Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus (HEV) can capture HEV particles in culture supernatant and serum but not those in feces
Monoclonal antibodies raised against the ORF3 protein of hepatitis E virus (HEV) can capture HEV particles in culture supernatant and serum but not those in feces
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DOI：
10.1007/s00705-008-0179-6
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发表时间：
2008-09-01
影响因子：
2.7
通讯作者：
Okamoto, Hiroaki
中科院分区：
文献类型：
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作者：
Takahashi, Masaharu;Yamada, Kentaro;Okamoto, Hiroaki
针对戊型肝炎病毒（HEV）ORF 3蛋白C-末端24个氨基酸部分的合成肽，制备了10株鼠单克隆抗体（MAbs），并进行了表征。使用抗ORF 3单克隆抗体的免疫荧光检测显示ORF 3蛋白在用ORF 3表达质粒转染或用细胞培养物产生的HEV接种的PLC/PRF/5细胞的细胞质中积累。抗ORF 3单克隆抗体可以捕获HEV颗粒在培养基和血清中的可变效率分别高达61%和49%，但不是在粪便中。通过ELISA中固定化和酶标记的抗ORF 3单克隆抗体之间的杂交，在培养基中检测到ORF 3抗原，HEV RNA滴度> 10（6）拷贝/ml，并且随着HEV载量的增加而平行增加。培养物上清液中的HEV后代，其表面上具有ORF 3蛋白，在蔗糖中以1.15 g/cm（3）的低浮力密度成带。代表性的抗ORF 3单克隆抗体（TA 0536）可以部分中和细胞培养系统中细胞培养产生的HEV感染。这些结果表明，ORF 3蛋白，至少其C-末端部分，存在于从感染细胞释放的HEV病毒粒子的表面上，并支持先前提出的假设，即ORF 3蛋白与从感染细胞释放的病毒有关。
Ten murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the well-conserved, C-terminal 24-amino acid portion of ORF3 protein of hepatitis E virus (HEV) were produced and characterized. Immunofluorescent assays using the anti-ORF3 MAbs revealed accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmid or inoculated with cell-culture-generated HEV. The anti-ORF3 MAbs could capture HEV particles in culture medium and serum at variable efficiency of up to 61 and 49%, respectively, but not those in feces. By sandwiching between immobilized and enzyme-labeled anti-ORF3 MAbs in ELISA, ORF3 antigen was detected in the culture media with an HEV RNA titer of > 10(6) copies/ml and increased in parallel with the increase in HEV load. HEV progenies in the culture supernatant, with ORF3 protein on the surface, banded at a low buoyant density of 1.15 g/cm(3) in sucrose. A representative anti-ORF3 MAb (TA0536) could partially neutralize the infection of cell-culture-generated HEV in a cell culture system. These results indicate that ORF3 protein, at least its C-terminal portion, is present on the surface of HEV virions released from infected cells and support a previously proposed assumption that ORF3 protein is associated with virus release from infected cells.