Highschooler North Allegheny Senior High School Wexford, Pennsylvania, United States
Introduction: Currently, over 90% of cancer patients succumb to cancer metastasis. Detecting cancer before it reaches this critical stage is imperative for effective treatment and prevention. During metastasis, tumors release various materials into circulation. Instead of relying on numerous types of imaging as the primary source of diagnosis, cancer can be detected from body fluids. Tumors can release extracellular vesicles into the bloodstream even before the tumor itself begins to metastasize. The majority of solid tumors are epithelial in nature. In addition, cells in the bloodstream should comprise mainly of mesenchymal and endothelial cells. Epithelial cell material found in the blood has a high likelihood of being cancerous. Thus, analyzing the contents of the blood for extracellular vesicles derived from epithelial cells can produce an accessible diagnosis with high specificity results, allowing for early diagnosis.
Materials and
Methods: Epithelial cell adhesion molecule (EpCAM) is a common epithelial surface antigen protein. A conjugation reaction was performed to link EpCAM antibodies onto nanomagnetic beads. The overall strategy of antibody conjugation to amine-terminated magnetic beads consisted of three steps. In the first reaction, the heterobifunctional crosslinker SM(PEG)24 was reacted with the amine groups on the magnetic beads through n-hydroxysuccinimide (NHS) chemistry. In a similar way, extra amine groups were passivated with mPEG-NHS. In the second reaction, some amine groups on the EpCAM antibody were converted to thiol groups with a low concentration of Traut’s reagent. In the final step, the maleimide end of the crosslinker reacted with the thiolated antibodies. This process was monitored through the use of Zetasizer to measure the surface electrical potential of these beads. Efficiency of antibody conjugation was evaluated by measuring the protein concentration of the supernatant after the conjugation reaction between the magnetic beads and the thiolated antibodies. This was done by using the Micro BCA kit, a colorimetric assay. To assess the ultimate performance of EpCAM antibody-conjugated magnetic beads for extracellular vesicle isolation, extracellular vesicles from two different cell lines (with one high and one low EpCAM expression) were spiked into artificial plasma samples. The conjugated beads were introduced to the spiked-in plasma and allowed to capture the extracellular vesicles. The resulting DNA concentration from the captured extracellular vesicles were used to determine isolation efficiency and specificity.
Results, Conclusions, and Discussions: To characterize the conjugation reactions, the zeta potential of the starting material, conjugation intermediate, and final product was measured. The zeta potential changed from positive to negative, as the positively charged surface amine groups were conjugated to the crosslinker and then to the negatively charged antibody. Furthermore, the Micro BCA measurement confirmed that the majority of the antibodies conjugated onto the beads.
Next, the isolation efficiency of cancer derived extracellular vesicles was measured. The initial attempts resulted in low isolation efficiencies from both cell lines. To boost conjugation reaction efficiency, the reactant concentration was increased through reducing the reaction volume, increasing the antibody amount, and increasing reaction time. These changes yielded results with high extracellular vesicle isolation efficiencies for the cell line with high EpCAM expression. However, the efficiency difference between the high and low EpCAM expression groups was not statistically significant—indicating a lack of isolation specificity. To improve, the ratio of the crosslinker and passivation agent in the first reaction was optimized, and the antibody amount in the third reaction was further increased. Mixing conditions were also optimized. The final results demonstrated both high isolation efficiency and specificity for EpCAM positive extracellular vesicles.
In conclusion, this project is widely applicable. Since each type of cancer has its cancer-specific antigens, this same process can be used with any cancer as long as its cancer-specific antigen is known and cancer-specific antibody is available. With the refinement of this procedure, cancer detection can be more accurate and accessible. Moreover, this method can be applied to other diseases related to proteins such as Alzheimer’s disease, cardiovascular diseases, and infectious diseases as long as a disease-specific receptor and ligand is known.
