Multiplexing Cancer Sample Preparation: Indirect Immunomagnetic Enrichment

描述（由申请人提供）：临床活性生物标志物对于靶向抗癌治疗开发至关重要，并且对于个体患者的护理越来越重要。在由表皮生长因子受体（EGFR）控制的原型酪氨酸激酶途径中，该途径的功能活性通过EGFR的磷酸化状态和下游信号传导中间体如磷酸化-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克隆抗体）可能导致这些下游中间体缺乏磷酸化。传统上，磷酸化蛋白质已通过对来自多达106个细胞的肿瘤蛋白质提取物进行蛋白质印迹来分析。提出了一种方法，可以利用小样本，以实现类似水平的检测EGFR途径中的磷酸蛋白。将从给予EGFR靶向治疗之前和之后获得的外周血中分离肿瘤细胞，并分析关键EGFR途径中间体的磷酸化状态。 所提出的样品制备方法基于两个分子识别事件：在携带受体以及“代码”的非磁性珠上捕获感兴趣的分析物（在这种情况下，肿瘤细胞或其裂解后的组分）;然后通过携带“反代码”的磁性珠结合这些珠。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与不同珠类型同时孵育来同时捕获多种分析物。然后通过连续暴露于各种类型的“解码”珠子来对它们进行分类。 在处理样品以同时捕获多个感兴趣的分析物后，样品将被加载到电润湿（EW）生物芯片上。样品将被细分为类似大小的液滴，并流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选过程成为可能。例如，不同等份的珠悬浮液可以与不同序列的磁珠批次反应，以避免由于非特异性结合引起的偏差。最终的优势是样品浓缩至少103倍，并去除背景材料。样品不需要细分，这增加了多重测定的灵敏度和速度，同时允许微创样品收集。此外，最终分析-免疫测定或PCR或RTPCR-可以在同一芯片上进行，利用这些液相测定的最终灵敏度和动态范围。Advanced Liquid Logic，Inc.将与杜克大学综合癌症中心的合作者合作执行这个项目。

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.