Sensitive and rapid quantification of C-reactive protein using quantum dot-labeled microplate immunoassay.
Sensitive and rapid quantification of C-reactive protein using quantum dot-labeled microplate immunoassay.
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使用量子点标记微孔板免疫测定法灵敏快速地定量 C 反应蛋白
DOI：
10.1186/1479-5876-10-24
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发表时间：
2012-02-06
影响因子：
7.4
通讯作者：
Fu W
中科院分区：
文献类型：
--
作者：
Luo Y;Zhang B;Chen M;Jiang T;Zhou D;Huang J;Fu W
超敏C反应蛋白（hs-CRP）检测对预测冠心病的危险性具有重要的临床意义。现有的超敏C反应蛋白检测要么需要复杂的操作或具有低吞吐量，不能常规实施在农村地区，由于有限的实验室resources.MethodsWe开发了一种新的超敏C反应蛋白检测能够同时定量超过90个临床样本，通过使用量子点标记的免疫测定在一个标准的96孔微孔板。通过采用两种靶向不同hs-CRP表位的单克隆抗体（mAb）分别作为包被抗体和检测抗体，增强了测定的特异性。在hs-CRP抗原的存在下，用酶标仪检测hs-CRP抗体夹心复合物的荧光强度，结果hs-CRP检测范围为0.001 ~ 100 mg/L，检出限为0.06（0.19）μg/L，检测时间为1.5 h。已确认拟定测定方法的准确度为低变异系数（CV），2.27%（测定内）和8.52%（测定间），回收率为96.7- 104.2%。104例临床样本的Bland-Altman图表现出良好的一致性之间的拟议的检测，商业高灵敏度ELISA，比浊法，表明新开发的hs-CRP检测作为替代现有的hs-CRP assays.ConclusionThe开发的检测方法的前景，满足快速，灵敏和高通量测定hs-CRP水平在短时间内使用最少的资源的需求。此外，所开发的测定还可用于通过固定特异性单克隆抗体来检测和定量其他诊断生物标志物。
BackgroundHigh-sensitivity C-reactive protein (hs-CRP) assay is of great clinical importance in predicting risks associated with coronary heart disease. Existing hs-CRP assays either require complex operation or have low throughput and cannot be routinely implemented in rural settings due to limited laboratory resources.MethodsWe developed a novel hs-CRP assay capable of simultaneously quantifying over 90 clinical samples by using quantum dots-labeled immunoassay within a standard 96-well microplate. The specificity of the assay was enhanced by adopting two monoclonal antibodies (mAbs) that target distinct hs-CRP epitopes, serving as the coating antibody and the detection antibody, respectively. In the presence of hs-CRP antigen, the fluorescence intensity of the mAb-Ag-mAb sandwich complex captured on the microplate can be read out using a microplate reader.ResultsThe proposed hs-CRP assay provides a wide analytical range of 0.001-100 mg/L with a detection limit of 0.06 (0.19) μg/L within 1.5 h. The accuracy of the proposed assay has been confirmed for low coefficient of variations (CVs), 2.27% (intra-assay) and 8.52% (inter-assay), together with recoveries of 96.7-104.2%. Bland-Altman plots of 104 clinical samples exhibited good consistency among the proposed assay, commercial high-sensitivity ELISA, and nephelometry, indicating the prospects of the newly developed hs-CRP assay as an alternative to existing hs-CRP assays.ConclusionThe developed assay meets the needs of the rapid, sensitive and high-throughput determination of hs-CRP levels within a short time using minimal resources. In addition, the developed assay can also be used to detect and quantify other diagnostic biomarkers by immobilizing specific monoclonal antibodies.
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影响因子：
10.2
作者：
McBride JD;Cooper MA
通讯作者：
Cooper MA
影响因子：
4.4
作者：
Williams, D. Keith, Jr.;Muddiman, David C.
通讯作者：
Muddiman, David C.
影响因子：
17.1
作者：
Di Corato, Riccardo;Bigall, Nadja C.;Pellegrino, Teresa
通讯作者：
Pellegrino, Teresa
影响因子：
7.4
作者：
Dixit, Chandra Kumar;Vashist, Sandeep Kumar;O'Kennedy, Richard
通讯作者：
O'Kennedy, Richard
影响因子：
37.8
作者：
Sabatine, MS;Morrow, DA;Braunwald, E
通讯作者：
Braunwald, E