LOW-COST AFFINITY SYSTEM FOR PREPARATION OF PLASMA/SERUM FOR BIOMARKER ANALYSIS

描述（由申请人提供）：项目总结/摘要：我们将开发一种一次性亲和毛细管[AffiTip]，能够在2分钟内从<5 uL未稀释血浆中去除99%的干扰散装蛋白，用于生物标志物[将作用机制和临床有效性联系起来的特定化合物]分析。该产品最终将以任何当前产品的10-50%的销售价格[每个分析样品]推出。存在对适合于高通量使用的分析前净化的需要，因为：（a）99%的血浆蛋白[例如，白蛋白，IgG]模糊了目标分子的精确测量;（B）分析方法如质谱法只能容忍组分浓度的100-1000倍差异;和（c）痕量水平的生物标志物难以测量。因此，虽然生物标志物的使用是为了提醒临床医生异常的生理状态是重要的，但它们目前在常规基础上是无法实现的。该提议基于以下方面几十年的经验：（a）对血液蛋白具有非常高的特异性和亲和力的细菌表面蛋白;（B）亲和层析;和（c）装置开发。产品路径包括：（a）优化具有所需特异性的已知生物受体，（B）以保持高亲和力的方式制备重组形式;（c）将足够的重组形式装入待使用的分离装置中;和（d）通过稀释输入溶液进行测试，直到实现所需的99%配体去除。初步数据确定了所提出的方法的可行性和方法的前景。主要针对白蛋白的原理验证研究将通过表征已克隆的配体以确定其与当前商业产品匹配来确定I期的可行性。在II期研究中，将通过增加除白蛋白外可从血浆中清除的物质的范围来改善这一状况。已确定最终制造和销售的合作伙伴。第一阶段的具体目标是：目标1：开发4种基于S.具有G组动物免疫球蛋白结合活性[特异性白蛋白> IgG]，具有多组氨酸片段以允许从大肠杆菌中快速纯化，通过包含特异性“侧翼片段”增强亲和力，并配备末端硫醇以允许容易地附着至固体基质;目的2：用BIACORE技术测试特异性目的1的候选产物以快速确定其对白蛋白的亲和力特征;以及目的3：确认特定目标2中的候选先导化合物固定在聚苯乙烯微珠上时从血浆中去除蛋白质的能力。RH Hammerstedt PI旁白：公共卫生将受益于疾病的早期发现和/或适当的处方和药物的使用。现在很清楚，生物标志物[将作用机制与临床有效性联系起来的特定化合物]的分析将有助于优化这些目标，但由于血浆中生物标志物的相对浓度较低，因此这种分析很困难。设想的产品[AffiTip]将从血浆中去除干扰物质，从而实现生物标志物方法的全部潜力。

DESCRIPTION (provided by applicant): Project Summary/Abstract: We will develop a disposable, affinity-based capillary tube [AffiTip] that is capable of removing 99% of the interfering bulk protein from <5 uL of undiluted plasma within 2 minutes for use prior to analysis for biomarkers [specific compounds that link mechanism of action and clinical effectiveness]. The product ultimately will be introduced at a sale price [per sample analyzed] of 10-50% of any current offering. A need exists for a preanalysis clean up suitable for high throughput use because: (a) 99% of the plasma proteins [e.g., albumin, IgG] obscure precise measurement of molecule(s) of interest; (b) only a 100-1000 fold difference in concentration of components can be tolerated by analytical methods such as mass spectrometry; and (c) trace levels of biomarkers are difficult to measure. Thus, while biomarker use is to alert clinicians to aberrant physiological status is important, they currently are unattainable on a routine basis. This proposal builds from several decades of experience in: (a) bacterial surface proteins with very high specificity and affinity for blood proteins; (b) affinity chromatography; and (c) device development. The path-to-product involves: (a) optimizing a known biological receptor with the desired specificity, (b) preparing a recombinant form in a manner that retains high affinity; (c) packing sufficient recombinant form into the separation device to be used; and (d) testing by diluting out the input solution until the desired 99% removal of ligand is achieved. Preliminary data establishes viability of proposed methods and promise of the approach. Proof-of-principle studies, focusing on albumin, will establish feasibility in Phase I by characterizing an already cloned ligand to establish that it matches current commercial offerings. In Phase II, that position will be improved by increasing the breadth of materials that can be removed from plasma beyond albumin. Partner(s) for final manufacturing and sales have been identified. The Specific Aims for Phase I are Aim 1: Develop 4 "cassette ligands", based upon S. zooepidemicus group G binding activity [specificity albumin> IgG], with poly-histidine segment to allow rapid purification from E coli, enhanced affinity through inclusion of specific "flanking segments", and equipped with a terminal thiol to allow facile attachment to the solid matrix; Aim 2: Test candidate products of Specific Aim 1 with BIACORE technologies to rapidly determine their affinity characteristics for albumin; and Aim 3: Confirm the ability of lead candidate(s) from Specific Aim 2 to remove proteins from plasma when immobilized to polystyrene beads. RH Hammerstedt PI NARRATIVE: Public health will benefit from early detection of disease and/or appropriate prescription and use of drugs. It is now clear that analysis of biomarkers [specific compounds that link mechanism of action to clinical effectiveness] will be useful to optimizing those goals, but such analyses are difficult because of the low relative concentration of biomarkers in blood plasma. The envisioned product [AffiTip] will remove interfering materials from plasma, allowing full potential of biomarker approaches to be realized.