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目的 建立一种快速、高通量检测痘病毒中和抗体的方法。方法 对荧光斑点检测时间、病毒-抗体（血清）中和反应时间等关键参数进行优化。确定最佳反应条件，并进行方法学的系统验证。结果 确定荧光斑点检测时间为24 h，病毒-抗体中和反应时间为1 h。微量中和试验方法能准确区分已知阳性和阴性血清，在1:50至1:800稀释范围内灵敏度良好，且与噬斑减少中和试验（Plaque reduction neutralization test, PRNT）结果具有高度一致性。结论本方法将检测时间从72 h缩短至24 h，样品用量减少至原来的1/4，操作流程更易标准化，适用于痘病毒中和抗体的快速、高通量筛查。
Abstract：Objective To establish a rapid and high-throughput method for the detection of poxvirusneutralizing antibodies.Methods Key experimental parameters, including the fluorescent focus detection time and the virus-antibody(serum) neutralization incubation time, were systematically optimized to determine optimal assay conditions. The established method was subsequently subjected to comprehensive methodological validation. Results The optimal fluorescent focus detection time was determined to be 24 h, and the virusantibody neutralization incubation time was 1 h. The microneutralization assay accurately distinguished known positive and negative sera, demonstrated good sensitivity over a serum dilution range of 1:50 to 1:800, and showed high concordance with results obtained using the plaque reduction neutralization test(PRNT).Conclusion Compared with conventional assays, this method shortens the detection time from 72 h to 24 h, reduces sample consumption to one-fourth of the original amount, and offers a workflow that is more amenable to standardization. It is therefore well suited for the rapid and high-throughput screening of poxvirus-neutralizing antibodies.
[1]Oliveira GP, Rodrigues RAL, Lima MT, et al.Poxvirus host range genes and virus-host spectrum:a critical review[J]. Viruses, 2017, 9(11):331. DOI:10. 3390/v9110331.
[2]Henderson DA. The eradication of smallpox—an overview of the past, present, and future[J]. Vaccine,2011, 29(Suppl 4):D7-D9. DOI:10. 1016/j.vaccine. 2011. 06. 080.
[3]Krylova O, Earn DJD. Patterns of smallpox mortality in London, England, over three centuries[J]. PLoS Biol,2020, 18(12):e3000506. DOI:10. 1371/journal.pbio. 3000506.
[4]WHO. WHO Director-General declares mpox outbreak a public health emergency of international concern[EB/OL].(2024-08-14)[2025-04-14]. https：//www. who.int/news/item/14-08-2024-who-director-generaldeclares-mpox-outbreak-a-public-health-emergency-ofinternational-concern.
[5]WHO. Global Mpox Trends[EB/OL].(2025-10-11)[2025-10-15]. https：//worldhealthorg. shinyapps. io/mpx_global/.
[6]石建州，王国川，王彦伟，等.猴痘病毒研究进展[J].病毒学报，2022, 38(5):1195-1205. DOI:10. 13242/j. cnki. bingduxuebao. 004188.
[7]Azami NAM, Moi ML, Takasaki T. Neutralization assay for chikungunya virus infection:plaque reduction neutralization test[J]. Methods Mol Biol, 2016, 1426:273-282. DOI:10. 1007/978-1-4939-3618-2_25.
[8]Kong HJ, Kim YJ, Kim D, et al. Plaque reduction neutralization test for smallpox vaccines:Laboratory optimization and validation method for immunogenicity assessment[J]. J Immunol Methods, 2025, 536:113787. DOI:10. 1016/j. jim. 2024. 113787.
[9]Haga K, Chen ZN, Himeno M, et al. Utility of an invitro micro-neutralizing test in comparison to a plaque reduction neutralization test for dengue virus, Japanese encephalitis virus, and zika virus serology and drug screening[J]. Pathogens, 2023, 13(1):8. DOI:10. 3390/pathogens13010008.
[10]Wang G, Huang P, Hong J, et al. Establishment of a rapid ELISPOT assay for influenza virus titration and neutralizing antibody detection[J]. J Med Virol, 2021,93(6):3455-3464. DOI:10. 1002/jmv. 26257.
[11]Liu L, Wen K, Li J, et al. Comparison of plaque-and enzyme-linked immunospot-based assays to measure the neutralizing activities of monoclonal antibodies specific to domain III of dengue virus envelope protein[J]. Clin Vaccine Immunol, 2012, 19(1):73-78. DOI:10. 1128/CVI. 05388-11.
[12]Zhou Z, Zhu R, Yang H, et al. Development of a rapid neutralization testing system for Rhinovirus C15 based on the enzyme-linked immunospot assay[J]. Front Microbiol, 2022, 13:983656. DOI:10. 3389/fmicb. 2022. 983656.
[13]Li S, Zhao H, Yang H, et al. Rapid neutralization testing system for zika virus based on an enzyme-linked immunospot assay[J]. ACS Infect Dis, 2020, 6(5):811-819. DOI:10. 1021/acsinfecdis. 9b00333.
[14]Jacobs BL, Langland JO, Kibler KV, et al. Vaccinia virus vaccines:past, present and future[J]. Antiviral Res, 2009, 84(1):1-13. DOI:10. 1016/j.antiviral. 2009. 06. 006.
[15]Teodoro LI, Ovsyannikova IG, Poland GA, et al.Examining homology between MPXV and immunogenic VACV-derived peptides[J]. Vaccine, 2025, 48:126708. DOI:10. 1016/j. vaccine. 2025. 126708.
[16]刘铮，刘颖，邵一鸣.正痘病毒基因结构及功能研究的几项进展[J].病毒学报，2013, 29(4):437-441. DOI:10. 13242/j. cnki. bingduxuebao. 002415.
[17]梁慧颖，李剑楠，刘洪涛，等.基于补体依赖的水痘-带状疱疹病毒中和抗体效价检测方法的建立及应用[J].中国生物制品学杂志，2023, 36(4):464-468. DOI:10. 13200/j. cnki. cjb. 003872.
[18]王雪琛，李涛，杨晓岚，等.猴痘病毒蛋白的原核表达及免疫效果评价[J].病毒学报，2024, 40(4):772-778. DOI:10. 13242/j. cnki. bingduxuebao. 004527.
[19]Shi Y, Wang S, Hao Y, et al. Isolation and characterization of E8 monoclonal antibodies from donors vaccinated with recombinant vaccinia vaccine with efficient neutralization of authentic monkeypox virus[J]. Vaccines, 2025, 13(5):471. DOI:10. 3390/vaccines13050471.
[20]Marchi S, Piccini G, Cantaloni P, et al. Evaluation of monkeypox-and vaccinia-virus neutralizing antibodies before and after smallpox vaccination:a seroepidemiological study[J]. J Med Virol, 2024, 96(6):e29728. DOI:10. 1002/jmv. 29728.
[21]Gao F, He C, Liu M, et al. Cross-reactive immune responses to monkeypox virus induced by MVA vaccination in mice[J]. Virol J, 2023, 20(1):126.DOI:10. 1186/s12985-023-02085-0.
[22]Otter AD, Jones S, Hicks B, et al. Monkeypox virusinfected individuals mount comparable humoral immune responses as Smallpox-vaccinated individuals[J]. Nat Commun, 2023, 14(1):5948. DOI:10. 1038/s41467-023-41587-x.
[23]Li E, Guo X, Hong D, et al. Duration of humoral immunity from smallpox vaccination and its crossreaction with Mpox virus[J]. Signal Transduct Target Ther, 2023, 8(1):350. DOI:10. 1038/s41392-023-01574-6.
[24]白文娣，李曦然，马勇，等.猴痘病毒治疗性抗体的研究进展[J].病毒学报，2023, 39(4):1118-1123. DOI:10. 13242/j. cnki. bingduxuebao. 004343.
[25]公玥，彭宗根.抗猴痘病毒药物的研究进展[J].病毒学报，2023, 39(6):1703-1712. DOI:10. 13242/j.cnki. bingduxuebao. 004411.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250361
中图分类号:R446.6;R511
引用信息:
[1]张俊,刘颖,申秀丽,等.一种基于荧光ELISPOT技术的高通量快速检测痘病毒中和抗体方法[J].病毒学报,2026,42(03):793-800.DOI:10.13242/j.cnki.bingduxuebao.250361.
基金信息:
国家重点研发计划(项目号:2024YFC2309500),题目:HIV-猴痘病毒共感染免疫特征和防控关键技术研究; 全国重点实验室激励课题(项目号:2024NITFID805),题目:复制型痘病毒载体疫苗诱导特异性猴痘体液免疫特征研究~~
2026-02-02
2026-02-02
2026-02-02