Our proposed study will demonstrate that we can produce a targeting vehicle capable of both tunable affinity and heat-stability through nanoscale engineering of Variable Lymphocyte Receptors (VLR) antibodies. We will demonstrate these targeting qualities through binding assays of VLR functionalized nano/microparticles. We will create multivalent VLR antibodies with a range of valencies, linker lengths, and that have suitable residues for attachment to nano/microparticles. The multivalent recombinant forms of VLRs will recognize the Bacillus anthracis spore coat protein called BclA. We will verify the proper valency and linker arrangement with biophysical techniques and single molecule imaging. We will measure the binding strength the VLR antibodies to the BclA antigen with surface plasmon resonance (SPR) and single-molecule binding measurements based upon atomic force microscopy force spectroscopy. We will measure the kinetic rates and the resulting affinity of the multivalent antibodies as a function of valency and linker length. We will also validate that antigen binding occurs even when raised to high temperatures. We will conjugate the multivalent VLRs to several micro/nanoparticles and validate adhesion to B. anthracis using high throughput flow cytometry. We will measure particle adhesion and agglutination as a function of a) VLR molecular valency, b) VLR linker spacing, c) particle size, and d) temperature.
We aim to broaden the usefulness of nanoparticle targeting of pathogens with antibodies by 1) identifying design principles that offer a rational method for controlling the molecular affinity through simple modification of valency and 2) utilizing an antibody molecule that is heat-stable up to temperatures of 70?C or greater. Successful completion of this proposal will open several fruitful paths of discovery which will result in improved sensors and countermeasures for pathogens and biowarfare agents. We anticipate the creation of nano/microparticles with tunable antigen affinity and heat stability. These two impacts will drastically improve the viability of antibody- based detectors, countermeasures, and diagnosis methods.
We propose to create nano/microparticles that are targeted by the newly discovered VLR antibody. We will create recombinant forms of VLRs that we anticipate will offer the following benefits to drug delivery, reagent development, and sensors, including rational adjustment of antibody affinity and temperature stability.
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