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

项目概述/摘要：我们将开发一种一次性的、基于亲和力的毛细管[afftip]，能够在2分钟内从< 5ul未稀释血浆中去除99%的干扰体蛋白，用于生物标记物（连接作用机制和临床有效性的特定化合物）分析之前。该产品最终将以现有产品的10-50%（每个分析样品）的销售价格推出。需要适合高通量使用的分析前清理，因为：(A) 99%的血浆蛋白[例如，白蛋白，IgG]无法精确测量感兴趣的分子；(b)质谱等分析方法只能容忍组分浓度相差100-1000倍；(c)痕量生物标志物难以测量。因此，虽然生物标志物的使用是提醒临床医生异常的生理状态是很重要的，但它们目前无法在常规基础上实现。这一建议建立在几十年的经验基础上：(a)细菌表面蛋白对血液蛋白具有非常高的特异性和亲和力；(b)亲和色谱；(c)设备开发。产品途径包括：(a)以所需的特异性优化已知的生物受体，(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用的分离装置中；(d)通过稀释输入溶液进行测试，直到达到所需的99%的配体去除率。初步数据确定了所提出方法的可行性和方法的前景。以白蛋白为重点的原理验证研究将在第一阶段通过描述已经克隆的配体来确定其与目前的商业产品相匹配，从而确定可行性。在II期，通过增加血浆中除白蛋白外可去除的物质的宽度，这一地位将得到改善。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“盒式配体”，基于动物流行病S. group G结合活性[特异性白蛋白> IgG]，具有多组氨酸片段，可从大肠杆菌中快速纯化，通过包含特定的“侧翼片段”增强亲和力，并配备末端硫醇，使其易于附着在固体基质上；Aim 2：用BIACORE技术检测特异性Aim 1候选产品，快速确定其对白蛋白的亲和力特征；和Aim 3：确认特异性Aim 2的主要候选物在固定在聚苯乙烯珠上时从血浆中去除蛋白质的能力。叙述：公共卫生将受益于疾病的早期发现和/或适当的药物处方和使用。现在很清楚，分析生物标记物（将作用机制与临床疗效联系起来的特定化合物）将有助于优化这些目标，但由于血浆中生物标记物的相对浓度较低，这种分析很困难。设想中的产品[afftip]将从等离子体中去除干扰物质，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.