A quantitative immunoassay for lung cancer biomarker CIZ1b in patient plasma.
A quantitative immunoassay for lung cancer biomarker CIZ1b in patient plasma.
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DOI：
10.1016/j.clinbiochem.2016.11.015
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发表时间：
2017-04
影响因子：
2.8
通讯作者：
White J
中科院分区：
文献类型：
--
作者：
Coverley D;Higgins G;West D;Jackson OT;Dowle A;Haslam A;Ainscough E;Chalkley R;White J
早期检测肺癌的非侵入性检查是一个重要的未满足的临床需求。CIZ1b血浆生物标志物可以区分高风险人群中的1期肺癌，具有临床有用的准确性，两个独立回顾性队列的ROC auc超过0.9，因此可以满足这一需求。我们的目的是表征生物标志物的天然状态并开发定量免疫测定。采用选择性变性、制备电泳和质谱法对生物标志物和相互作用伙伴进行表征。构建夹心ELISA，检测肺癌患者血浆中CIZ1b生物标志物的特异性。CIZ1b生物标志物是一种抗变性复合物，位于CIZ1的c端片段和纤维蛋白原α链之间，该片段携带由外显子14选择性剪接指定的CIZ1b表位。用纯化的纤维蛋白原和CIZ1b重建生物标志物表位，而不是CIZ1a（非选择性剪接外显子14），证实了结果的特异性。内源性复合物在肺癌血浆中高度稳定，可以通过CIZ1b外显子连接特异性抗体与纤维蛋白原检测配对来定量。将这种夹心酶联免疫吸附试验应用于前瞻性收集的血浆发育集，其准确性与用于验证生物标志物区分能力的western blot方法相同。天然血浆中CIZ1b分子结构异常，导致免疫分析设计复杂，这一有前景的生物标志物翻译延迟。然而，现在可以使用高通量、医院友好的夹心ELISA格式来测量CIZ1b，克服了进一步临床开发和应用这种早期肺癌血液检测的重要障碍。CIZ1b是一种有前景的早期检测肺癌的血液生物标志物。CIZ1b生物标志物是一个跨越癌症特异性外显子边界的CIZ1片段。癌症患者血浆中CIZ1b片段与纤维蛋白原α链络合。一种夹心免疫分析法可以测量癌症患者血浆中的复合物水平。稳定性和特异性使其适用于肺癌的常规检测。
Non-invasive tests for early detection of lung cancer are an important unmet clinical need. CIZ1b plasma biomarker can discriminate stage 1 lung cancer from within high-risk groups with clinically useful accuracy, with ROC AUCs in excess of 0.9 for two independent retrospective cohorts, and could therefore meet this need. Our aim was to characterise the native state of the biomarker and develop a quantitative immunoassay. Selective denaturation, preparative electrophoresis and mass spectrometry of human plasma were used to characterise the biomarker and interaction partners. A sandwich ELISA was generated, and specificity for CIZ1b biomarker tested on lung cancer patient plasma. CIZ1b biomarker is a denaturation-resistant complex between a C-terminal fragment of CIZ1 bearing the CIZ1b epitope specified by alternative splicing of exon14, and fibrinogen alpha chain. Reconstitution of the biomarker epitope with purified fibrinogen and CIZ1b, but not CIZ1a (non-alternatively spliced exon 14) confirmed the specificity of the results. The endogenous complex is highly stable in lung cancer plasma and can be quantified by pairing of a CIZ1b exon-junction specific antibody with detection of fibrinogen. Application of this sandwich ELISA to a prospectively collected development set of plasmas reveals the same level of accuracy as the western blot used to validate the discriminatory capability of the biomarker. Unexpected and unusual molecular structure of CIZ1b in native plasma has complicated immunoassay design, and delayed translation of this promising biomarker. However, CIZ1b can now be measured using a high-throughput, hospital-friendly sandwich ELISA format, overcoming an important barrier to further clinical development and application of this blood test for early stage lung cancer. CIZ1b is a promising blood biomarker for early detection of lung cancer. CIZ1b biomarker is a CIZ1 fragment that spans a cancer-specific exon boundary. CIZ1b fragment is complexed with fibrinogen alpha chain in cancer patient plasma. A sandwich immunoassay for complex can measure levels in cancer patient plasma. Stability and specificity make it suitable for routine detection of lung cancer.