A high throughput neutralization test based on GFP expression by recombinant rabies virus

The effectiveness of rabies vaccination in both humans and animals is determined by the presence of virus neutralizing antibodies (VNAs). The Rapid Fluorescent Focus Inhibition Test (RFFIT) is the method traditionally used for detection and quantification of VNAs. It is a functional in vitro test for assessing the ability of antibodies in serum to bind and prevent infection of cultured cells with rabies virus (RABV). The RFFIT is a labor intensive, low throughput and semi-quantitative assay performed by trained laboratorians. It requires staining of RABV-infected cells by rabies specific fluorescent antibodies and manual quantification of fluorescent fields for titer determination. Although the quantification of fluorescent fields observed in each sample is recorded, the corresponding images are not stored or captured to be used for future analysis. To circumvent several of these disadvantages, we have developed an alternative, automated high throughput neutralization test (HTNT) for determination of rabies VNAs based on green fluorescent protein (GFP) expression by a recombinant RABV and compared with the RFFIT. The HTNT assay utilizes the recombinant RABV ERA variant expressing GFP with a nuclear localization signal (NLS) for efficient quantification. The HTNT is a quantitative method where the number of RABV-infected cells are determined and the images are stored for future analysis. Both RFFIT and HTNT results correlated 100% for a panel of human and animal positive and negative rabies serum samples. Although, the VNA titer values are generally agreeable, HTNT titers tend to be lower than that of RFFIT, probably due to the differences in quantification methods. Our data demonstrates the potential for HTNT assays in determination of rabies VNA titers.人和动物的狂犬病疫苗接种有效性，取决于病毒中和抗体（virus neutralizing antibodies，VNAs）的存在。传统上用于检测和定量病毒中和抗体的方法为快速荧光灶抑制试验（Rapid Fluorescent Focus Inhibition Test，RFFIT），其是一种功能性体外试验，可评估血清中抗体结合并阻止狂犬病病毒（rabies virus，RABV）感染培养细胞的能力。RFFIT属于劳动强度大、通量较低且半定量的检测手段，需由经过专业培训的实验人员操作：需使用狂犬病特异性荧光抗体对RABV感染的细胞进行染色，并通过人工计数荧光灶以确定抗体效价。尽管实验过程中会记录每个样本观测到的荧光灶数量，但并未留存或采集对应的图像以供后续分析。为规避上述诸多缺陷，本研究开发了一种基于重组RABV表达绿色荧光蛋白（green fluorescent protein，GFP）的自动化高通量中和试验（high throughput neutralization test，HTNT）以测定狂犬病病毒中和抗体效价，并将其与RFFIT进行了对比。该HTNT检测方法利用携带核定位信号（nuclear localization signal，NLS）的重组RABV ERA变体株表达GFP，以实现高效定量。HTNT属于定量检测方法，可统计RABV感染细胞的数量并留存图像以供后续分析。针对一组人类与动物的狂犬病阳性、阴性血清样本，RFFIT与HTNT的检测结果完全相关。尽管病毒中和抗体效价的总体数值较为一致，但HTNT测得的效价往往低于RFFIT，这可能源于两种检测方法的定量原理存在差异。本研究数据证实了HTNT检测方法在狂犬病病毒中和抗体效价测定中的应用潜力。