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目的建立一种人巨细胞病毒(Human cytomegalovirus,HCMV)中和抗体效价检测方法。方法系列稀释的待检样品与等体积HCMV混合,中和反应1 h后,接种MRC-5细胞,37℃孵育过夜,依次加入HCMV anti-IE1单抗、Biotin-SP-Goat antiMouse IgG、Peroxidase-Labeled Streptavidin及底物,对HCMV感染细胞的细胞核进行特异性染色和定位,采集显微图像,计数每孔中感染细胞数。采用Reed-Muench计算样品的中和效价。并对建立的方法进行精密性、耐用性和专属性验证。结果感染HCMV的MRC-5细胞,出现典型的CPE,且其细胞核出现特异性着色颗粒,而未感染的细胞则未出现着色颗粒。该方法重复性检测的变异系数为3.26%,中间精密性检测的变异系数为1.05%;样品与等体积中和病毒液于37℃中和反应55、60和65 min,中和抗体效价差异无统计学意义(P>0.05);水痘病毒感染的MRC-5细胞未出现特异性着色颗粒,10份不同类型的样品与MRC-5细胞共同孵育后,也未出现特异性着色颗粒。结论已建立了一种HCMV中和抗体效价检测方法,该方法快速、客观,精密性高,耐用性好,专属性强,可用于血浆和IVIG中HCMV中和抗体效价的检测。
Objective To develop a method for detecting neutralizing antibody titer of human cytomegalovirus (HCMV). Methods Serial diluted samples were mixed with equal volume of HCMV. After 1 h neutralization reaction, MRC-5 cells were inoculated and incubated overnight at 37 ℃. HCMV anti-IE1 monoclonal antibody, Biotin-SP-Goat antiMouse IgG and Peroxidase- Labeled Streptavidin and substrate were used to specifically stain and locate the nuclei of HCMV-infected cells. Micro-images were collected and the number of infected cells in each well was counted. Neutralization titers of samples were calculated using Reed-Muench. The method is validated for its precision, durability and specificity. Results MRC-5 cells infected with HCMV showed typical CPE with specific colored particles in their nuclei, while no particles appeared in uninfected cells. The coefficient of variation of the method was 3.26% for repeatability detection and 1.05% for the intermediate precision detection. The neutralization antibody titers of the samples were neutralized with the equal volume of neutralizing virus solution at 37 ℃ for 55, 60 and 65 min. There was no statistical significance (P> 0.05). No specific colored particles were found in MRC-5 cells infected with varicella virus. No specific colored particles were observed after incubated with MRC-5 cells in 10 different types of samples. Conclusions A detection method of HCMV neutralizing antibody titer has been established. The method is rapid, objective, accurate, robust and specific. It can be used to detect the titer of HCMV neutralizing antibody in plasma and IVIG.