BNP和IL-6双标记时间分辨免疫荧光检测方法的建立及初步应用

Establishment and preliminary application of BNP and IL-6 double-labeled time-resolved immunofluorescence detection method

目的研制一种脑钠肽(BNP)、白细胞介素-6(IL-6)的双标记时间分辨免疫荧光分析方法(TRFIA)并对其性能进行初步评价。方法将抗BNP和IL-6单克隆抗体(MAb)作为包被抗体包被在96孔板,用Eu3+和Sm3+分别标记另一配对抗体作为检测抗体,建立双抗体夹心TRFIA分析法并制备成试剂盒。进行灵敏度、准确度、重复性、特异性、稳定性和临床样本比对等实验评价其检测性能。结果制备的双标记时间分辨免疫荧光试剂盒对BNP检测的线性范围为10 pg/mL~5000 pg/mL,灵敏度为8.65 pg/mL,对IL-6检测的线性范围为1 pg/mL~1000 pg/mL,灵敏度为5.36 pg/mL。BNP的回收率在91.49%~99.16%,批内CV在5.73%~8.96%,批间CV在8.42%~11.73%;IL-6的回收率在93.95%~111.50%,批内CV在7.32%~10.07%,批间CV在9.40%~11.30%。与血清中常见的脓毒血症检测指标均无明显的交叉反应。试剂盒能够在4℃稳定保存半年,37℃稳定保存7 d。对临床样本的检测发现,该方法参考区间Cut-off值BNP:46.01 pg/mL,IL-6:13.62 pg/mL;检测结果与临床诊断结果一致,准确性较好。结论建立的BNP和IL-6双标记TRFIA方法具有高灵敏度、高特异性、高准确率、方便快捷等优点,可作为预警脓毒症发生、预测脓毒症病情程度及临床预后的一种新的实验室检测方法。

Objective To develop a dual-labeled time-resolved fluoroimmunoassay(TRFIA)for the detection of brain natriuretic peptide(BNP)and interleukin-6(IL-6)levels.Methods Anti-BNP and anti-IL-6 monoclonal antibodies(MAb)were used as coating antibodies to coat 96-well plates,and Eu3+and Sm3+were used to label the other paired antibodies as detection antibodies to establish a double antibody sandwich TRFIA method.Sensitivity,accuracy,repeatability,specificity,stability and clinical sample comparison experiments to evaluate its detection performance.Results The linear range of BNP was 10 pg/mL~5000 pg/mL,sensitivity was 8.65 pg/mL,and the linear range of IL-6 was 1 pg/mL~1000 pg/mL,sensitivity was 5.36 pg/mL.The recovery rate of BNP was 91.49%~99.16%,the intra-assay CV was 5.73%~8.96%,the inter-assay CV was 8.42%~11.73%;the recovery rate of IL-6 was 93.95%~111.50%,and the intra-assay CV was 7.32%~10.07%,the inter-assay CV was 9.40%~11.30%.There is no obvious cross-reaction with common serum interferents.The kit can be stored stably at 4℃ for half a year and at 37℃ for 7 days.The clinical samples detection found that the reference interval(Cut-off value)of BNP was 46.01 pg/mL,and IL-6 was13.62 pg/mL;the results were consistent with the clinical diagnosis results,indicating the accuracy of this TRFIA was good.Conclusion The established BNP and IL-6 dual-labeled TRFIA method has the high sensitivity,high specificity,high accuracy and convenience,which can be used as a new laboratory test for early warning of sepsis,predicting the severity of sepsis and clinical prognosis.