人巨细胞病毒pp65抗原血症检测方法的建立和初步临床应用

Development and optimization of a laboratory assay for the human cytomegalovirus pp65 antigenemia and its preliminary clinical application

目的 建立人巨细胞病毒抗原 pp6 5 (HCMVpp6 5 )血症特异、灵敏和低成本的酶免组化和荧光免疫组化方法 ,以利于临床推广应用。方法 以HCMV AD16 9感染的人胚肺成纤维细胞 (HELF)爬片作阳性对照 ,选择试剂和材料组建试剂盒 ,并与商品化的同类试剂盒作比较 ,建立人外周血多形核白细胞 (PMNLs)中HCMVpp6 5抗原阳性细胞的检测方法 ,然后再用荧光定量PCR(FQ PCR)方法做相应考评。 结果 我们建立的HCMVpp6 5抗原血症检测法能成功地检出PMNLs中的 pp6 5阳性细胞。其可检测限分别为 10 2 pfu/ml的AD16 9攻击 2 4h后的HELF细胞爬片和 3～ 5个 pp6 5阳性细胞 / 2× 10 5PMNLs ;用高相对分子质量 (5 0 0 0 0 0 )的葡聚糖能快速、有效地分离PMNLs ;用 4 %中性多聚甲醛固定的细胞片 ,于 - 2 0°C或以下可长期保存 (≥ 6个月 ) ,而且 pp6 5抗原性稳定。 结论 自建的试剂盒与同类进口试剂盒相比 ,检测 pp6 5的结果差异无显著性 ,并与FQ PCR的检测结果相一致 ,其检测特异、灵敏、操作简单、费用低廉 ,适宜临床实验室推广应用。

Objective To develop a sensitive, specific, and economic HCMVpp65 antigenemia assay for clinical application. Methods We took human embryonic lung fibroblast(HELF) infected with HCMV AD169 as positive control, and selected appropriate reagents to combine a new kit. Peripheral polymorphonuclear leukocytes (PMNLs) were isolated by sedimentation with a 6% dextran solution in 0.9% saline. The fixation was using 4% formalin. Slides were stained by an indirect immunoperoxidase staining with a commercially available mixture of monoclonal antibodies C 2/C 6. Then compared with a commercial indirect immunofluorescence assay kit. And then we carried a prospective, paralled, and blind study on 100 blood samples in order to compare our result with a fluorescent quantitative PCR assay. Results Our kit could successfully detect positive PMNLs for pp65. Its sensitivity corresponded with HELFs infected by 10 2 pfu/ml AD169 for 24 hours or 3 5 positive cells/2×10 5 PMNLs. Dextran with high molecular weight could rapidly and efficiently extract PMNLs from peripheral blood. Cytospins fixed with 4% formalin could be stored (-20 °C ) for a long time with stability of pp65 antigen. Conclusions The optimized kit in our laboratory had no statistical difference in the detection of pp65 antigenemia comparing with congeneric commercial kits. And its result coincided with that of FQ PCR. But our kit is simpler, less expensive, and more applicable for clinical applications.