Antibody quantum dot conjugates developed via copper-free click chemistry for rapid analysis of biological samples using a microfluidic microsphere array system.
Antibody quantum dot conjugates developed via copper-free click chemistry for rapid analysis of biological samples using a microfluidic microsphere array system.
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DOI：
10.1021/bc500139u
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发表时间：
2014-07-16
影响因子：
4.7
通讯作者：
Achilefu, Samuel
中科院分区：
文献类型：
--
作者：
Kotagiri, Nalinikanth;Li, Zhenyu;Xu, Xiaoxiao;Mondal, Suman;Nehorai, Arye;Achilefu, Samuel
基于抗体的蛋白质组学是一种使能技术，其对癌症生物标志物发现、诊断筛选、疾病状态的预后和药效学评估以及靶向治疗具有重要意义。基于量子点的荧光免疫缀合物具有实现这一目标的有前景的特征，例如高光稳定性、亮度和多光谱可调谐性。然而，目前产生这种缀合物的策略充满了复杂性，例如抗体的抗原结合位点的不适当取向、聚集和稳定性问题。我们报告了一种简便而有效的策略，使用无铜点击反应将抗表皮生长因子受体（EGFR）抗体偶联到量子点上，并将其与使用传统策略制备的类似构建体进行了比较，如琥珀酰亚胺基-4-（N-马来酰亚胺甲基）环己烷-1-羧酸酯（SMCC）和生物素-链霉亲和素方案。与通过传统方案产生的那些相比，缀合物的Fc和Fab区保留其结合潜力。我们进一步将缀合物应用于测试一种新的微球阵列装置，该装置被设计用于通过荧光免疫测定进行癌症生物标志物的灵敏检测。使用纯化的EGFR，我们确定了显微镜中心系统的检测限为12.5 ng/ml。通过使用肿瘤细胞裂解物以及来自乳腺癌患者的人血清，在计算机上成功地测试和验证了生物测定，并将结果与正常血清进行了比较。观察到与已建立的临床数据一致的模式，这进一步验证了所开发的缀合物的有效性及其在体外以及计算机荧光免疫测定中的成功实施。结果表明，基于相对于血液中正常参考水平的EGFR表达水平预测患者癌症类别的高通量计算机模拟范例的潜在发展。
Antibody-based proteomics is an enabling technology that has significant implications for cancer biomarker discovery, diagnostic screening, prognostic and pharmacodynamic evaluation of disease state, and targeted therapeutics. Quantum dot based fluoro-immunoconjugates possess promising features towards realization of this goal such as high photostability, brightness and multispectral tunability. However, current strategies to generate such conjugates are riddled with complications such as improper orientation of antigen binding sites of the antibody, aggregation and stability issues. We report a facile yet effective strategy to conjugate anti epidermal growth factor receptor (EGFR) antibody to quantum dots using copper-free click reaction, and compared them to similar constructs prepared using traditional strategies such as succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and biotin-streptavidin schemes. The Fc and Fab regions of the conjugates retain their binding potential, compared to those generated through the traditional schemes. We further applied the conjugates in testing a novel microsphere array device designed to carry out sensitive detection of cancer biomarkers through fluoroimmunoassays. Using purified EGFR, we determined the limit of detection of the microscopy centric system to be 12.5 ng/ml. The biological assay, in silico, was successfully tested and validated by using tumor cell lysates, as well as human serum from breast cancer patients and the results were compared to normal serum. A pattern consistent with established clinical data was observed, which further validates the effectiveness of the developed conjugates and its successful implementation both in vitro as well as in silico fluoroimmunoassays. The results suggest the potential development of a high throughput in silico paradigm for predicting the class of patient cancer based on EGFR expression levels relative to normal reference levels in blood.
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