(275e) Using Phage and Phage Genomes for Generating Highly Amplified Signals for Protein Sensing

For early detection of many diseases, it is critical to be able to diagnose small amounts of biomarkers in blood or serum. One of the most widely used sensing assays is the enzyme-linked immunosorbent assay (ELISA), which typically uses detection monoclonal antibodies conjugated to enzymes to produce colorimetric signals. In order to increase the overall sensitivities of these sensors, in the first half of the talk, I will describe our use of a dually-modified version of filamentous bacteriophage that produces significantly higher colorimetric signals in ELISAs than what can be achieved using antibodies alone. Since only a few proteins at the tip of the micron-long bacteriophage are involved in antigen binding, the  ~4000 other coat proteins can be augmented - either by chemical functionalization or genetic engineering - with hundreds to thousands of functional groups. In this work, we demonstrate the use of bacteriophage that bear a large genomic fusion which allows it to bind specific antibodies on coat protein 3 (p3) and multiple biotin groups on coat protein 8 (p8) to bind to avidin-conjugated enzymes. In direct ELISAs, the anti-rTNFa conjugated bacteriophage show about 3 to 4-fold gains in signal over that of anti-rTNFa, demonstrating their use as platforms for highly sensitive protein detection.

In the second half of the talk, I will highlight our recent efforts of using the genetic information of the M13 bacteriophage to generate highly amplifiable signals in a single solution isothermally. While one method for producing amplified signals in the presence of antigens has been to use oligonucleotide-conjugated antibodies followed by real-time or isothermal PCR to amplify the DNA strands, the DNA conjugation methods used can affect an antibody’s ability to bind its protein target, and many rounds of linear amplification are often required for sufficient signal generation. In contrast, because the viruses naturally contain a large, 9.5 kilobase genome, combining isothermal rolling circle amplification (RCA) with multiple priming sites can yield statistically significant positive signals at sub-femtomolar antigen concentrations.  The phage therefore provides a convenient container to carry the detection genome to the analyte. I will show that since the genome can be released without or minimal heat denaturation of the phage, and that the number of DNA amplification steps (primer annealing, extension, and dehybridization) are limited, massive amounts of DNA can be detected in real time by luminescence in a much shorter time, in a single reaction tube, at room temperature, and without a specialized PCR instrument.  I will also show that the virus genome can be easily modified with restriction sites at different locations so different analytes can be identified through digestion with a single restriction enzyme and gel electrophoresis.