A new monoclonal antibody (Cox mAB 31A2) detects VP1 protein of coxsackievirus B3 with high sensitivity and specificity
A new monoclonal antibody (Cox mAB 31A2) detects VP1 protein of coxsackievirus B3 with high sensitivity and specificity
复制标题
DOI：
10.1007/s00428-016-2008-8
复制
发表时间：
2016-11-01
期刊：
影响因子：
3.5
通讯作者：
Klingel, Karin
中科院分区：
文献类型：
--
作者：
Ettischer-Schmid, Nicole;Normann, Andrea;Klingel, Karin
人类肠道病毒，例如柯萨奇病毒可诱发多种严重的急性和慢性疾病，包括心肌炎、脑膜炎和 1 型糖尿病。为了可视化肠道病毒感染并具有诊断目的，许多研究应用了市售抗体（抗 CVB5 VP1，克隆 5-D8/1，Dako，汉堡，德国）来识别不同肠道病毒血清型的 VP1。然而，已发现许多抗体与心肌细胞和间质空间中的蛋白质非特异性结合，导致免疫组织化学中的非特异性染色。在本文中，我们表明抗CVB5 VP1抗体可识别柯萨奇病毒的VP1并广泛用于诊断和研究，与人类和小鼠心脏（和胰腺）中的细胞蛋白表现出强烈的交叉反应性，这需要更特异性的抗体用于诊断目的。当使用克隆 5-D8/1 时，我们通过对人类心脏组织样品和 HeLa 细胞的裂解物进行蛋白质印迹分析观察到两个交叉反应条带。肽质量指纹图谱 (MALDI-TOF) 将这些蛋白质鉴定为肌酸激酶（B 型）和微管蛋白，证实该 mAb 除病毒 VP1 外还可检测细胞蛋白质。为了克服 VP1 染色假阳性的问题，我们生成了一种新的高度特异性和敏感的单克隆抗体 (Cox mAB 31A2)，可以从 CVB3 中识别 VP1。新抗体经过表征，发现在免疫组织化学、免疫荧光染色、蛋白质印迹、ELISA 和 FACS 分析中表现良好。
Human enteroviruses, e.g. coxsackieviruses, induce a variety of severe acute and chronic forms of disease, including myocarditis, meningitis and diabetes mellitus type 1. To visualize enterovirus infection with a diagnostic intent, many studies have applied a commercially available antibody (anti-CVB5 VP1, clone 5-D8/1, Dako, Hamburg, Germany) that identifies VP1 of different enteroviral serotypes. Many antibodies, however, have been found to bind non-specifically to proteins of cardiomyocytes and in the interstitial space, resulting in non-specific staining in immunohistochemistry. In this paper we show that the anti-CVB5 VP1 antibody, recognizing VP1 of coxsackieviruses and widely used in diagnostics and research, shows strong cross-reactivity with cellular proteins in the heart (and pancreas) of humans and mice, which calls for a more specific antibody to be used for diagnostic purposes. We observed by Western blot analyses of lysates from human heart tissue samples and HeLa cells two cross-reactive bands when using clone 5-D8/1. Peptide mass fingerprinting (MALDI-TOF) identified these proteins as creatine kinase (B-type) and tubulin, confirming that this mAb detects cellular proteins in addition to viral VP1. In order to overcome the problems of false positive VP1 staining we generated a new highly specific and sensitive monoclonal antibody (Cox mAB 31A2) that recognizes VP1 from CVB3. The new antibody was characterized and was found to function well in immunohistochemistry, immunofluorescence staining, Western blotting, ELISA and FACS analyses.