To bring AtheroSHuffle to life, two main components are needed. First, the antibodies that to be used in test strip construction must be successfully produced in E. coli SHuffle. Second, the LFA test strip must be able to use oxLDL specifically as the antigen of interest. Team Virginia has made strides on both components.
Restriction digest verifications for all cloned plasmids matched expected results acquired from virtual digests conducted on the Benchling software. Therefore, all expression plasmids have been successfully cloned into the pSB3K3 backbone. This is further supported by consistent sequencing results. See Results Page for more information and in-depth analysis.
Results from SDS-PAGE gels indicate successful production of our desired antibodies by SHuffle. See Results Page for more information.
Moving forward, Western blots and ELISA’s will be conducted to confirm that the antibodies are produced with functional structures and that the sandwich complex can truly form.
We are developing a lateral flow assay (LFA) pilot using commercially-available antibodies able to detect commercially-available oxLDL. The purpose of the pilot is, among other things, to test the feasibility of oxLDL as an analyte. oxLDL’s large size has the potential to prevent it from flowing down a nitrocellulose membrane. We observed the solution diffuses up the membrane, and the solution appears red due to the presence of gold nanoparticles, which conjugates with the commercially available antibodies that bind to oxLDL epitopes. The red solution’s diffusion confirms that oxLDL fails to remain immobile and can be used as an analyte. (See Results Page for more information).
Once all antibodies are confirmed to be correctly produced in SHuffle, we will construct the entire LFA and run more pilots to verify that each component, especially those involved in sandwich complexes, functions properly on a LFA. Blood samples will be tested to confirm that the entire device can be used by patients.
We designed and 3D printed an LFA cassette that takes less than 20 minutes to print and uses just over 0.5 meters of material, proving that a suitable cassette can be printed quickly and cheaply - the two major requirements for making the production of an accessible test strip feasible. See Results Page for more information.
We are confident that a successful LFA using our own antibodies is just around the corner, especially since we have already tackled the cloning process and optimizing the physical build of the LFA cassette and test strip itself. With more tests to confirm valid antibody production and LFA functions, our complete device can be realized.