人巨细胞病毒IgM抗体生物素-亲和素单克隆抗体捕获时间分辨荧光免疫法检测试剂盒的研制与评价

Preparation and evaluation of a detection kit of biotin-avidin monoclonal antibody capture time-resolved fluoroimmunoassay for IgM antibody to human cytomegalovirus

目的建立人巨细胞病毒IgM(human cytomegalovirus IgM,HCMV IgM)抗体生物素-亲和素单克隆抗体捕获时间分辨荧光免疫法(biotin-avidin monoclonal antibody capture time-resolved fluoroimmunoassay,BA Mac TrFIA)检测试剂盒,并对其性能进行评价。方法采用鼠抗IgMμ链单克隆抗体(monoclonal antibody,McAb)作为固相反应板的包被抗体,生物素(biotin)标记的HCMV基因重组抗原作为桥接抗原,链酶亲和素(SA)标记铕(Eu3+)作为示踪物,配制以β-萘甲酰三氟丙酮为主要成分的增强液,应用BA Mac TrFIA法进行HCMV IgM抗体的检测,对连续制备的3批试剂盒的最低检测限、重复性、阴性参考品符合率、阳性参考品符合率、热稳定性进行评价,并与巨细胞病毒IgM抗体诊断试剂盒同时对1 020份血清或血浆样本进行临床研究比对试验,采用Kappa检验进行一致性分析。结果选择4μg/ml作为鼠抗IgMμ链McAb最佳包被浓度,1∶10 000稀释作为HCMV-Bio最佳工作浓度,1∶1 500稀释作为SA-Eu3+最佳工作浓度,试剂盒参考值建议为阴性对照品检测荧光值(negative control counter,NCx)×2.1;连续制备的3批试剂盒最低检测限、重复性、阴性参考品符合率、阳性参考品符合率均达到国家检定标准;于37℃恒温箱放置6 d后,检测性能无明显改变;临床研究比对试验Kappa指数为1,一致程度达100%。结论成功制备了HCMV IgM BA Mac TrFIA法检测试剂盒,该试剂盒灵敏度高,精密性较好,与同类产品检测结果相关性好,为临床HCMV lgM抗体的检测提供科学、可靠的诊断依据。

Objective To develop a detection kit of biotin-avidin monoclonal antibody capture time-resolved fluoroim- munoassay（BA Mac TrFIA） for IgM antibody to human cytomegalovirus（HCMV IgM） and evaluate its performance. Methods Mouse anti-human IgM μ chain monoclonal antibody（McAb）was coated onto microplates,while the recombinant HCMV antigen labeled with biotin was served as bridging antigen,and streptavidin（SA）-labeled Europium（Eu3＋）as a tracer. The mainly component of enhancement solution was β-naphthalene thrifluoroacetone. The developed BA Mac TrFIA kit was used for detection of HCMV IgM. Three consecutive batches of kits were prepared and evaluated for minimum detection limit, reproducibility,coincidence rates with positive and negative references and heat stability. A total of 1 020 serum or plasma samples were detected by the developed kit,and the results were compared with those by diagnostic kit for HCMV IgM,while the consistency was analyzed by Kappa test. Results The optimal coating concentration of mouse anti-human IgM μ chain McAb was 4 μg / ml,while the optimal working concentration of HCMV-Bio was 1 ∶ 10 000,and that of SA-Eu3＋was 1 ∶ 1 500. The reference value of prepared kit was recommend as negative control counter（NCx） × 2. 1. The minimum detection limit,reproducibility as well as coincidence rates of negative and positive references of three consecutive batches of kits met the national requirements,while the performance showed no significant change after storage at 37 ℃ for 6 d. The Kappa index of clinical comparative research was 1,indicating a consistency of 100%. Conclusion The BA Mac TrfIA kit for HCMV IgM was successfully prepared and showed high sensitivity and precision,of which the detection result was highly correlated to those by the kits of same kind. It provided a scientific and reliable diagnostic basis for detection of HCMV IgM in clinic.