Wang Shixia, Sakhatskyy Pavlo, Chou Te-Hui W, Lu Shan
Laboratory of Nucleic Acid Vaccines, Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Lazare Research Building, Worcester, MA 01605-2397, United States.
J Immunol Methods. 2005 Jun;301(1-2):21-30. doi: 10.1016/j.jim.2005.03.008. Epub 2005 Apr 25.
Accurate assessment of neutralizing antibody activities is important either for patients infected with Severe Acute Respiratory Syndrome (SARS) or for animals and volunteers immunized with the experimental vaccines against the SARS associated coronavirus (SCV). However, the current assay based on the cytopathic effect (CPE) which has been frequently cited in literature has several limitations. The CPE assay relies on the visual observation on the damage of SCV infected target cells under a microscope. It is subjected to observer variations and it is difficult to generate a quantitative determination of neutralizing activities based on the level of CPE. In the current study, we established the utility of two additional assays to measure the neutralizing activities against SCV: the plaque reduction (PR) and the neutral red staining (NRS) assays. The PR assay described in this study was modified from the traditional viral plaque reduction assay by using an improved crystal staining method to achieve better plague formation in SCV infected Vero E6 cells. The NRS neutralization assay was adopted from a similar system used for detecting neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1). In this assay, the protective effect of neutralizing antibodies was determined by the cell viability which is measured by the uptake of neutral red dye at A540. The neutralizing antibody titers can be easily determined with either of the two new assays. In this report, we described the utility of these two new neutralization assays in measuring the neutralizing activities against SCV infection from rabbit sera immunized with various forms of spike protein of SCV.
准确评估中和抗体活性对于感染严重急性呼吸综合征(SARS)的患者,以及用抗SARS相关冠状病毒(SCV)实验疫苗免疫的动物和志愿者而言都至关重要。然而,目前基于细胞病变效应(CPE)的检测方法(该方法在文献中经常被引用)存在若干局限性。CPE检测依赖于在显微镜下对SCV感染的靶细胞损伤进行肉眼观察。它受观察者差异的影响,并且难以基于CPE水平对中和活性进行定量测定。在本研究中,我们确立了另外两种检测方法用于测量针对SCV的中和活性的实用性:蚀斑减少(PR)法和中性红染色(NRS)法。本研究中描述的PR法是在传统病毒蚀斑减少检测方法的基础上进行改良,通过使用改进的结晶染色方法,以在SCV感染的Vero E6细胞中实现更好的蚀斑形成。NRS中和检测法是借鉴用于检测针对1型人类免疫缺陷病毒(HIV-1)中和抗体反应的类似系统。在该检测方法中,中和抗体的保护作用通过细胞活力来确定,细胞活力通过在A540处对中性红染料的摄取来测量。使用这两种新检测方法中的任何一种都可以轻松测定中和抗体滴度。在本报告中,我们描述了这两种新的中和检测方法在测量用各种形式的SCV刺突蛋白免疫的兔血清针对SCV感染的中和活性方面的实用性。