HCMV pp65 antigenemia assay using indirect alkaline phosphatase staining method
HCMV pp65 antigenemia assay using indirect alkaline phosphatase staining method
复制标题
使用间接碱性磷酸酶染色法测定 HCMV pp65 抗原血症
DOI：
10.2220/biomedres.16.125
复制
发表时间：
1995
影响因子：
1.2
通讯作者：
A. Ito
中科院分区：
文献类型：
--
作者：
T. Kurihara;J. Hayashi;Tohru Matsuoka;A. Ito
本研究旨在探讨间接碱性磷酸酶染色法检测外周血多形核白细胞中人巨细胞病毒（HCMV）抗原血症的几种影响因素。用抗人巨细胞病毒（HCMV）低基质蛋白（pp 65）的单克隆抗体混合物检测离心血白细胞中的HCMV抗原。影响灵敏度的主要因素是孵育温度、洗涤液、单克隆抗体的稀释缓冲液和二抗。特别是，利用碱性磷酸酶标记的山羊抗小鼠抗体作为第二抗体，在背景染色方面得到了显著更好的结果。在优化的条件下使用该测定，在150，000个抗原阴性的多形核白细胞中可检测到单个阳性细胞。对36例骨髓移植受者的83份血标本进行了检测，其中50份血标本的间接碱性磷酸酶染色和常规碱性磷酸酶-抗碱性磷酸酶（APAAP）染色均为阴性，30份血标本均为阳性。间接碱性磷酸酶染色与APAAP法比较，敏感性为96.7%（30/31），特异性为96.1%（50/52）。间接染色和APAAP染色检测的阳性细胞数之间存在显著相关性（rS = 0.917，斯皮尔曼检验）。当每1.5 × 105个细胞中感染的多形核白细胞数大于3个时，两种染色方法均得到一致的阳性结果。该方法快速、灵敏，可用于HCMV抗原的无背景检测
The purpose of this study was to investigate several factors affecting the sensitivity of human cytomegalovirus (HCMV) antigenemia assay using indirect alkaline phosphatase staining method for HCMV antigen in peripheral blood polymorphonuclear leukocytes. A mixture of two monoclonal antibodies directed against the HCMV lower matrix protein (pp65) was used for detecting HCMV antigen in cytocentrifuged blood leukocytes. Major factors affecting the sensitivity were incubation temperature, washing solution, diluent buffer for monoclonal antibodies, and second antibody. In particular, utilization of alkaline phosphatase-labeled goat anti-mouse antibody as second antibody gave significantly better results in terms of background staining. Using the assay under the optimized condition, one single positive cell among 150,000 antigen-negative polymorphonuclear leukocytes was detectable. Of 83 blood samples from 36 bone marrow transplant recipients examined, 50 samples were concordantly negative and 30 concordantly positive for indirect alkaline phosphatase staining and conven-tional alkaline phosphatase-anti-alkaline phosphatase (APAAP) staining. The sensitivity and specificity of indirect alkaline phosphatase staining against APAAP method were 96.7% (30/31) and 96.1% (50/52), respectively. A significant correlation was found between the number of positive cells detected by indirect staining and APAAP staining (rS = 0.917, Spearman test). When the number of infected polymorphonuclear leukocytes per 1.5 >< 105 cells was more than 3, both staining methods gave concordant positive results. This rapid and sensitive method will be valuable for detection of HCMV antigen without any background