Shailen Patel's CIAP Aptamer Project

An ELISA with Calf Intestinal Alkaline Phosphatase to Test for Colon Cancer

Introduction & Background

Colon cancer is the fourth most common cancer among all people of the world with about 140,000 new cases diagnosed per year.1 Cancers are dangerous to the patients who suffer from them and thus the fourth most common cancer is a clear danger to society.2 Colon cancer starts out benign and has only a few controllable symptoms, such as constipation or diarrhea. However, it becomes dangerous when the tumors begin to metastasize. Alkaline phosphatase is shown to have a direct correlation with the metastatic ability of colon cancer. Thus the severity of the cancer increases as the level of alkaline phosphatase increases in the body.2

Alkaline phosphatase is an enzyme that catalyzes the hydrolysis of phosphate groups from the 5’ end of DNA, RNA, ribonucleoside triphosphates and deoxyribonucleoside triphosphates in alkaline environments.3,6 By removing phosphate groups from substances the alkaline phosphatase maintains the basic pH in which it works best. Alkaline phosphatase levels are currently measured to test for bone and liver diseases. If the alkaline phosphatase level is high that is indicative of serious maladies of the liver and bone, such as Paget’s disease and cirrhosis.4 Alkaline phosphatases are found mostly in the liver and bones, but also in the intestines, placenta, and the kidney. They can also be found in the blood, but they do not originate here and are simply released into the stream by the area from which they originate. The molecular weight of CIAP is 140,000 kilodaltons and it is a dimeric glycoprotein.7 The size and structure of CIAP and human intestinal alkaline phosphatase is similar and they have the same catalytic properties and thus the same protein core and active sites.5 Currently a common test is performed to determine the levels of alkaline phosphatase in the blood to test for liver and bone diseases. Increased liver alkaline phosphatase in the blood is indicative of disease and the same goes for bone alkaline phosphatase.4

Aptamers are sequences of DNA or RNA that have high specificity for their targets which are usually proteins or enzymes. Aptamers have the ability to form 3D shapes and thus have the potential to bind to many different proteins.

Aptamers have many applications such as therapeutics, diagnostics, and drug delivery. In drug delivery systems aptamers have been selected for a specific target that needs to be affected by a drug. The aptamer was attached to the drug and inserted into the body. The complex moves through the body until the aptamer binds to its target. Once the aptamer binds the drug is released and thus it specifically affects that target and no other cells or tissues in the body. This application would be most useful in delivering chemotherapy drugs to the specific cancer cells. The aptamer would be selected for a specific antigen on the membrane of the cancer cell that is not found in other body cells. Once the aptamer binds the drugs are released affecting only the cancer cells and no other cells which is currently a major problem with chemotherapy. In our case the aptamer will serve as a diagnostic tool through the creation of an ELISA.

An ELISA is an enzyme-linked immuno assay which is typically used to detect different antibodies in the body.9 There are different types of ELISAs but for this purpose we will use a sandwich ELISA. A sandwich ELISA works by attaching a capture antibody to a plate and this plate is exposed to a blood sample and CIAP binds to the antibody. Then an aptamer with an affinity for CIAP is added to the wells followed by horseradish peroxidase which is a reporter molecule. Tetramethylbenzidine (TMB) is then added to the wells which is oxidized by horseradish peroxidase (HRP) and produces a color change that can be quantified.12 Before this ELISA is performed a sensitivity assay can be performed to determine how much TMB is oxidized by HRP over certain lengths of time and in certain amounts (moles.) The concentration of TMB can be compared to this sensitivity assay to determine how much CIAP there was in the sample. Figure 2 shows a sandwich assay in which the red represents a capture antibody, the green represents CIAP, blue represents the aptamer, and yellow represents HRP.

CIAP contains 2 free sulfhydryl groups per dimer and 4 atoms of zinc per mole of protein. Other than that there is nothing more than polysaccharide and peptide sequences.7 CIAP is known to be stable at basic pHs, hence the name alkaline phosphatase. However because we are testing for it in the blood we will use a buffer that is closer to the pH of blood around 7.3-7.4. The buffer that best represents this is phosphate-buffered saline which is 7.4. In aptamer selection an ideal protein will be positive so as to avoid repelling DNA and RNA which have negative charges. CIAP has an isoelectric point of 5.7.11 The charge of the protein is negative and continues to be negative in PBS, which could prove to be a problem in aptamer selection.

CIAP is stored in Tris-HCl, but PBS is better because PBS functions best at the pH we desire. An aptamer has been found for this target by Vincent Huynh of the Aptamer lab at the University of Texas at Austin. Mr. Huynh has developed an aptamer to create an ELISA. An RNA aptamer selection is being run and the current results show that there were no potential aptamers in the pool though the researchers believe that a problem may have occurred during one of the steps after selection causing no results to appear. This experiment aims to find an aptamer that will bind with a high specificity to CIAP and later to human intestinal alkaline phosphatase. An ELISA can be created to create a diagnostic tool for colon cancer using the aptamer and CIAP.

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Citations