MOE Key Laboratory of Protein Science, School of Life Sciences, Tsinghua University, Beijing 100084, PR China; AnyGo Technology, D1117 New China International Square, 89 Dayangfang Rd, Chaoyang District, Beijing, PR China.
National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention.155 Changbai Rd, Changping District,Beijing, PR China.
Vaccine. 2019 Sep 24;37(41):6060-6067. doi: 10.1016/j.vaccine.2019.08.043. Epub 2019 Aug 27.
Vaccination provides protection against infection by inducing VNAs mainly against RABV surface GP. The measurement of VNAs to RABV is commonly used to assess the level of immunity in humans and animals after vaccination. A VNA titer of ≥ 0.5 IU/mL of sera indicates adequate response to vaccination. Here, we report the development and validation of a RABV GP serology ELISA kit for semi-quantitative measurement of VNA titers in sera of vaccinated human subjects.
Using a recombinant RABV GP expressed in mammalian cells as the capture antigen, the ELISA method was established using HuMAb NM57 reference initially and HRIG reference subsequently. The limit of detection (LOD), linear range, reproducibility, and precision of the method were examined. Specificity and sensitivity were established to assess the diagnostic accuracy.
RABV GP for ELISA plate coating and optimal dilution of human serum sample was 1 µg/mL and 1:20, respectively. Multiple assays were carried out by different technicians at different laboratories for assay standardization. Using the HRIG reference, the LOD was found to be 0.02-0.06 IU/mL and the linear range was 0.2-10.0 IU/ mL. The inter-assay CVs were in the range of 6.60-10.79%, indicating the reproducibility. None of the 12 known negative human sera, tested positive by ELISA, highlighting the specificity. A total of 415 unknown positive human sera were double-blind tested by the RFFIT and ELISA. The VNA titer cut-off value of ELISA was set at 1.5 IU/mL to ensure no false-positive. The diagnostic specificity and sensitivity were 100% and 91.1%, respectively.
The validation data characterize this ELISA as a suitable method for semi-quantitative measurement of VNA titers in human serum samples to assess vaccination status. The ELISA kit can offer simplicity, speed, low cost and high throughput, making it a practical tool for monitoring the immune response following vaccination.
疫苗接种通过诱导主要针对 RABV 表面 GP 的 VNA 来提供针对感染的保护。测量针对 RABV 的 VNA 通常用于评估人类和动物接种疫苗后的免疫水平。血清中的 VNA 效价≥0.5 IU/mL 表明对疫苗接种有足够的反应。在这里，我们报告了一种用于半定量测量接种人类受试者血清中 VNA 效价的 RABV GP 血清学 ELISA 试剂盒的开发和验证。
使用在哺乳动物细胞中表达的重组 RABV GP 作为捕获抗原，使用 HuMAb NM57 参考品最初和 HRIG 参考品随后建立 ELISA 方法。检查了方法的检测限（LOD）、线性范围、重现性和精密度。建立了特异性和敏感性来评估诊断准确性。
RABV GP 用于 ELISA 板包被和人血清样品的最佳稀释度分别为 1μg/mL 和 1:20。由不同的技术员在不同的实验室进行多次测定以进行测定标准化。使用 HRIG 参考品，LOD 为 0.02-0.06 IU/mL，线性范围为 0.2-10.0 IU/mL。批内 CV 范围为 6.60-10.79%，表明重现性良好。在 ELISA 检测中，没有 12 份已知的阴性人血清呈阳性，表明特异性良好。共有 415 份未知的阳性人血清由 RFFIT 和 ELISA 进行双盲检测。ELISA 的 VNA 效价截断值设定为 1.5 IU/mL 以确保无假阳性。ELISA 的诊断特异性和敏感性分别为 100%和 91.1%。
验证数据表明，该 ELISA 是一种适合半定量测量人血清样品中 VNA 效价的方法，用于评估接种状态。该 ELISA 试剂盒具有简单、快速、低成本和高通量的特点，是监测接种后免疫反应的实用工具。
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