Abstract
We report a novel, modular approach to immuno-detection based on antibody recognition and PCR read-out that employs antibody-conjugated bacteriophage and easily-manipulated non-pathogenic viruses as affinity agents. Our platform employs phage genetically tagged for in vivo biotinylation during phage maturation that can easily be linked, through avidin, to any biotinylated affinity agent, including full-length antibodies, peptides, lectins or aptamers. The presence of analyte is reported with high sensitivity through real-time PCR. This approach avoids the need to clone antibody-encoding DNA fragments, allows the use of full-length, high affinity antibodies and, by having DNA reporters naturally encapsulated inside the bacteriophage, greatly reduces nonspecific binding of DNA. We validate the efficacy of this new approach through the detection of Vascular Endothelial Growth Factor, a known angiogenic cancer biomarker protein, at attomolar concentrations in bronchoalveolar lavage fluid.
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Acknowledgments
We are grateful to Dr. Brian Kay for providing the phage and plasmid reagents. This research was funded in part by grants from the Welch foundation (Grant E-1264), the Cancer Prevention & Research Institute of Texas (Grant RP110360), the Norman Hackerman Advanced Research Program (Grant 003652-0186-2009) and NIAID/NIH (Grant U54 AI057156), and by the Huffington-Woestemeyer Professorship. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the RCE Programs Office, NIAID, or NIH. A postdoctoral scholarship for AEVH from the Olle Engkvist Byggmästare Foundation is gratefully acknowledged.
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Litvinov, J., Hagström, A.E.V., Lopez, Y. et al. Ultrasensitive immuno-detection using viral nanoparticles with modular assembly using genetically-directed biotinylation. Biotechnol Lett 36, 1863–1868 (2014). https://doi.org/10.1007/s10529-014-1555-9
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DOI: https://doi.org/10.1007/s10529-014-1555-9