Cloning and expression of two human recombinant monoclonal Fab fragments specific for EBV viral capsid antigen
Cloning and expression of two human recombinant monoclonal Fab fragments specific for EBV viral capsid antigen
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DOI：
10.1093/intimm/dxl150
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发表时间：
2007-03-01
影响因子：
4.4
通讯作者：
Umehara, Hisanori
中科院分区：
文献类型：
--
作者：
Dong, Lingli;Masaki, Yasufumi;Umehara, Hisanori
EB病毒(EBV)病毒衣壳抗原(VCA)抗体滴度与淋巴系增生性恶性肿瘤呈正相关，如Burkitt‘s淋巴瘤和Hodgkin’s淋巴瘤。我们从1例血清抗EBV-VCA抗体滴度较高的干燥综合征边缘带B细胞淋巴瘤患者体内构建了噬菌体展示抗体Fab文库。以包被于酶标板上的EBV-VCA蛋白为底物，筛选Fab片段，并对Fab克隆进行酶联免疫吸附试验、免疫印迹、间接免疫荧光和免疫组织化学鉴定。我们建立了两个Fab克隆，Fab-aVCA1和Fab-aVCA21，分别用EL ISA和WB特异性识别EBV-VCA。抑制性ELISA竞争表明，两个克隆均能显著降低抗EBV-VCA单抗与其相关蛋白的结合。此外，这两个Fab克隆可以在EBV阳性的P3HR1和Daudi细胞系以及EBV感染的淋巴系统恶性肿瘤患者的组织样本中定位VCA蛋白。这些结果表明，我们的两个Fab克隆是针对EBV-VCA蛋白的新的人源单抗，可能对开发EBV相关淋巴系统恶性肿瘤的新的诊断和治疗方法具有潜在的益处。
Serum titers of antibody to Epstein-Barr virus (EBV) viral capsid antigen (VCA) have been positively correlated with malignancies of lymphoid proliferation, such as Burkitt's lymphoma and Hodgkin's lymphoma. We have constructed a phage display combinatorial antibody Fab library from a patient with marginal zone B cell lymphoma associated with Sjogren's syndrome and carrying high serum anti-EBV-VCA IgG titer. Fab fragments were selected by panning against EBV-VCA protein coated onto ELISA plates, and selected Fab clones were characterized by ELISA, western blotting (WB), indirect immunofluorescence assay and immunohistochemistry. We have established two Fab clones, Fab-aVCA1 and Fab-aVCA21, which specifically recognize EBV-VCA by ELISA and WB. Inhibition ELISA competition showed that both clones could significantly reduce the binding of specific anti-EBV-VCA mAb to its relevant proteins. Furthermore, these two Fab clones could localize VCA protein in the EBV-positive P3HR1 and Daudi cell lines, as well as in tissue samples from patients with EBV-infected lymphoid malignancies. These results indicate that our two Fab clones are novel human mAbs specific for EBV-VCA protein and may have potential benefits for development of novel diagnostic and therapeutic approaches in EBV-related lymphoid malignancies.