肝炎E病毒荧光减少中和测试用于测量中和抗体滴度
May 22nd, 2026
目前尚无针对非细胞病理阳性RNA病毒E型肝炎病毒（HEV）的标准病毒中和检测方法。这里介绍了一种基于荧光标记病毒衣壳蛋白定量的荧光减少中和试验（FRNT），用于评估抗HEV抗体的疗效。
基于荧光还原的中和试验的主要目的是量化疫苗候选物和抗HEV抗体的中和潜力。HEV已知的细胞病理性质限制了PRND测定。该方案侧重于基于免疫的PRND训练，以便观察感染细胞，随后观察我的细胞——首先，统计新鲜分裂的HuH-7细胞。
然后，将显微镜级玻璃盖片放入12孔板中，在1毫升完整DMEM中播种20万个细胞，补充10%FBS。在37摄氏度与5%二氧化碳的温度下孵育细胞过夜。次日，向种子细胞中添加500微升无血清培养基，含1.8乘以10的幂次，按四个纯化g1-HEV基因组的幂次方。
在37摄氏度与5%二氧化碳下孵育一小时。使用500微升DPBS清洗电池三次。向孔中加入2毫升DMEM，并补充2%FBS。
将细胞与5%二氧化碳在37摄氏度下孵育72小时。用500微升DPBS洗涤细胞三次，每次洗涤间隔在室温下培养五分钟。向细胞中加入500微升4%对甲醛，并在室温下培养20分钟。
然后，按照之前的演示，用DPBS清洗细胞。洗涤后，在室温下用DPBS孵育30分钟。在DPBS中加入250微升阻断液，含5%普通山羊血清、0.5%牛血清白蛋白和0.3%Triton X100。
在室温下放入摇杆孵化一小时。去除阻隔液，用500微升DPBS清洗一次。然后以1比500稀释加入250微升初级抗体溶液。
在四摄氏度下孵育细胞一夜。第二天，按照之前的步骤，用DPBS清洗细胞三次。加入100微升二级抗体，稀释比例为1：1,000，并在室温下在腐殖箱中孵育一小时。
按照之前演示的洗涤孵育周期进行。在玻璃载玻片上加入10微升的DAPI防褪入剂。将盖片装上，电池面向试剂，同时避免气泡。
用指甲油封住封面，以便共聚焦显微镜观察。数数新鲜分裂的HuH-7细胞。然后，将显微镜级玻璃盖片放入12孔板中，每孔用一毫升完整DMEM播种20万个细胞。
次日，在无血清培养基中制备抗ORF2和兔子免疫球蛋白G抗体的十倍序列稀释，起始抗体浓度为每毫升25微克，最终稀释至10微克的负五次方。最终稀释液的体积应为100微升。然后，将4份纯化g1-HEV基因组的幂次加1.8乘以10，对每次抗体稀释。
将抗体病毒混合物与5%二氧化碳在37摄氏度下孵育一小时。用500微升的DPBS清洗细胞。将抗体病毒混合物加入细胞中。
在37摄氏度与5%二氧化碳下孵育一小时。用500微升缓冲液清洗细胞。加入含2%FBS的DMEM后，在37摄氏度、含5%二氧化碳的环境下培养72小时。
如前所述，对细胞进行免疫荧光染色处理。获取荧光图像后，打开ImageJ软件。选择文件，打开图片。
然后，选择图像，类型，点击8位，将RGB图像转换为8位格式。通过选择图片并选择调整，再选择阈值，将下级设置为40，上级设置为255来调整阈值。选择分析，选择分析粒子，并启用显示结果、清除结果、总结、排除边缘和复合感兴趣区域。
然后点击确定以获取荧光斑点计数。计算中和百分比和NT50作为Log10 NT50的反对数，使用提供的公式。绘制所有字段均值和标准差的图表。
与模拟感染细胞相比，感染细胞中检测到的g1-HEV核糖核酸水平显著。使用抗ORF2抗体进行免疫荧光测定，确认感染的HuH-7细胞中ORF2表达。抗ORF2抗体的50%中和滴度估计为2.1乘以10的3次方。
代表性图像显示，在荧光还原中和测试中出现了模拟感染和g1-HEV感染的HuH-7细胞。该方案使研究人员能够研究抗HEV抗体的中和潜力，并测量其50%中和滴度。该方案的关键步骤是分离传染性E型肝炎病毒，并进行适当的细胞染色和高分辨率图像获取。
可用RT-qPCR和ELISA等替代方法测定中和滴度，但存在一些局限性。
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Overview
This article describes a fluorescence reduction neutralization test (FRNT) designed to measure the neutralization potential of antibodies against Hepatitis E virus (HEV). The method provides a valuable alternative to plaque reduction neutralization tests (PRNT), which are not feasible for non-cytopathic viruses like HEV. The FRNT enables researchers to estimate the 50% neutralization titre (NT50) of anti-HEV antibodies, supporting vaccine and therapeutic antibody development.
Key Study Components
Area of Science
- Virology
- Immunology
- Vaccine development
Background
- Virus neutralization assays assess the efficacy of antibodies or vaccines against specific viruses.
- PRNT is commonly used but unsuitable for non-cytopathic viruses such as HEV.
- HEV is a major cause of acute viral hepatitis and lacks widespread vaccine or therapeutic antibody availability.
- Reliable neutralization assays are needed to evaluate HEV vaccine candidates and antibody responses.
Purpose of Study
- To develop and describe a fluorescence-based neutralization assay for HEV.
- To provide a method for estimating the NT50 of anti-HEV antibodies.
- To facilitate the evaluation of vaccine candidates and antibody efficacy against HEV.
Methods Used
- HEV was neutralized with anti-HEV ORF2 antibodies or rabbit IgG.
- Neutralized virus was used to infect Huh7 cells.
- Immunofluorescent staining targeted the viral ORF2 protein.
- Fluorescent positive cells were quantified using ImageJ software.
- NT50 values were calculated based on fluorescence reduction.
Main Results
- The FRNT successfully estimated the NT50 of anti-HEV antibodies.
- The NT50 was determined to be 2.1 x 103 for the tested antibodies.
- The method is applicable to sera from immunized mice, HEV-recovered individuals, and laboratory-produced antibodies.
Conclusions
- The FRNT provides a practical and effective assay for measuring HEV-neutralizing antibodies.
- This method supports the evaluation of vaccine candidates and antibody-based therapies for HEV.
- It addresses the limitations of PRNT for non-cytopathic viruses.
Frequently Asked Questions
Executive Industry Relevance
Quantitative measurement of neutralizing antibody titers against Hepatitis E virus (HEV) is critical for vaccine candidate evaluation and therapeutic antibody development, especially given the lack of cytopathic effect in standard cell culture. The fluorescence reduction neutralization test (FRNT) enables robust assessment of antibody efficacy where traditional plaque assays are not feasible, directly impacting early discovery and translational research pipelines. This capability supports portfolio decisions by providing actionable data on immunogenicity and neutralization potential for HEV interventions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct quantification of antibody-mediated HEV neutralization for target validation.
- Supports mechanistic de-risking by confirming functional antibody-virus interactions.
- Provides predictive confidence for advancing vaccine and antibody candidates.
Screening & Assay Development
- Delivers a standardized, reproducible assay for measuring neutralizing titers in non-cytopathic virus systems.
- Facilitates quantitative comparison of candidate antibodies and sera from immunized or recovered subjects.
- Prepares validated biological systems for downstream screening and optimization workflows.
Translational & Preclinical Research
- Aligns neutralization data with translational biomarker strategies for HEV.
- Enables continuity from discovery-stage antibody screening to preclinical efficacy assessment.
- Supports risk-adjusted advancement of vaccine and therapeutic antibody programs.
Pipeline & Workflow Integration
The FRNT method integrates into the discovery-to-preclinical continuum by providing a quantitative, reproducible readout of antibody neutralization for HEV, bridging early candidate selection and translational validation.
- Discovery Biology: Supports hypothesis testing and biological de-risking by quantifying neutralization capacity.
- Screening: Offers assay readiness and reproducibility for evaluating multiple antibody or serum samples.
- Analytics: Provides quantitative NT50 measurements to compare neutralizing potency across conditions.
- Translational Research: Links in vitro neutralization to preclinical biomarker strategies for HEV.
- Enterprise Reuse: Establishes a reusable platform for neutralization testing in non-cytopathic virus research.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in HEV antibody evaluation.
- Operational Value: Standardizes neutralization testing for non-cytopathic viruses, improving reproducibility and scalability.
- Strategic Value: Enables informed go/no-go decisions and capital-efficient advancement of HEV vaccine and antibody assets.
- Portfolio Impact: Supports risk-adjusted prioritization of candidates based on quantitative neutralization data.
Implementation Considerations
- Requires expertise in immunofluorescence and quantitative image analysis.
- Needs access to Huh7 cell culture, immunostaining reagents, and fluorescence microscopy.
- Demands cross-team standardization for assay setup and data interpretation.
- Adaptation to other non-cytopathic viruses may require protocol optimization.
- Dependent on availability of validated anti-HEV antibodies and reference sera.