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基于病毒细胞病变的禽肾炎病毒滴定和中和的分光光度微孔板检测法。

Spectrophotometric microplate assay for titration and neutralization of avian nephritis virus based on the virus cytopathicity.

机构信息

Nippon Institute for Biological Science, 9-2221-1 Shin-machi, Ome, Tokyo, 198-0024, Japan.

Nippon Institute for Biological Science, 9-2221-1 Shin-machi, Ome, Tokyo, 198-0024, Japan.

出版信息

J Virol Methods. 2022 Jan;299:114303. doi: 10.1016/j.jviromet.2021.114303. Epub 2021 Oct 2.

Abstract

INTRODUCTION

Plaque assay (PA) is a gold standard for virus titration and neutralization of various cytopathic viruses, including avian nephritis virus (ANV), the etiological agent associated with kidney disorders in chickens. In this study, as an alternative to the labor-intensive PA, we developed a spectrophotometric microplate assay (MA) for ANV titration and neutralization based on the virus cytopathicity to primary chicken kidney (CK) cells.

METHODS

CK cells were infected with ANV in the presence or absence of chicken serum in a 96-well microplate, and the virus-induced cytolysis was quantified by measurement of neutral red uptake using a spectrophotometer. The absorbance values obtained were subjected to a sigmoidal four-parameter logistic regression analysis for the virus titer determination and serum neutralization assessment. Accuracy and reliability of the serum neutralization MA in comparison to the standard PA was statistically evaluated.

RESULTS

The ANV-MA was capable of quantifying infectious virus titers based on a virus dose-dependent cytolysis of CK cells, and serum neutralization could be assessed as an inhibition of the virus-induced cytolysis accordingly. Statistical evaluation using a 2 × 2 contingency table and receiver-operating characteristic analyses showed 82 % sensitivity, 99 % specificity and 0.97 area under the curve, supporting an overall diagnostic accuracy of the neutralization MA.

CONCLUSION

The newly developed MA using simplified experimental procedures in the microplate format and direct spectophotometric data readout is readily applicable to general laboratories for high-throughput screening of serum neutralization of ANV.

摘要

简介

噬斑测定(PA)是测定各种细胞病变病毒滴度和中和作用的金标准,包括致鸡肾炎病毒(ANV),该病毒是引起鸡肾脏疾病的病原体。在这项研究中,作为替代劳动密集型 PA 的方法,我们开发了一种基于原发性鸡肾(CK)细胞对病毒细胞病变作用的分光光度微孔板测定(MA)来测定 ANV 的滴度和中和作用。

方法

在存在或不存在鸡血清的情况下,将 CK 细胞用 ANV 感染于 96 孔微量滴定板中,通过分光光度计测量中性红摄取量来定量病毒诱导的细胞溶解。将获得的吸光度值进行四参数逻辑斯蒂回归分析,以确定病毒滴度和血清中和评估。通过统计学评估,比较标准 PA,评估血清中和 MA 的准确性和可靠性。

结果

ANV-MA 能够根据 CK 细胞的病毒剂量依赖性细胞溶解来定量传染性病毒滴度,并且可以根据病毒诱导的细胞溶解的抑制来评估血清中和作用。使用 2×2 列联表和接收者操作特征分析的统计评估显示,敏感性为 82%,特异性为 99%,曲线下面积为 0.97,支持中和 MA 的总体诊断准确性。

结论

新开发的 MA 使用简化的实验程序和直接分光光度数据读取,可在微孔板格式中易于应用于普通实验室,用于高通量筛选 ANV 的血清中和作用。

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