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

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。

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-αand hs-CRP monoclonal antibodies were coated on the 96-well plate,meanwhile prepared the europium(Eu^(3+))and samarium(Sm^(3+))-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 TNFαand hs-CRP levels by TRFIA was successfully established and assembled into a kit.The linear range of the prepared TRFIA kit for TNF-αwas 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-αwas 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-αCV 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-αwas 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-αand 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.