QUALITATIVE AND QUANTITATIVE-ANALYSES OF EPSTEIN-BARR VIRUS EARLY ANTIGEN DIFFUSE COMPONENT BY WESTERN BLOTTING ENZYME-LINKED IMMUNOSORBENT-ASSAY WITH A MONOCLONAL-ANTIBODY
QUALITATIVE AND QUANTITATIVE-ANALYSES OF EPSTEIN-BARR VIRUS EARLY ANTIGEN DIFFUSE COMPONENT BY WESTERN BLOTTING ENZYME-LINKED IMMUNOSORBENT-ASSAY WITH A MONOCLONAL-ANTIBODY
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DOI：
10.1128/jvi.53.3.793-799.1985
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发表时间：
1985-01-01
影响因子：
5.4
通讯作者：
PAGANO, JS
中科院分区：
文献类型：
--
作者：
LIN, JC;CHOI, EI;PAGANO, JS
报道了使用抗EB病毒（Epstein-Barr virus，EBV）早期抗原扩散成分的单克隆抗体（anti-EA-D）定性和定量分析化学诱导剂激活的各种人淋巴母细胞系中EA-D的表达。P3 HR-1、B 95 -8和拉莫斯/AW细胞中EA-D的合成动力学相似，均在诱导后第5天达到合成高峰。在诱导的BJAB/GC细胞系中未发现EA-D的表达。EBV阴性细胞系BJAB和拉莫斯对EA-D呈阴性。Raji细胞没有检测到EA-D，但对诱导反应迅速，在第3天达到峰值。Raji细胞的超感染也导致EA-D的显著诱导，其在感染后8至12小时之间达到平台。蛋白质印迹结合酶联免疫吸附试验[ELISA]被用来鉴定代表EA-D的多肽。分子量为46，000（46 K蛋白）、49，000、52，000和55，000的4种多肽家族被鉴定为与单克隆抗EA-D抗血清反应。EA-D多肽在各细胞系中的表达模式不同。特别令人感兴趣的是大量的46 K蛋白在诱导的Raji和P3 HR-1细胞中的表达，但在超感染的Raji细胞中没有。在活化的p3 HR-1、B 95 -8和拉莫斯/AW细胞中以及在超感染的Raji细胞中表达49 K双联体。两种不同的52 K和55 K多肽在诱导的拉莫斯/AW和超感染的Raji细胞中表达。在BJAB/GC、BJAB、拉莫斯和模拟感染的Raji细胞中没有检测到这些EA-D多肽。为了近似细胞提取物中EA-D的相对浓度，通过使用纯化的EA-D组分之一来构建标准曲线，采用ELISA和免疫印迹斑点法。根据细胞系，估计. apprx.最大诱导细胞的总蛋白中有1-3%（通过ELISA测定）和0.8-1.6%（通过免疫印迹点测定）是EA-D。这些结果表明，EA-D多肽的差异表达可能在EBV感染状态的诊断中具有重要意义。
The use of monoclonal antibody against the early antigen diffuse component (anti-EA-D) of Epstein-Barr virus (EBV) to analyze, both qualitatively and quantitatively, the expression of EA-D in various human lymphoblastoid cell lines activated by chemical inducers is reported. The kinetics of synthesis of EA-D in P3HR-1, B95-8 and Ramos/AW cells were similar in that they all reached the peak of synthesis on day 5 after induction. No expression of EA-D was found in induced BJAB/GC, an EBV-genome-containing cell line. EBV-negative cell lines, BJAB and Ramos, were negative for EA-D. Raji cells had no detectable EA-D but responded rapidly to induction, reaching on peak on day 3. Superinfection of Raji cells also resulted in marked induction of EA-D, which reached a plateau between 8 to 12 h postinfection. Western blotting coupled with the enzyme-linked immunosorbent assay [ELISA] was employed to identify polypeptides representing EA-D. A family of 4 polypeptides with MW of 46,000 (46K protein), 49,000, 52,000 and 55,000 were identified to be reactive with monoclonal anti-EA-D antiserum. The pattern of EA-D polypeptides expressed in each cell line was different. Of particular interest was the expression of a large quantity of 46K protein both in induced Raji and P3HR-1 cells, but not in superinfected Raji cells. A 49K doublet was expressed in activated p3HR-1, B95-8 and Ramos/AW cells and in superinfected Raji cells. Two distinct 52K and 55K polypeptides were expressed in induced Ramos/AW and superinfected Raji cells. None of these EA-D polypeptides was detectable in BJAB/GC, BJAB, Ramos and mock-infected Raji cells. To approximate relative concentrations of EA-D in cell extracts, ELISA and immunoblot dot methods were employed by using one of the purified EA-D components to construct a standard curve. Depending on the cell lines, it was estimated that .apprx. 1-3% (determined by the ELISA) and 0.8-1.6% (determined by immunoblot dot) of total proteins from maximally induced cells were EA-D. These results suggest that differential expression of EA-D polypeptides could be of importance in the diagnosis of state of EBV infection.