Research Project
9175644
SUMMARY/ABSTRACT Blood-based multi-biomarker panels hold great promise for effective early cancer detection and population-wide screening. For example, panels consisting of three or more biomarkers which exhibit high sensitivity and specificity have been reported for several different cancers including ovarian, lung, breast and colorectal cancer. A variety of miniaturized solid-phase immunoassay platforms have also been developed to perform high throughput and low- cost multiplex biomarker detection and quantification. These include instruments based on microarrays, microfluidics and micro-bead technology. A high priority is now to transition promising blood-based multi-biomarker cancer panels to multiplex platforms for large-scale biomarker validation and ultimate use in the clinic. Multiplex assay technologies are especially important to provide the cost-effectiveness and high throughput capacity necessary for population-wide screening. However, a major problem with multiplex immunoassays is the so-called ?matrix effect?. Compared to most conventional single-plex assays such as ELISA, miniaturized multiplex assays are highly susceptible to interference caused by the presence of the more abundant, non-target agents in blood. Such interference can originate from a variety of mechanisms including: i) low specificity heterophile antibodies; ii) matrix-induced bead aggregation (e.g. in Luminex® assays) and iii) specific or non-specific binding of non-target matrix components to any component of the biomarker assay. In addition, high viscosity of the sample matrix (e.g. from high total protein concentration) can interfere with the microfluidics commonly used for multiplex assays. Importantly, the matrix effect not only limits assay sensitivity, but reduces linearity and quantitative accuracy. During Phase I we will evaluate a new approach to multiplex serological cancer assays termed PC-PURE? which is designed to eliminate the matrix effect. This technology is based on the use of novel photocleavable (PC) linkers developed by AmberGen which are incorporated into affinity capture agents such as aptamers or antibodies. The photocleavable capture agents are then tethered to micro-beads, affinity resins or other surfaces and used to isolate the target biomarker. The biomarker-[capture agent] complexes are gently and rapidly photo-released in minutes under non-denaturing conditions by low-intensity near-UV light into a well-defined buffer, enabling simultaneous pre-purification and concentration of the target biomarkers prior to multiplex immunoassay. Unlike conventional approaches using blocking buffers, diluents and selected depletion, specific to particular matrix components, PC- PURE? eliminates all matrix effects by rapidly pre-purifying the biomarkers of interest. In Phase I we will evaluate the application of PC-PURE? to improve the multiplex detection of blood-based panels of tumor-shed protein biomarkers. Tumor-shed biomarkers have great potential for high cancer specificity but are found at extremely low abundance in the blood and hence suffer most from the matrix effect. A model 5-biomarker protein panel for ovarian cancer diagnosis will be tested. Both spike-in samples with known concentrations of the biomarkers and ovarian cancer patient blood samples will be analyzed on a multiplex Luminex® MagPix® platform and in Phase II on a Bio-Plex 2200 platform designed for high-throughput clinical testing. This research will be conducted in collaboration with Dr. Gheorghe Doros, Associate Professor of Biostatistics and Director of the Biostatistics Consulting Group at the Boston University School of Public Health, who will provide expert guidance for statistical analysis of the data. We will also work closely with Dr. Bill Jackson, Founder and CSO of Base Pair Biotechnologies, a leading expert on aptamers. To accelerate commercialization of PC-PURE?, we will work closely with Luminex®, one of the leading manufacturers of multiplex assay platforms (see letters of support).
