SBIR-STTR Award

The real-time polymerase chain reaction (PCR) is the most commonly used method for quantitative gene expression profiling; it allows extremely sensitive measurement of hundreds of mRNA transcripts in parallel. We propose a method by which protein abundances can be quantitated using the same technology. This is achieved by implementing a sandwich immuno-assay format in which the detection antibodies are labeled with different DNA sequences. Real-time PCR is then used to quantify all of the DNA labels in parallel, thus revealing the quantities of the cognate proteins in the biological samples. Preliminary experiments show that real-time PCR using such antibody-DNA conjugates gives over two orders of magnitude more sensitive detection and almost three orders of magnitude increased dynamic range in comparison to standard ELISA, and standard deviations are very low (CV approximately 5%). Initial evidence supporting the ability to multiplex this method has also been obtained.

Thesaurus Terms:
high throughput technology, immunologic assay /test, polymerase chain reaction, protein quantitation /detection, technology /technique development biotechnology

Numerous proteomics platforms for measuring multiple proteins in parallel have been developed, but adoption has been limited by the fact that they all are orders of magnitude less sensitive than standard ELISA assays. By contrast, methods for quantitatively measuring the levels of multiple mRNAs in parallel are very robust and sensitive. The real-time polymerase chain reaction (PCR) is the most sensitive and commonly used method for quantitative gene expression profiling; it allows extremely sensitive measurement of hundreds of mRNA transcripts in parallel. We have now developed a proprietary method for using the real-time-PCR technology to measure protein concentrations, also in a multiplexed fashion and with very high sensitivity and reproducibility. The method, Multiplexed Real-time-lmmuno-PCR (MRI-PCR), consists of a sandwich immuno-assay in which the detection antibodies are labeled with different DMA sequences. Real-time PCR is then used to quantify all of the DNA labels in parallel, thus revealing the quantities of the cognate proteins in the biological samples. Phase I data show that multiplexed protein measurements employing MRI-PCR assays provides ~2 orders of magnitude more sensitive detection and >2 orders of magnitude increased dynamic range in comparison to standard ELISA assays, along with very low standard deviations (CV <10%) and high specificity. In Phase II, we propose to develop two commercial-ready panels of dodecaplexed assays for 12 cytokines and 12 adipokines, targeting research needs in inflammatory and metabolic diseases, respectively