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

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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.