一种人巨细胞病毒中和抗体效价快速检测方法的建立

Development of A Method for Rapid Detection of Neutralizing Antibody Titer against Human Cytomegalovirus

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 detection of neutralizing antibody titer against human cytomegalovirus（HCMV）.Methods Serially diluted test samples were mixed with HCMV at an equal volume respectively for neutralization for 1 h,inoculated to MRC-5 cells and incubated at 37℃ overnight,then added with anti-HCMV IE1 McAb,Biotin-SP-Goat anti Mouse IgG,Peroxidase-Labeled Streptavidin and substrate in turn.The nuclei of cells infected with HCMV were stained specifically and observed for location.The infected cells in each well were counted by microscopy.The neutralizing titers of test samples were calculated by Reed-Muench method.The developed method was verified for precision,serviceability and specificity.Results MRC-5 cells infected with HCMV showed typical CPEs,of which the nuclei were stained specifically,while no stained particles were observed in uninfected cells.The variation coefficient of detection result by the developed method in reproducibility test was 3.26%,while that in intermediate precision test was 1.05%.After the test samples were neutralized with an equal volume of neutralizing virus liquid at 37℃ for 55,60 and 65 min,the neutralizing antibody titers showed no significant difference（P 〉 0.05）.No specifically stained particles were observed in the MRC-5 cells infected with varicella virus,or in the plasma,IVIG or CMV-IVIG samples after co-incubation with MRC-5 cells.Conclusion A method for detection of neutralizing antibody titer against HCMV was developed,which was rapid,objective,and of high precision,serviceability and specificity,and might be used for the detection of neutralizing antibody titer against HCMV in plasma and IVIG.