TNF-α/hs-CRP双标记时间分辨荧光免疫法用于脓毒症早期筛查诊断的研究

doi:10.11958/20220176

天津医药 ›› 2022, Vol. 50 ›› Issue (8): 868-872.doi: 10.11958/20220176

TNF-α/hs-CRP双标记时间分辨荧光免疫法用于脓毒症早期筛查诊断的研究

李云鹏¹(), 郝培远², 曹雪明², 闫兆月¹, 王恩锋¹, 黄书满¹, 代荣钦¹^,^Δ()

1河南省人民医院外科重症监护病房（邮编450003）
2华中阜外医院心内科

收稿日期:2022-01-30 修回日期:2022-03-18 出版日期:2022-08-15 发布日期:2022-08-12
通讯作者: 代荣钦 E-mail:498648994@qq.com;drqin@163.com
作者简介:李云鹏（1980）,男,主治医师,主要从事重症医学方面研究。E-mail： 498648994@qq.com
基金资助:
国家自然科学基金资助项目(U1704167);国家卫计委河南省联合共建项目(SB201902033);河南省科技厅科技攻关项目(182102310525)

The study on the TNF-α/hs-CRP double-labeled time-resolved fluorescence immunoassay for early screening and diagnosis of sepsis

LI Yunpeng¹(), HAO Peiyuan², CAO Xueming², YAN Zhaoyue¹, WANG Enfeng¹, HUANG Shuman¹, DAI Rongqin¹^,^Δ()

1 Central ICU (Surgery Intensive Care Unit), Henan Provincial People's Hospital, Zhengzhou 450003, China
2 Department of Cardiology, Central China Fuwai Hospital

Received:2022-01-30 Revised:2022-03-18 Published:2022-08-15 Online:2022-08-12
Contact: DAI Rongqin E-mail:498648994@qq.com;drqin@163.com

李云鹏, 郝培远, 曹雪明, 闫兆月, 王恩锋, 黄书满, 代荣钦. TNF-α/hs-CRP双标记时间分辨荧光免疫法用于脓毒症早期筛查诊断的研究[J]. 天津医药, 2022, 50(8): 868-872.

LI Yunpeng, HAO Peiyuan, CAO Xueming, YAN Zhaoyue, WANG Enfeng, HUANG Shuman, DAI Rongqin. The study on the TNF-α/hs-CRP double-labeled time-resolved fluorescence immunoassay for early screening and diagnosis of sepsis[J]. Tianjin Medical Journal, 2022, 50(8): 868-872.

摘要/Abstract

摘要：

目的建立一种双标记时间分辨荧光免疫法（TRFIA）用于定量检测血清中肿瘤坏死因子-α（TNF-α）和超敏C-反应蛋白（hs-CRP）水平。方法将抗TNF-α和hs-CRP单克隆抗体包被在96孔板,制备铕（Eu³⁺）和钐（Sm³⁺）检测抗体偶联物,建立双抗体夹心TRFIA法并组装成试剂盒,评价此试剂盒的灵敏度、线性范围、加标回收率和各项检测性能。结果建立了TRFIA检测血清TNF-α和hs-CRP水平的新方法并组装成试剂盒,此试剂盒对TNF-α检测的线性范围为0~100 ng/L,灵敏度为0.05 ng/L,对hs-CRP检测的线性范围为0~100 mg/L,灵敏度为0.02 mg/L;对TNF-α检测的加标回收率92.00%~107.00%,对hs-CRP检测的加标回收率95.00%~106.82%,与其他常见的血清干扰物质无明显的交叉反应;对TNF-α检测的批内CV 4.57%~9.24%,批间CV 5.13%~9.27%;对hs-CRP检测的批内CV 3.57%~7.69%,批间CV 6.07%~10.00%;试剂盒能够在4 ℃稳定保存6个月,37 ℃下可稳定保存7 d以上。TNF-α的检测阈值为0.44 ng/L,hs-CRP的检测阈值为1.41 mg/L。该试剂盒检测判定结果与临床情况相一致,符合率100%。结论双标记TRFIA法可定量检测TNF-α和hs-CRP水平,具有灵敏度和准确度高、特异性强、方便快捷等优点,可为脓毒症临床样品的早期筛查、疗效评价和预后评估提供一种新的检测方法。

关键词: 脓毒症, 肿瘤坏死因子-α, 超敏C-反应蛋白, 双标记时间分辨荧光免疫法

Abstract:

Objective To investigate the value of double-labeled time-resolved fluorescence immunoassay (TRFIA) for quantitatively detecting serum levels of tumor necrosis factor-α (TNF-α) and high-sensitivity C-reactive protein (hs-CRP). Methods Anti-TNF-α and hs-CRP monoclonal antibodies were coated on the 96-well plate, meanwhile prepared the europium (Eu³⁺) and samarium (Sm³⁺)-detection antibody conjugates, and then established the double-antibody sandwich TRFIA method and assembly into a kit, and finally evaluated the detection performance of this kit, such as sensitivity, linear range, spike recovery rate. Results A new method for detecting serum TNF-α and hs-CRP levels by TRFIA was successfully established and assembled into a kit. The linear range of the prepared TRFIA kit for TNF-α was 0-100 ng/L, sensitivity was 0.05 ng/L, the linear range for hs-CRP was 0-100 mg/L, sensitivity was 0.02 mg/L. The spiked recovery rate of TNF-α was between 92.00% and 107.00%, and that of hs-CRP was between 95.00% and 106.82%. There was no obvious cross-reaction with the symptom detection indexes. The TNF-α CV of intra-assay was between 4.57% and 9.24%. The inter-assay was between 5.13% and 9.27%. The hs-CRP CV of intra-assay was between 5.12% and 7.69%, and the inter-assay was 6.07%-10.00%. Additionally, the kit can be stored stably at 4 ℃ for half a year and at 37 ℃ for 7 days. The detection threshold of TNF-α was 0.44 ng/L, and the detection threshold of hs-CRP was 1.41 mg/L. The test results of the kit were consistent with the clinical situation, and the coincidence rate reached 100%. Conclusion The double-labeled TRFIA method can quantitatively detect TNF-α and hs-CRP levels, which has the advantages of high sensitivity, high specificity, convenience and fast. This TRFIA kit provides a new detection method for the early screening, efficacy evaluation and prognostic assessment of clinical samples of sepsis.

Key words: sepsis, tumor necrosis factor-alpha, high-sensitivity C-reactive protein, time-resolved fluorescence immunoassay

中图分类号:

R446

图/表 4

图1 TRFIA试剂盒检测TNF-α和hs-CRP水平的标准曲线

Fig.1 Standard curves of TRFIA kit for detecting TNF-α and hs-CRP levels

表1 TRFIA试剂盒准确度和精密度测定结果（n=10）

Tab.1 The accuracy and precision results of TRFIA kit

表2 TRFIA试剂盒特异性试验结果

Tab.2 The specific test results of TRFIA kit

表3 TRFIA试剂盒稳定性实验结果

Tab.3 The stability results of TRFIA kit

参考文献 16

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TNF-α/hs-CRP双标记时间分辨荧光免疫法用于脓毒症早期筛查诊断的研究

The study on the TNF-α/hs-CRP double-labeled time-resolved fluorescence immunoassay for early screening and diagnosis of sepsis

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