SBIR

Aptamers (nucleic acid binding species) can be selected against a wide variety of analytes. Aptamers are not only interesting as affinity and diagnostic reagents but could prove to be useful as therapeutics. Accacia proposed to generate aptamer receptors and develop aptamer-based diagnostic assays such as the proximity ligation assay (PLA) that have much greater sensitivity than conventional ELISAs. Goals included selection of aptamers with low nanomolar Kds against ten targets (one aptamer/target), produce extended pools and generate high affinity "bivalent" aptamers. Paired aptamers that are superior to antibodies in both ELISAs and PLA assays will then be generated.

The goal of this project is to develop single-chain variable-fragment antibodies (scFV) against cancer-related proteins. Although monoclonal antibodies are the most widely used reagents for detecting and quantifying proteins, their development is time consuming and expensive. Furthermore, many antigens are non-immunogenic and therefore unusable for antibody generation. The large size of the antibodies may also limit their use in cases where more than one antibody competes for closely juxtaposed epitopes. Therefore scFV can serve as alternative capture reagents with high specificity, high affinity, and small size that can be easily and economically produced. The Company proposed to screen (against 10 cancer antigens) a large scFV antibody library composed of genes encoding rationally-designed complimentarity-determining region sequences with designed codons mimicking natural human antibody diversity. The isolated antibodies will be characterized by Western Blot, ELISA and microarray platforms.

The objective of this contact in phase I research is to develop and validate multiplex mass spectrometric immunoassays (MSIA) for detection and quantification of cancer-related proteins with intrinsically low bodily fluids concentrations. In the first specific aim, the company will develop, optimize, test, and validate a MSIA assay for cancer-related protein whose intrinsic concentration in a bodily fluid (i.e., plasma) is in the 1-10 ng/mL range. A quantitative MSIA assay utilizing a standard curve approach will be developed, and the limits of detection and quantifications will be determined. The assay will be tested with at least a dozen different human plasma samples, in triplicate, and the CVs of the assay will be determined. Usability testing of the assay will be performed with three representative users, and their feedback will be used to make modifications and improvements. The results obtained with the MSIA assay will be compared to a commercially available ELISA assay. In the second specific aim, the company will create a multiplexed MSIA assay that will be able to detect and profile five plasma proteins simultaneously, with a concentration range of 1 ng/mL - 1 mg/mL. Various ratios of antibodies will be immobilized in the affinity pipettes and the assay will be tested on a standard solution of antigens in their physiological concentration to determine the best ratio of antibodies that produces comparable signals in the mass spectra for all proteins, without signal suppression issues. The limit of detection and CVs of the multiplex assay will be determined.

The overall objective of the phase II research is to develop a fully functional quantitative, automated, high-throughput, multiplex affinity protein capture technology platform and Multiplex Mass Spectrometric Immunoassays for analysis of low abundance cancer related proteins/peptides from bodily fluids. The company will start by identifying potential protein and peptide targets and their corresponding affinity reagents. Depending on the availability of such reagents, they will compile a list of more than 50 cancer-related proteins and peptides, and initiate development of individual Mass Spectrometric Immunoasays for each target. The performance of the individual assays will be assessed in regards to their sensitivity and reproducibility Those targets for which the assays pass the performance criteria will be evaluated for grouping into 10 sets of Multiplex Mass Spectrometric Immunoassays, each targeting 5 protein/peptide analytes. The multiplex assays will also include an internal reference for quantification purposes. The multiplex assays, which represent the final product of this research, will be evaluated and validated in regards to their limits of detection, reproducibility, sensitivity, and specificity. As a final task of the project the company will assemble kits containing the multiplex assays, reagents, and protocols, and evaluate other product-related variables such as packaging and storage conditions.

The company proposed to develop a quantitative, automated, high-throughput, multiplexed immunoassay platform. This platform will be able to simultaneously measure up to 75 biomarkers using 30 ul of body fluid samples. The features of this platform include ultrasensitive detection capabilities (< 1 pg/ml), a dynamic range of 108, and a throughput of 60–300 samples per day. A consumable kit will be provided containing lyophilized detection antibodies, reconstitution assay diluents, and a 96-well plate containing the immobilized capture antibodies. It was projected that this platform will facilitate the measurements and verification of cancer diagnostic biomarkers.

During the phase I period, PPM will couple immunoaffinity capture (IC) of prostate cancer biomarker candidate proteins with ion mobility spectrometry (IMS) - mass spectrometry (MS). The approach is unique in that the IC-IMS combination significantly reduces chemical noise therapy allowing greater utilization of the mass spectrometer. That is, higher sensitivity measurements will be achieved. There are three specific aims for the proposed work which include: 1) developing the prototype instrumentation and sample preparation method; 2) validating the analytical platform; and 3) performing comparative proteomics profiling studies for a larger number of control and disease samples. The latter aim will be accomplished by developing an automated, high-throughput analytical platform and is directly focused on demonstrating the applicability of the technique for clinical analyses. The proteins that have been selected for immunoaffinity enrichment include putative biomarker candidates for prostate cancer diagnosis as well as the determination of disease progression and metastasis. Thus, the overall goal of this work is to not only produce an efficacious biomarker panel for prostate cancer but also develop instrumentation that can be used directly in the clinical laboratory. The company is planning to start the project in the near future.

The company proposed to develop an array of 10-100 microfabricated disposable nano-immmunoaffinity chromatography (NIAC) columns originating from a central 10-50 ul well that can be used to selectively capture and enrich 10-100 cancer markers from bodily fluids as it flows into the column array from the central well where the sample is deposited. Quadraspec predicted that as sample flows into NIAC columns, this loading process will enrich and extend the dynamic range as well as enhance sensitivity by a million-fold while minimizing non-specificity. Detection is performed using conventional monitoring methods such as conjugated secondary antibodies and enzyme amplification. The goal was to develop a simple chip that can be used in clinical set ups.

Protein phosphorylation is a central mechanism of cellular regulation. Aberrant phosphorylation activities can cause human cancers. Highly specific capturing reagents are needed to detect and monitor these activities with the aim of detecting cancer early. To generate, characterize and validate these capturing reagents, sufficient amounts of functional phosphoproteins are needed. The long-term goal of this project is to establish a cell-free protein synthesis system that can be used to produce large quantities of site-specifically phosphorylated proteins. The system will feature low cost, high reproducibility, high quality and high yield. The key advancement of the new method is made possible by incorporating phosphoamino acids through site-directed nonsense suppression. Specific reagents will be developed to activate phosphoamino acids efficiently and continuously in the translation reaction. Translation termination at the selected nonsense codon will be manipulated to achieve high suppression efficiency. This cell-free protein sysnthesis system will comprise of both prokaryotic and eukaryotic translation systems. In phase I, the objective is to develop an E.coli cell-free system that is able to produce proteins containing phosphotyrosines in a yield up 0.5 mg per 1 ml of translation reaction.

The company proposed to develop a quantitative, automated, 5-plex immunoassay for the rapid detection of low abundance cancer-related proteins in phase I. In phase II, the Company proposes to validate the screening platform developed in phase I by screening an additional 45 targets. Therefore, a 50-plex immunoassay for low abundance cancer-related proteins will be developed.

The company proposed a novel approach to protein detection and quantification in bodily fluids that integrates three technologies with which Sequenom, Inc. and their collaborators have expertise in: immuno-PCR, competitive-PCR, and mass spectrometric analysis using Sequenom’s MassARRAY platform.