Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology
Efficient recovery and enrichment of infectious rotavirus using separation with antibody-integrated graphite-encapsulated magnetic nanobeads produced by argon/ammonia gas plasma technology
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DOI：
10.2147/ijn.s191784
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发表时间：
2019-03
影响因子：
8
通讯作者：
R. Yamashiro;A. Sakudo;M. Nagatsu
中科院分区：
文献类型：
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作者：
R. Yamashiro;A. Sakudo;M. Nagatsu
研究背景轮状病毒是引起婴幼儿严重急性胃肠炎的代表性病原。轮状病毒的一个特征是感染剂量低和病毒粒子坚固，这表明卫生和个人卫生的影响很小。因此，疫苗的开发应被优先考虑。有效捕获感染性病毒是产生疫苗的重要步骤。以前，已经开发了抗体整合的磁性纳米珠（MNB）用于病毒捕获。本研究考察了该方法用于感染性轮状病毒回收和富集的适用性。材料和方法用射频（RF）激发的Ar/NH3气体混合物等离子体处理石墨封装的MNB以将氨基引入到它们的表面上。使用偶联剂N-琥珀酰亚胺基3-（2-吡啶基二硫代）丙酸酯（SPDP）通过这些氨基将轮状病毒病毒蛋白7（VP 7）抗体连接到MNB的表面。将抗体整合的MNB与轮状病毒感染的细胞裂解物一起孵育，然后通过施加磁场从上清液中分离。彻底洗涤后，回收轮状病毒，并通过聚合酶链反应（PCR）、免疫层析和使用MA 104细胞的感染分析进行富集分析。结果和讨论免疫层析和PCR表明，抗轮状病毒抗体整合的MNP有效地捕获轮状病毒的衣壳蛋白和病毒RNA。PCR法和感染分析法的回收率分别为80.2%和90.0%，富集倍数分别为7.9倍和6.7倍。此外，使用整合有抗登革病毒抗体的MNB作为阴性对照，证实了不存在与整合有抗体的MNP的非特异性结合。结论该方法是一种有效的轮状病毒分离富集方法。此外，当与合适的病毒测定组合时，该捕获程序可以增加轮状病毒检测的灵敏度。因此，这种捕获方法是用于产生疫苗以及用于开发用于病毒的灵敏检测系统的有价值的工具。
Background Rotavirus is the representative cause of severe acute gastroenteritis in young children. A characteristic feature of rotavirus is low infectious dose and robustness of the virion, suggesting sanitation and hygiene will have little impact. Thus, development of a vaccine should be given priority. Efficient capture of infectious viruses is an important step in generating a vaccine. Previously, antibody-integrated magnetic nanobeads (MNBs) have been developed for virus capture. This study examines the applicability of this method for infectious rotavirus recovery and enrichment. Materials and methods Graphite-encapsulated MNBs were treated with radio frequency (RF) excited Ar/NH3 gas mixture plasma to introduce amino groups onto their surfaces. Rotavirus viral protein 7 (VP7) antibody was attached to the surface of MNBs via these amino groups using a coupling agent, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The antibody-integrated MNBs were incubated with rotavirus-infected cell lysate and then separated from the supernatant by applying a magnetic field. After thorough washing, rotavirus was recovered and enrichment analysis done by polymerase chain reaction (PCR), immunochromatography, and an infection analysis using MA104 cells. Results and discussion Immunochromatography and PCR indicate that anti-rotavirus antibody-integrated MNPs efficiently capture rotavirus with the capsid protein and viral RNA. The estimated recovery rate was 80.2% by PCR and 90.0% by infection analysis, while the concentrating factor was 7.9-fold by PCR and 6.7-fold by infection analysis. In addition, the absence of non-specific binding to the antibody-integrated MNPs was confirmed using anti-dengue virus antibody-integrated MNBs as a negative control. Conclusion These results suggest that this capture procedure is a useful tool for recovery and enrichment of infectious rotavirus. Moreover, when combined with a suitable virus assay this capture procedure can increase the sensitivity of rotavirus detection. Therefore, this capture method is a valuable tool for generating vaccines as well as for developing sensitive detection systems for viruses.