantavirus antibody in the serum of experimentally infected rats. The WB-determined IgG antibody titer was about 10 times higher than that determined by the IFA. No background staining was observed by WB even at a 1:10 dilution of serum. The selected rat sera with strong background staining or confusing staining patterns by IFA, but not focus reduction neutralization test titers, could be interpreted as test-negative because they did not have a specific reaction to virus antigen by WB. Thus the specificity of WB was higher than that of the IFA. Moreover, WB can distinguish specific from nonspecific reactions by the detection of the specific antigen on the WB membrane. Therefore the IFA or enzyme-linked immunosorbent assay followed by WB is recommended for serologic confirmation of hantavirus infection.
汉坦病毒重组核衣壳蛋白（rNP）通过杆状病毒载体在家蚕血淋巴中表达，并用作蛋白质印迹法（WB）中的抗原。rNP在家蚕血淋巴中以不溶性形式表达；因此，通过低速离心用磷酸盐缓冲盐水简单洗涤不溶性部分，即可制备用于WB的纯化抗原。rNP具有与汉坦病毒真实NP抗原相似的株特异性和汉坦病毒共同表位，转移至膜上后很稳定。为检测实验感染大鼠连续采集血清中的抗体，WB能够在感染后3天检测到IgM抗体，这比间接免疫荧光抗体试验（IFA）检测至少早2天。因此，在检测实验感染大鼠血清中的汉坦病毒抗体时，WB比IFA具有更高的灵敏度。WB测定的IgG抗体效价比IFA测定的高约10倍。即使血清稀释至1:10，WB也未观察到背景染色。通过IFA检测呈强背景染色或染色模式混乱，但焦点减少中和试验效价正常的大鼠血清，因对病毒抗原无WB特异性反应，可判定为检测阴性。因此，WB的特异性高于IFA。此外，WB可通过检测WB膜上的特异性抗原区分特异性和非特异性反应。因此，建议采用IFA或酶联免疫吸附测定法，随后进行WB，以进行汉坦病毒感染的血清学确认。
