Ji Yae (Euince) Lee's GOx Aptamer Selection Project (2018)

Detection of HER-2 Positive Breast Cancer: A Glucose Oxidase Aptamer ELISA

Introduction and Background

Breast cancer is the most widespread cancer in females, not only accounting for 23% of new cancer cases but also accounting for 14% of total cancer-related deaths (Jemal A. et al., 2011). And about 20% of breast cancers are HER-2 positive breast cancer that is induced by the excess amount of human epidermal growth factor receptor-2 (HER-2) protein (Moynihan, T. J., 2018). As seen in Figure 1, HER-2 positive breast cancer occurs when the HER-2 gene is overexpressed that increases the amount of HER-2 receptors in cells. The increased number of HER-2 receptors would send more signals that allows the cells to grow faster. Therefore, HER-2 positive breast cancer should be diagnosed in the early stage by using an accurate diagnostic test. Since the increase of HER-2 protein can be an indicator for the HER-2 positive breast cancer, cancer can be diagnosed by detecting the increase of protein by looking at the whole cell. By utilizing Enzyme-linked immunosorbent assay (ELISA) method, the aptamer against glucose oxidase can work as a reporter molecule that detects and conjugates the aptamer or the antibody of HER-2 protein.

An aptamer is an oligonucleotide molecule that has a high affinity to a specific target. By various applications, the aptamer can be used as diagnostic, therapeutic, and drug delivery for selected diseases. The aptamer against glucose oxidase will serve as a diagnostic application. Specifically, the aptamer will be utilized to diagnose and detect the HER-2 positive breast cancer via ELISA. As seen in Figure 3, sandwich Enzyme-linked immunosorbent assay (ELISA) is utilized with a secondary antibody that has specificity for the primary antibody. The aptamer has in-vitro capability and high selectivity. In addition, since the aptamer has a better cost efficiency and smaller sizes than the antibody, the antibody could be replaced by the aptamer in the ELISA (Toh S. et al., 2014). When there is an increased amount of HER-2 protein in the human body, the proteins will bind to the capture aptamer, and the primary aptamer which is the aptamer against HER-2 protein, will also bind to the HER-2 protein. In addition, the secondary aptamer which is the aptamer against glucose oxidase, will act as a reporter molecule that detects the HER-2 protein, and determines the concentration of HER-2 protein through the colorimetric system. This secondary aptamer should only have specificity for the primary aptamer, not for the capture aptamer. In this way, by utilizing the biopsy, the increase of HER-2 protein can be detected by the aptamer against glucose oxidase and can allow an early detection and diagnosis of the HER-2 positive breast cancer.

The aptamer selection was done by the Sequential Evolution of Ligands by Exponential Enrichment (SELEX) process. The process started with the target immobilization and went through the binding and selection. After collecting the unbound pool and bound pool, reverse transcription was performed. Then the polymerase chain reaction (PCR) was conducted to amplify the sample. Finally, the amplified DNA went through transcription to generate RNA from the DNA template, and the RNA was purified by polyacrylamide gel electrophoresis (PAGE). The SELEX process is repeated several rounds to narrow down the RNA pool and to find the aptamer.

Round one for the RNA aptamer selection against glucose oxidase was performed from the binding and selection to the cycle course PCR. The result of ccPCR indicated that the selection was stringent and that 30 cycles would be the optimal number for amplification. Round one will be finished and additional round for the aptamer selection against glucose oxidase will be conducted. Finally, a binding assay will be performed after finishing 6 rounds of aptamer selection to find the aptamer that would attribute to the detection of HER-2 protein, and to diagnose the HER-2 positive breast cancer for women.

Click here for Final Report

Reference and Citations

Bartlett, P. N., & Al-Lolage, F. A. (2017). There is no evidence to support literature claims of direct electron transfer (DET) for native glucose oxidase (GOx) at carbon nanotubes or graphene. Journal of Electroanalytical Chemistry. doi:10.1016/j.jelechem.2017.06.02

Goodsell D (May 2006). "Molecule of the Month: Glucose Oxidase". RCSB Protein Data Bank. doi:10.2210/rcsb_pdb/mom_2006_5.

Herceptin Prescribing Information. Genentech, Inc. 2017

Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. (2011). Erratum: Global Cancer Statistics. CA: A Cancer Journal for Clinicians, 61(2), 134-134. doi:10.3322/caac.20115

Moynihan, T. J. (2018, March 29). HER2-positive breast cancer: What is it? Retrieved April 01, 2018, from https://www.mayoclinic.org/breast-cancer/expert- answers/faq-20058066

Recondo, G. J., Dìaz Canton, E., de la Vega, M., Greco, M., Recondo, G. S., & Valsecchi, M. E. (2014). Therapeutic options for HER-2 positive breast cancer: Perspectives and future directions. World Journal of Clinical Oncology, 5(3), 440–454. http://doi.org/10.5306/wjco.v5.i3.440

Thermo Fisher Scientific Inc. (2015). “Enzyme-linked Immunosorbent Assay (ELISA) Overview." Overview of ELISA

Toh, S., Citartan, M., Gopinath, S., and Tang, H. (2014). “Aptamers as a replacement for antibodies in Enzyme-linked Immunosorbent Assay” Elsevier (64). 392-403