The exploration of ELISA detection assays using L1CAM as a biomarker in malignant cancer
Date
2015
Authors
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Publisher
University of Delaware
Abstract
Metastasizing cells are generally the primary basis of malignancy in cancer. There is an array of signaling mechanisms that influences cancer cell migration and metastasis such as RTK (FGFR) and integrin signaling. Cancer cells manipulate signaling cascades such as these by multiple mechanisms, including the expression of proteins that otherwise would not be expressed, which results in aberrant behavior such as increased proliferation and motility. Abnormal proteins expressed on the cell surface of, or released by, migrating cancer cells can be considered biomarkers specifically to detect that metastasis is occurring. In this project, I have focused on the documented abnormally expressed cancer biomarker, L1CAM (L1; CD171). Several different cancer types such as breast, colon, myeloma, uterine, ovarian, pancreatic, and glioma have all been reported to have L1-positive tumors. Interestingly, clinical studies that have monitored survival outcomes of persons who have had L1-positive and L1-negative tumors, show that persons with L1-positive tumors have significantly lower survival rates compared to their counterparts. Therefore, the ability to detect L1 not only determines the presence of, or potential for, metastasis, but provides a better assessment of each cancer prognosis and the initial rigor of therapy to be administered. Toward this goal of better L1 detection, two commercial monoclonal antibodies, UJ127.11 and 5G3, were used in chromogenic capture and indirect poly-ornithine (p-orn)-ELISAs to detect L1 in cell lysates/extracts, conditioned media, and both PBS and human serum spiked with known concentrations of L1. I found that in indirect (non-capture) p-orn-ELISAs, UJ127 detects L1 in PBS spiked with purified L1fc, and conditioned media from U118L1Le cell lines, but does not from 293TL1fc conditioned media or human serum spiked with purified L1fc. On the other hand, capture p-orn-ELISAs were capable of detecting L1 from all L1-containing samples with generally very low background. To optimize assay conditions, I tested both nitrocellulose and poly-ornithine for increased binding capacity, and poly-ornithine was selected as the best material to accomplish this. I also compared p-orn-coated ELISAs to the more commonly used high-pH buffer coated ELISAs, and found that p-orn-ELISAs were more sensitive for detecting L1. Using the p-orn-coated capture ELISAs, I was able to detect purified L1fc diluted in PBS as well as with human serum present, down to 0.4 ng/ml. This suggests that an improved L1 ELISA detection assay could be of benefit in a clinical setting to evaluate the potential degree of morbidity in cancer cases.