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

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。

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

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.