两种HSV-1及猴B病毒相关抗体测定方法的比较

目的以人单纯疱疹病毒（HSV-1）做为抗原,利用空斑法和IFA法比较猴BV和人HSV-1阳性血清两种不同血清的中和能力的差异,建立一种实用、准确、可靠的病毒毒力的检测方法。方法首先,将HSV-1病毒悬液作连续的10倍稀释,取1 mL接种于已经长成单层的Vero-E6细胞上,用1%甲基纤维素覆盖,待其出现蚀斑后计数,算出病毒悬液中每毫升所含蚀斑单位,即滴定出HSV-1的TC ID50。同时,用免疫荧光方法（IFA）对猴和人疱疹阳性血清进行滴定,得到其血清的效价。其次,用滴定出的病毒液分别与两种阳性血清体外中和后,接种到单层的Vero-E6细胞上,用1%甲基纤维素覆盖,待其出现蚀斑后计数。最后,计算出其蚀斑减少率。结果用1%甲基纤维素作覆盖层的蚀斑数量平均为10-5PFU,能形成115-116个/mL蚀斑,形状呈黍米大小的规则圆形,其蚀斑边缘清晰。IFA滴定的人HSV-1阳性血清与猴BV阳性血清的中和抗体均为1∶80。人HSV-1和猴BV两种阳性血清的空斑减少率均为100%。结论确定了利用1%甲基纤维素做为覆盖层可得到清晰可靠的蚀斑,由此方法检测到用人HSV-1可以代替猴B病毒,筛查猴B病毒抗体。且为将来进行药物筛选和中和实验中利用病毒空斑法建立方便、可靠的方法。

Comparison of Two Method about Antibody against Herpesvirus Saimiri from Rhesus Macaques

Objective To compare the serological detection sensitivity of sera against HSV-1 and BV from humans and rhesus monkeys with PR（ plaque reduction） and IFA （ immunofluorescence assay）. Methods The PR assay described in this study was modified from the traditional viral plaque reduction assay by using with 1 mL of DMEM medium containing 1% （weight/volume） of methylcellulose （4000 cP; Sigma, MC） ,and an improved crystal staining method to achieve better plague formation in HSV-1 infected Veto E6 cells. The neutralizing antibody titer was determined as the highest serum dilution which achieved 50% plaque reduction. Results In this assay,HSV-1 infected Vero E6 cells formed clear plaques with the density of （ 115 - 116） dots/mL. The positive serum neutralizing antibodies titers and ratios were 1：80 and 100% both of HSV-1 and BV. Conclusions The PR assay described in this study will be used not only for serological detection but also for medical selection in cell model.