Scalable, semi-automated fluorescence reduction neutralization assay for qualitative assessment of Ebola virus-neutralizing antibodies in human clinical samples

Antibody titers against a viral pathogen are typically measured using an antigen binding assay, such as an enzyme-linked immunosorbent assay (ELISA), which only measures the ability of antibodies to identify a viral antigen of interest. Neutralization assays measure the presence of virus-neutralizing antibodies in a sample. Traditional neutralization assays, such as the plaque reduction neutralization test (PRNT), are often difficult to use on a large scale due to being both labor and resource intensive. Here we describe an Ebola virus fluorescence reduction neutralization assay (FRNA), which tests for neutralizing antibodies, that requires only a small volume of sample in a 96-well format and is easy to automate. The readout of the FRNA is the percentage of Ebola virus-infected cells measured with an optical reader or overall chemiluminescence that can be generated by multiple reading platforms and the readout is compatible with lytic and non-lytic viruses. Using blinded human clinical samples (EVD survivors or contacts) obtained in Liberia during the 2013–2016 Ebola virus disease outbreak, we demonstrate that FRNA-measured antibody titers are highly correlated with those measured by the Filovirus Animal Non-clinical Group (FANG) ELISA - the current standard for anti-EBOV antibody measurement with the important distinction of providing information on the neutralizing capabilities of the antibodies.

针对病毒病原体的抗体滴度通常采用抗原结合试验进行检测，例如酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA），该方法仅能测定抗体识别目标病毒抗原的能力。中和试验则用于检测样本中病毒中和抗体的存在。传统中和试验如空斑减少中和试验（plaque reduction neutralization test, PRNT）往往因劳动强度大、资源消耗高，难以大规模开展。本文介绍了一种埃博拉病毒荧光减少中和试验（Ebola virus fluorescence reduction neutralization assay, FRNA），该方法用于检测中和抗体，仅需少量样本，采用96孔板格式，且易于实现自动化。该试验的读数结果为通过光学读数仪或多平台均可生成的化学发光法检测的埃博拉病毒感染细胞占比，其读数方式兼容裂解性与非裂解性病毒。本研究使用2013–2016年埃博拉病毒病（Ebola virus disease, EVD）疫情期间在利比里亚获取的盲法人类临床样本（埃博拉病毒病幸存者及接触者）进行验证，结果显示FRNA测得的抗体滴度与当前抗埃博拉病毒（Ebola virus, EBOV）抗体检测的金标准——丝状病毒动物非临床工作组（Filovirus Animal Non-clinical Group, FANG）ELISA的检测结果高度相关，且其独特优势在于可提供抗体中和能力相关信息。