Background

Plaque Reduction Neutralization Test (PRNT) is the standard assay used for measuring neutralizing antibody responses to Herpes simplex virus type-2 (HSV-2). The PRNT is a cumbersome, time-consuming and laborious assay. The development of a faster, high throughput microneutralization assay (MNA) for HSV-2 viruses carried out in a 96-well format will allow for rapid testing of large numbers of samples for drug and vaccine development.

Methods

We describe the generation of a MNA that utilizes a pair of anti-HSV human monoclonal antibodies (mAbs) for virus detection in HSV-2 infected Vero cells. Antibodies were generated by B-cell cloning from PBMC’s isolated from HSV-1 negative/HSV-2 positive donors. We describe the selection and characterization of the antibodies used for virus detection by ELISA with purified, recombinant anti-HSV glycoproteins, antibody binding in infected cells, and Western Blot. We determine the anti-HSV-2 neutralizing titers of immune sera from mice by MNA and PRNT and compare these results by linear regression analysis.

Results

We show that neutralization titers for HSV-2, determined by the 96-well MNA correlate with titers determined by a PRNT completed in 24-well plates in both the absence (R² = 0.8250) and presence (R² = 0.7075) of complement.

Conclusions

We have successfully developed an MNA that can be used in place of the burdensome PRNT to determine anti-HSV-2 neutralizing activity in serum. This MNA has much greater throughput than the PRNT, allowing many more samples to be processed in a shorter time saving ∼90 % of the time required by the laboratory scientist to complete the task as compared to the traditional PRNT.

中文翻译：

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HSV-2 微量中和试验的开发

背景

斑块减少中和试验 (PRNT) 是用于测量对 2 型单纯疱疹病毒 (HSV-2) 的中和抗体反应的标准检测方法。PRNT 是一种繁琐、耗时且费力的检测方法。以 96 孔形式进行的 HSV-2 病毒的更快、高通量微量中和试验 (MNA) 的开发将允许对大量样品进行快速测试以用于药物和疫苗的开发。

方法

我们描述了利用一对抗 HSV 人单克隆抗体 (mAb) 在 HSV-2 感染的 Vero 细胞中检测病毒的 MNA 的产生。通过从分离自 HSV-1 阴性/HSV-2 阳性供体的 PBMC 中克隆 B 细胞产生抗体。我们描述了通过 ELISA 与纯化的重组抗 HSV 糖蛋白、感染细胞中的抗体结合和蛋白质印迹法检测病毒所用抗体的选择和表征。我们通过 MNA 和 PRNT 确定小鼠免疫血清的抗 HSV-2 中和滴度，并通过线性回归分析比较这些结果。

结果

^{我们表明，由 96 孔 MNA 确定的 HSV-2 中和滴度与在补体不存在 (R 2} = 0.8250) 和存在 (R ² = 0.7075) 的情况下在 24 孔板中完成的 PRNT 确定的滴度相关。 .

结论

我们成功开发了一种 MNA，可用于代替繁重的 PRNT 来确定血清中的抗 HSV-2 中和活性。这种 MNA 的吞吐量比 PRNT 高得多，与传统的 PRNT 相比，允许在更短的时间内处理更多的样品，节省了实验室科学家完成任务所需时间的 90%。