Sample and reagent preparation process for ELISA assay

Author: Abdussaboor Muhammad | Major: Biomedical Engineering

Abdussaboor Muhammad is an honors college student in the College of Engineering studying Biomedical Engineering. He researched the relationship that a certain type of protein, MMP14, has with gene expression of angiogenesis in chemotherapy-treated tumors.

During the Spring 2022 semester, I performed research in the Biomedical Engineering Department under my advisor, Dr. Timothy Muldoon. The main goal of my research was to understand how MMP14, a membrane-type matrix metalloproteinase, plays a role in angiogenesis and how it is affected by 5-FU chemotherapy treatment. It is known that MMP14 significantly contributes to angiogenesis regulation by cleaving extracellular matrix (ECM) molecules in addition to being a key effector in the production of pro-angiogenic factors. I specifically researched how the maximum tolerated dose (MTD) group of 5-FU treatment affects the relative gene expression of vascular endothelial growth factor (VEGF), a pro-angiogenic factor, and hypoxia- inducible factor 1 (HIF-1), which induces hypoxia, as well as quantification of MMP14.

The methods for this research begin with the animal model. For this study, we used an azoxymethane (AOM) mouse model. This process includes three phases: the AOM tumor inducing phase, rest phase to allow for tumor development, and the colonoscopy phase to extract tumor biopsies. Once we had collected all the biopsies necessary, we performed the appropriate tests to quatify protein as well as gene expression. Enzyme-linked immunosorbent assay (ELISA) was used to quantify MMP14 protein quantification, and real time quantitative polymerase chain reaction (qPCR) was used to measure and quantify gene expression of VEGF and HIF-1. For both tests, it was necessary to dilute the samples appropriately so that the results could be referenced using the standard calibration curve for each respective test. Then, the respective protocol was followed from the manual for each test. Once raw data was collected, further analysis was performed to translate the data into interpretable data to find correlation between MMP14 protein quantification vs relative VEGF expression and MMP14 protein quantification vs relative HIF-1 expression. We also researched the relationship between Nestin-1 quantification in end of study colon tissue slides and MMP14 protein quantification.

There were many challenges and road blocks during this whole process, however, Dr. Muldoon guided me throughout the whole process of this research study. All the way from the beginning when I was trying to figure out my research topic to when I was putting together my Honors Thesis poster and paper. He would make sure that I am always on track to finishing my work and properly understanding the research I am performing. Dr. Ariel Mundo, a former PhD student in Dr. Muldoon’s lab, also greatly helped me throughout this whole process. He let me extract biopsies his AOM mouse study and use them for my ELISA MMP14 quantification. I am really grateful to the mentorship I received from both Dr. Muldoon and Dr. Mundo.

Now that I have graduated with my B.S. in Biomedical Engineering, I plan to take a gap year and then apply to medical school. During my gap year, I hope to find a job to gain experience in the health field. I also plan to study for and take the MCAT, and then work on my applications to medical schools.