Elizabeth Herrin's GOx Aptamer Project

Hepatitis A is a viral disease that manifests in an individual’s liver. It is spread primarily in areas that don’t have access to clean water and have poor sanitation conditions. While there is a vaccine that prevents the contraction of Hepatitis A, many remain unvaccinated. These unvaccinated people are the ones who spread the disease, because they contract it when they come in contact with contaminated food, water, and feces. Once these unvaccinated individuals contract the disease, they are able to give it to other unvaccinated people. While death is not imminent for those who carry HAV (Hepatitis A virus), the disease is known to cause both liver failure and disease.

There is also no current cure for HAV. Because of this, the severity of the disease, and the fact that the government cannot mandate that everyone gets the vaccination, reliable and cost efficient means of detection are crucial to prevent the spread of HAV.

A reliable and affordable method of detection is Aptamer-based detection via ELISA. During an ELISA, antibodies are typically bound to a target protein. The use of antibodies can be both costly and inefficient. The goal of this experiment is to find an aptamer that binds to glucose oxidase. This aptamer has the ability to conjoin to another aptamer during an ELISA and allow for the detection of specific antigens. The antigen in this assay is Hepatitis A. The use of an aptamer would improve the ELISA method, making it more reliable and cost efficient

A direct ELISA (see figure 1) tests for the presence of an antigen in a solution. Antibodies or aptamers that are specific to the antigen are released into the solution, and bind to the antigen if it is present.2 However, this is not a visible result. So, a substrate and enzyme are added to the solution. If the antigen is present, the solution changes color. If the solution does not contain the antigen, there is no color change. This process is observable because the enzyme catalyzes its substrate’s reaction to a differently colored product.1 The strength of the color change and the concentration of the primary antibody/aptamer have a direct corelation.2

By performing a direct ELISA test on possibly contaminated HAV infected food or water, the researcher would be able to see if HAV was present in the solution. Glucose oxidase and its aptamer would conjoin to the antigen (HAV) and its aptamer. The conjunction of the two aptamers calls for a direct ELISA test as opposed to a sandwich ELISA test.2

An RNA aptamer selection against glucose oxidase is currently underway, and the potential aptamers have already undergone cycle-course PCR. If an aptamer is found in this species, it can be conjugated with an aptamer to an antigen and a direct ELISA test can be performed. This would help improve the ELISA method by replacing the antibodies with aptamers as well as allow for possible detection of antigens such as HAV.

Glucose oxidase is a highly stable enzyme, even in a solution, and can be obtained at a relatively low cost. It changes color in the presence of glucose and peroxidase, which can be measured by spectrophotometry, and can thus be used as a reporter molecule. Glucose oxidase can be found in humans, fungi, and bacteria. It is found in a person’s blood and weighs 160 kDa as a dimer. Primarily, glucose oxidase is used in glucometers to detect glucose levels in diabetic’s blood.4