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Aptamer Against CIAP for Monitoring of Osteosarcoma
Osteosarcoma is the most common type of bone cancer, and tends to affects children and young adults most. Although osteosarcoma is not a common cancer, it is very aggressive, and can vary in severity (World Health Organization, 2014). Tumor markers are a substance produced by cancer cells or other cells in response to the cancer cells. They are usually present in the body already, but appear in higher concentrations in cancerous situations, and the most reliable tumor marker varies for each type of cancer (Tumor Markers, 2015). A reliable tumor marker for osteosarcoma is alkaline phosphatase (ALP) (What is Osteosarcoma?). ALP is produced in all tissues of the body, but is most concentrated in the bone and liver; due to this, elevated levels of ALP can be an indication of various diseases of the bone and liver (Figure 2). It has been found that ALP levels are an effective biomarker of prognosis for osteosarcoma (Ren, 2015). While elevated levels of ALP can be an indication for other diseases, monitoring the levels of ALP is commonly used as a way to measure the prognosis of osteosarcoma after various detection methods.
Currently, the diagnostic for osteosarcoma consists of several steps. Initially, an imaging test such as an x-ray is done. If cancer is suspected, an MRI scan of the bones involved must be taken. After this, a core needle biopsy must be done, which involves using a wide, hollow needle to remove a tissue sample. This tissue sample then undergoes lab testing to determine if it is cancerous. If osteosarcoma is diagnosed, the patient must the undergo surgery, chemotherapy, or a combination of both as treatment (World Health Organization, 2014). During treatment, the cancer’s prognosis can be monitored by measuring the levels of alkaline phosphatase (Levine, 1979). The cost of all these medical treatments add up quickly, and this research hopes to ease the burden on patients by attempting to create a new way to monitor the prognosis of osteosarcoma using aptamers.
An enzyme-linked immunosorbent assay, or ELISA, is a technique used for the detection of various substances, such as proteins and hormones (Overview of ELISA). An ELISA test is able to detect a number of diseases based off the presence of an antigen. Although there are different types, the typical ELISA procedure involves the antigen to be immobilized, then exposed to antibodies specific to that antigen. Figure 1 depicts the components that go into an indirect ELISA. The plate is then incubated with a substrate to produce a measurable detection method, such as color change (Indirect ELISA, conventional but efficient). However, due to the use of multiple antibodies, ELISA tests have a high cost. Antibodies are costly to produce; kits to perform these tests cost hundreds of dollars. The antibodies are also easily denatured and are only produced in vivo using living organisms. This is not only costly, but takes large amounts of time and space. An Enzyme-Linked Oligonucleotide Assay (ELONA) could be used instead to monitor the prognosis of osteosarcoma by measuring the change in ALP levels after the diagnosis of cancer.
Aptamers are single stranded oligonucleotides that have a high binding affinity and specificity. Aptamers can bind to various targets, ranging from small inorganic molecules to entire cells (Lakhin, 2013). Aptamers are significantly cheaper to produce than antibodies, since aptamers can be produced in vitro. Antibodies also cannot be modified without changing the activity of the protein, while aptamers can be easily modified. Aptamers also have a greater degree of thermal stability and can refold after denaturation. Since aptamers can have the same functions as antibodies, they can be used to replace antibodies for the same diagnostic purposes while significantly lowering the cost.
Calf intestinal alkaline phosphatase (CIAP) is known as a reporter molecule, responsible for a coloremic response in ELISA tests. However, this project focuses on CIAP for its similarities to human Alkaline Phosphatase. Alkaline phosphatase is found in the liver and bones and is involved in the removal of phosphates from the 5’ ends of DNA and RNA (CIAP (Calf Intestinal Alkaline Phosphatase), 20 U/μL). If an aptamer is found against CIAP in this research, it could replace the secondary antibody in an ELISA to reduce the cost and increase the binding specificity to the target.
The goal of this research is to find an aptamer against CIAP that can be used in an indirect ELISA in place of the secondary antibody. The aptamer would bind to the human alkaline phosphatase detected in blood, and use a reporter molecule such as horseradish peroxidase in order to produce a colorimetric response (Figure 3). The amount of substrate produced would be dependent of the amount of ALP present in the sample. The research will be done using the SELEX method using biotinylated CIAP and magnetic bead-based partitioning (Figure 4). This involves selection of binding species, amplification, and purification using the N71 RNA pool, as demonstrated in Figure 4 (Zhuo, 2017). This cycle in completed multiple times, as the iterative rounds enrich the binding species in the pool. An RNA pool contains variations of nucleotides that make up different aptamers; as many at 10^15 variations can appear in the pool. Once an aptamer against CIAP is identified, it will be sequenced and then used in place of the secondary antibody in indirect and sandwich ELONA tests.
The current selection is in the early stages, and is currently on the amplification portion of the second round. CcPCR has been completed, and lsPCR is the next step. In the future, more rounds of selection will be completed before a binding assay can be completed. The binding assay will determine if the aptamer is suitable for use in an osteosarcoma diagnostic, of if modification would be necessary. If the aptamer can be adapted for use in an ELONA, then it can be used to lower the cost of monitoring the prognosis of osteosarcoma.
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References
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