Multiplexing Cancer Sample Preparation: Indirect Immunomagnetic Enrichment

描述(由申请人提供)：临床活动的生物标记物对于有针对性的抗癌疗法的开发至关重要，并且对个别患者的护理正变得重要。在由表皮生长因子受体(EGFR)调控的典型酪氨酸激酶通路中，该通路的功能活性由EGFR的磷酸化状态以及下游的信号中介如磷酸化ERK和磷酸化Akt来评估。阻断EGFR功能的干预措施(如酪氨酸激酶抑制剂或单抗)可能会导致这些下游中间体缺乏磷酸化。传统上，磷酸化蛋白是通过对多达106个细胞的肿瘤蛋白提取液进行Western blotts分析的。提出了一种可以利用小样本实现对EGFR途径中的磷蛋白进行类似水平的检测的方法。肿瘤细胞将从给予EGFR靶向治疗前后的外周血中分离出来，并将分析关键的EGFR途径中间产物的磷酸化状态。 拟议的样品制备方法基于两个分子识别事件：在携带受体和“密码”的非磁珠上捕获感兴趣的分析物(在这种情况下，是肿瘤细胞或其裂解后的成分)；然后用携带“反密码”的磁珠结合这些珠。密码和反密码可以简单地是两条互补的DNA链。与单步磁珠捕获不同，所提出的方法允许通过同时与不同类型的磁珠孵育来同时捕获多个分析物。然后，通过连续接触各种类型的“解码”珠子对它们进行分类。 在对样品进行处理以同时捕获多个感兴趣的分析物之后，样品将被加载到电湿(EW)生物芯片上。样本将被细分为类似大小的液滴，并经过含有解码磁珠的液滴。EW芯片快速处理多个液滴的能力使分拣过程成为可能。例如，不同等量的珠悬浮液可以以不同的顺序与批次的磁珠反应，以避免由于非特异性结合而产生的偏置。最终的优势是样品浓缩至少103倍，并去除背景物质。样品不需要细分，这提高了多重分析的灵敏度和速度，同时允许微创样品收集。此外，最终的分析--免疫分析，或称聚合酶链式反应或逆转录聚合酶链式反应--可以在同一芯片上进行，利用这些液相分析的极限灵敏度和动态范围。先进液体逻辑公司将与杜克大学综合癌症中心的合作者合作执行这一项目。

DESCRIPTION (provided by applicant): Biomarkers of clinical activity are critical for targeted anti-cancer therapy development and are becoming important for the care of individual patients. In a prototypical tyrosine kinase pathway, governed by the epidermal growth factor receptor (EGFR), functional activity of the pathway is assessed by the phosphorylation status of EGFR and downstream signaling intermediaries such as phospho-ERK and phospho-Akt. Interventions that block the function of EGFR (such as tyrosine kinase inhibitors or monoclonal antibodies) may lead to lack of phosphorylation of these downstream intermediaries. Traditionally, phosphorylated proteins have been analyzed by Western blots performed on tumor protein extracts from as many as 106 cells. A method is proposed that can utilize small sample to achieve a similar level of detection of phopshoproteins in the EGFR pathway. Tumor cells would be isolated from peripheral blood obtained before and after administration of an EGFR targeted therapy and the phosphorylation status of key EGFR pathway intermediates would be analyzed. The proposed approach to sample preparation is based on two molecular recognition events: capture of analytes of interest (in this case, tumor cells, or their components after lysis) on non-magnetic beads carrying receptors as well as "codes"; and then binding these beads by magnetic beads carrying "anticodes". The codes and anti-codes can simply be two complementary DNA strands. Unlike single-step magnetic-bead capture, the proposed method allows simultaneous capture of multiple analytes by incubation with different bead types at the same time. They are then sorted by consecutive exposure to various types of "decoding" beads. After processing the samples to simultaneously capture multiple analytes of interest, the sample will be loaded onto electrowetting (EW) biochip. The sample will be subdivided into droplets of similar size and run past droplets containing decoding magnetic beads. The ability of EW chip to rapidly process multiple droplets enables the sorting procedure. For example, different aliquots of bead suspension can be reacted with the batches of magnetic beads in different sequences, to avoid bias due to non-specific binding. The ultimate advantage is sample concentration by at least 103x and removal of background material. The sample need not be subdivided which increases the sensitivity and speed of multiplexed assays while allowing minimally-invasive sample collection. Moreover, the final analysis - immunoassay, or PCR or RTPCR, - can be performed on same chip, taking advantage of the ultimate sensitivity and dynamic range of these liquid-phase assays. Advanced Liquid Logic, Inc. will team with collaborators at Duke University's Comprehensive Cancer Center to execute this project.