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Signal Amplification to Enable Attomolar Quantitation in Slide-Based or ELISA Biomarker Immunoassays

Accurate detection of specific markers is crucial for the diagnosis of malignant disease, monitoring drug therapy and patient screening. The development of sensitive and reliable strategies for the detection of biomarkers at ultra-low concentration is of particular importance to cancer medicine.  Efforts have been made to develop techniques for the amplification of signals, but there is still a paucity of novel approaches used specifically to improve the simplicity, selectivity and sensitivity of cancer biomarker assays, especially for the interrogation of tumor tissue.
The current work focus of this SBIR topic is for incorporation of an existing or novel signal amplification system  into  high value cancer biomarker antibody based  assays  (ELISA type or slide-based  IFA/IHC) to enable an increase in accuracy and sensitivity (at least 100-fold) for  detection of the specific analyte at the lowest possible  concentration level. Detection at attomolar/sub-femtomolar concentrations is desired. These assays must be optimized for performance on tumor tissues and tissue extracts, which requires a fundamentally different approach than those commonly used for high-sensitivity serum assays already in use in the field.
In vitro immunoassays are probably the most common, simple and relatively inexpensive methods used in clinical laboratories for the diagnosis and management of disease. Despite continued efforts to improve the performance of immunoassays, there is an urgent need for assays with increased sensitivity (i.e., attomolar sensitivity) and accuracy for detection of low-level disease markers specifically in tumor tissue.
There are many amplification systems that have been published in the past 10-15 year but most have limited effect,  demonstrating improved sensitivity < 10 fold,  and have not been widely adopted in the clinical lab.  One issue is low signal-to-noise ratio; noise often increases with signal amplification.  There is a big technological gap as we learn more and more about signaling molecules in cancer; many are tightly regulated with low levels of protein expression. Simply put, the existing assays are not of sufficient sensitivity to detect low abundance biomarkers.  Finally, the most commonly requested utility for these existing assays is on serum or cell line lysates, not solid tumor tissues. There is a great need for the development of highly sensitive/specific antibody based assays that can detect analytes in tumor lysates (ELISA) and in tumor tissue sections (IFA/IHC).
The current techniques used in molecular tag detection assays use radiolabels, electrical, light scattering, fluorescent, and chemiluminescent molecules?see table below. Most of the commercially available labels have inherent limitations in signal strength. These assay limitations lead to numerous drawbacks in our ability to measure low abundant biomarkers to improve the clinical care of the patient ? to include:

The assay is limited (i.e., the sensitivity of an assay is not sufficient to detect biomarkers in majority of clinical specimens) to the measurements of biomolecules in clinical situations where they are present in high abundance (e.g., protein overexpression or high gene copy number)
The assays can only be applied to biological samples where biomolecules are more abundant such as tumor tissue, but not to more easily obtained non-invasive specimens, such as blood, where the biomolecules are often present but in extremely low- abundance.
The current lower sensitivity of antibody based assays limits detection of many signaling molecules, and where they can be detected, often it will not be possible to detect a drug-mediated decrease in the signal (e.g., for pharmacodynamics [PD] applications).
If the biomarker is of low abundance, then the reliable measurement via an ELISA assay requires the consumption of large amounts of biological samples (e.g., tumor needle biopsies)  to bring up the protein level  high enough to quantitate biomarkers at the  limits of detection (i.e., in the process,  large amount of precious tumor biopsy is lost to further molecular analyses) .

*David A. Giljohann & Chad A. Mirkin.  Nature 462, 461-464, 2009
Recently, a few new technologies have been described in the literature that is reported to achieve 102-106 fold improvement in signal strength.  Many of the technologies bring together large aggregates of immune complexes to produce amplified detection signals several magnitudes greater than reagents in which unitary labels are coupled directly to the secondary antigen or antibody without using multi-label scaffolds.   A short description of some of the more promising technologies is listed below with greater details found in Appendix A. These technologies described below are representative, but not exclusive; offerors are welcome to propose solution technologies not listed below, provided they address the aims of this solicitation.
Plasmonic absorbers, especially in the infrared (IR) range, have potential applications for biochemical sensing, imaging, energy conversion, and other medical diagnostics. One technology referred to as ?Nanobar shaped disk-coupled dots-onpillar antenna-array? (Bar-D2PA) claims up to 1 million fold improvement of immune sensitivity. ? see DocLINK. The D2PA is composed of an array of dense three-dimensional nanoantennas that can be layered with immunological reagents to create a specific assay. The benefits of the bar-D2PA technology are:
(a) low manufacturing costs;
(b) multiple identifying resonance peaks;
(c) tunable transmission with high absorption;  and
(d) high-field regional cross-section for analyte detection.  The bar-D2PA structure shows unique mid-IR light response with polarization-dependent plasmonic resonances.
Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins have attomolar sensitivity. This ultrasensitive method for detecting protein analytes is referred to as the bio-barcode method and has been shown to amplify the signal from extremely low levels of protein or oligonucleotide in solution as published by Mirkin and colleagues (Nam et al., 2003). The principle is to get low-abundant proteins to bind to particles containing a target specific antibody ? this particle is also tagged with thousands of identical single strands of DNA to the target analyte gene which is used to amplify the signal. A magnetic particle tagged with another analyte specific antibody (different epitope) pulls the analyte-particle complex out of solution, and the DNA is separated from the captured particle and quantified.
Another approach of amplification technology is 3DNA? developed by Genisphere which is a 3-dimensional structure made entirely out of DNA. For many applications, a four-layer arrangement is used, which has an average of 280 +/- 20 arms per molecule. The arms are modified with labels and targeting moieties.  As examples, the labels can be fluorescent, enzymatic (HRP, AP), nanogold, or a hapten (biotin, FITC, DIG); and the targeting moiety may be an antibody, peptide, specified RNA/DNA sequence, aptamer, PNA, or a hapten (biotin, FITC, DIG). Mixing and matching a variety of labels and targets on the same 3DNA core creates a highly customized reagent. Genisphere?s 3DNA technology has been used to improve the limit of detection by up to 100-fold in a variety of assay platforms, including microarray, ELISA, bead-based flow cytometry, and lateral flow.
Another novel signal amplification strategy in lateral flow immunoassay (LFIA) utilizes three amplification steps: (a) biotin-streptavidin amplification; (b) polylysine amplification; and (c) fluorescence dye signal amplification.  The resulting conjugates achieved a detection limit 100-fold lower than that of the magnetic beads-based ELISA and gold-based LFIA.
The Enzyme-cascade-amplification strategy (ECAS-CIA) allows detection of low-abundance proteins by coupling with enzyme cascade amplification strategy (DocLINK).  In the presence of target analyte, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3_,5,5_-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification.
It is believe that these technologies and other new technologies could have broad applicability to improving the sensitivity of ELISA and slide-based immunoassays for target proteins as well as for nucleic acid detection assay platforms. However, most of these reagents and associated instrumentations are not commercially available to laboratory researcher or adapted to clinical quality assays.
David A. Giljohann & Chad A. MirkinNature 462, 461-464, 2009
Liangcheng Zhou; Fei Ding; Hao Chen; Wei Ding; Weihua Zhang; Stephen Y. Chou; Anal. Chem.  2012, 84, 4489-4495.
Chao Wang, Qi Zhang, Yu Song, and Stephen Y. Chou*.  Plasmonic Bar-Coupled Dots-on-Pillar
Cavity Antenna with Dual Resonances for Infrared Absorption and Sensing: Performance and Nanoimprint Fabrication.  ACS Nano. VOL. 8 ? NO. 3 ? 2618?262, 2014 DocLINK
Nam JM, Thaxton CS, Mirkin CA. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science. 2003 Sep 26;301(5641):1884-6. PubMed.
Chem. Commun., 2012, 48, 12207?12209 Nanogold-based bio-bar codes for label-free immunosensing of proteins coupling with an in situ DNA-based hybridization chain reaction.
Jun Zhou, Mingdi Xu, Dianping Tang,* Zhuangqiang Gao, Juan Tang and Guonan Chen*
Expert Rev Mol Diagn. 2006 Sep;6(5):749-60.Signal amplification systems in immunoassays: implications for clinical diagnostics. Dhawan S
Sensors (Basel). 2012; 12(9): 11684?11696. A Fast and Sensitive Quantitative Lateral Flow Immunoassay for Cry1Ab Based on a Novel Signal Amplification Conjugate Chunxiang Chen1 and Jian Wu2
Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein Zhuangqiang Gao,1, Li Hou,1, Mingdi Xu1, & Dianping Tang1, Scientific Reports4, Article number:3966Volume
Project Goals
With rapid advances in biomedical research and the growing biotechnology industry, development of highly sensitive and economical assays will meet an unmet need and serve to promote the use of biomarkers in the personalized care of patients.  These enhanced detection technologies can also be directed towards designing fully automated, highly sensitive assays to identify multiple disease markers in a single clinical specimen using multiple assay platforms and to improve the amount of molecular information that a clinician obtains for more precise care of the cancer patients.
The increasing demand for screening assays at the early stage of disease development and from minimally-sized specimens calls for ultrasensitive detection of biologically relevant biomarkers at an extremely low level of expression. To keep pace with expectations in clinical assays, there is still the quest for more flexible, yet highly sensitive, quantitative, and easy-to-use methods.  This SBIR topic is to support assay development that pushes the level of analyte detection to the absolute maximum.
The goal of this SBIR topic is to incorporate recent advances in signal amplification methods into the development of quantitative ELISA and/or slide-based antibody assays (IFA/IHC) to low abundance but high value cancer biomarkers. The amplification system could be chemical, bio-chemical, nano-particle, or any other component based.  The desired aim is to improve the sensitivity of an assay by at least 100-fold and preferably thousand-fold.  Demonstrating the diagnostic potential of existing or novel amplification systems (i.e., platform, tagging, enrichment)  in antibody based assays to protein analytes will provide the foundation for their use in other molecular assays (FISH, RNA in situ, etc) ? i.e. this SBIR will support a significant technological advance in molecular test development in general by promoting the integration of emerging technology into the diagnostic paradigm.
There are two objectives:
Objective 1. Select an appropriate signal enhancing system to improve the signal strength /sensitivity of an ELISA or slide-based antibody assays (IFA/IHC)  using tumor tissue by 102-106 fold (preferably at attomolar levels) for two high value cancer biomarkers, preferably a NCI designated target listed below .


Assay Type


N-Terminal or C-Terminal non-phosphorylated epitopes and coupled with specific phosphorylated sites (pY1234/pY1235, pY1235, pY1236, pY1349 or pS1009)


ERK1 and ERK2 specific epitopes and phosphorylated sites (ERK1-pY202pT204, and ERK2-pY185pT187)


AKT 1, 2, & 3 specific epitopes and  phosphorylated sites (pT308 or pS473)

Apoptosis Biomarkers


HIF1 alpha

Use the polyclonal/monoclonal provided in the DUOSet IC ELISA Kit, for detection of human/mouse total HIF-1 alpha (R&D Systems, Inc., Cat#: DYC1935-5 or DYC1935E)

NCI may advise on the appropriate assay reagents and non-clinical models.   In special cases, NCI may provide reagents to selected PD biomarkers and/or the associated xenograft tumors or cell line models to awardees. 
Objective 2. The enhanced sensitivity system must maintain the integrity of ELISA and/or slide-based antibody assay designs and be easily adaptable to widely used formats/platforms in clinical laboratories (i.e.,  96 well microtiter plate assay, detection instrumentation, bead based detection, automated slide strainers, flurosecent microscopes, etc.).
Phase I Activities and Deliverables
?       The amplification technology must provide a significant improvement in assay sensitivity (102-106 fold) to high value low abundant cancer biomarkers using tumor tissue. Two assays are to be developed with the new amplification system. 
?       The amplification technology must be consistently manufacturable, and if new instrumentation is required, size and cost of prototype instrumentation should be within reach of a clinical lab.
?       Any alteration in the assay design or assay protocol as an attempt to increase the sensitivity of the assay constitutes a critical issue and introduces bias resulting from the changes made. The assay design must be adjusted to optimize the analytic performance of the assay for clinical utility while ensuring analytic validity using proper controls to minimize false results. Suggested activities to test for optimal assay performance are:
o   Optimize the assay to increase signal over background noise and maintain the optimal kinetics of the assay (in fact faster assays with improved kinetics should be possible with a strong amplification system). Appropriate controls and calibrators are to be used.
o   Evaluate the specificity of the higher sensitivity assay to make sure that it is unchanged by challenging the system with interfering substance es and related proteins.
o   The reproducibility and precision of the enhanced antibody based assay are to be evaluated by calculating the intra- and inter-batch variation coefficients (CVs of the assays using the same batch of signal enhancing reagent should be <20%).
o   The batch-to-batch reproducibility using at least 2 different batches of the signal enhancing reagent, performed on different days with different operators should have CV <20%.
?       Assay performance must be tested in the appropriate non-clinical models for the chosen immunoassay analytes.  NCI will recommend at least two models and expects that at least 6 separate specimen preparations from the models will be tested for statistical determinations.
?       The assay, associated methodology, and if applicable, instrumentation will be independently verified by an external laboratory (NCI may be available for this activity). 
Phase II Activities and Deliverables
?       Expand and ?optimize? enhanced detection for the selected ELISA or slide-based antibody assays (at least 100 fold to bring analysis in attomolar range) to at least 4 high value oncology biomarkers, preferably from NCI?s list of desired analytes.  Test in appropriate models using target specific calibrators and controls. The assays/associated equipment must be affordable and easily adaptable to standard clinical laboratories.
?       Reproducibly manufacture signal amplification reagents for the 4 high value oncology biomarkers (3 lots) and do at least 6 months stability testing under different storage conditions. If new instrumentation or equipment is required, then the design and manufacture must be optimized.
?       Show reproducibly/robustness of the antibody-based assay quantitation of the designated biomarkers in selected models/tumor specimens performed by 2 users on 3 different days.
?       Statistical quantification of the signal should be provided to demonstrate the enhanced sensitivity and the reliability of the technique.
?       The assay, associated methodology, and if applicable, instrumentation should be independently verified by an external laboratory (NCI may be available for this activity).
?       Show adaptability of the signaling reagents for use in other assay formats (e.g., FISH). 
?       Provide the program and contract officers with a letter of commercial interest. 

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