Eric Lumanog's HRP Aptamer Project (2017)

A Diagnostic Tool for Alzheimer’s disease with the Utilization of Aptamers

Introduction and Background

Alzheimer’s disease is considered the most prevalent form of dementia, totaling an average of seventy percent of all dementia patients. The disease is characterized typically by memory loss. Patients affected by this disease have difficulty with the recollection of short term events. Over time, this chronic condition can worsen, and symptoms such as severe confusion about time and location, as well as an unprecedented suspicion of close family and relatives can happen. Although it is known to result from an increasing age, this disease is not considered to be natural for onset to occur. Alzheimer’s disease currently does not have a cure, but treatments can be performed to help lessen the onset of dementia symptoms from occurring. Alzheimer’s also does not have a distinct and qualitative way to diagnose its onset. With the use of Horseradish Peroxidase, Alzheimer’s could potentially be detected and diagnosed by visualizing the appearance of the hypothesized disease biomarker, Beta-Amyloid, through an ELISA test. An ELISA is a technique that quantifies the concentration of a specific substrate by tagging that enzyme with an antibody. After washing the unbound substances way, the solution is presented with an enzyme that activates on a substrate which causes a color change that can be detected visually. This change is quantified by how much has color change has occurred in the sample.

At the moment, The Amyloid Cascade Hypothesis is the most commonly accepted theory as to how the onset of AD occurs in the brain. The theory postulates that Beta Amyloid, a long chained peptide, folds and builds up as plaque on nerve endings, or dendrites. The idea stems from the overload of Beta Amyloid, or the inactivity of Beta Amyloid enzymes. (Huang 2011)

These plaque build ups, as depicted in Figure (2), deteriorate the endings and causes severe neural damage overtime. The amyloid plaque that covers the large nerve endings in the brain are a hallmark of Alzheimer’s disease. The problem with the hypothesis arises in that research believes that “Since AD is multifunctional, a single biomarker like amyloid does not make for an accurate diagnosis” (Patel, 2001) and would not prove to be conclusive for an AD diagnostic. While this is still being researched, the main focus of this paper will focus on finding an oligonucleotide that precisely binds to Horseradish Peroxidase, and later connect through sequencing to an aptamer or antibody for Beta amyloid biomarker.

Horseradish Peroxidase is an enzyme found primarily in the roots of horseradish, whose main function is most efficiently when in conjunction with a substrate that visualizes changes visually. HRP is a 44kDa protein that can be conjugated to another molecule.

These aptamers will be found by utilizing the SELEX process. This process is a cycle that utilizes a large library of nucleic acid sequences and concentrates them to a specific sequence that binds tightly and efficiently to a designated target, such as proteins or other large molecules.

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References

Bio Rad Laboratories, https://www.bio-rad-antibodies.com/elisa-types-direct-indirect-sandwich-competition-elisa-formats.html

Clark M. F., Adams A. N., (1977). Characteristics of the Microplate Method of Enzyme-Linked Immunosorbent Assay for the Detection of Plant Viruses. Journal of General Virology, 34: 475-483.

Craig S. Atwood,Mark E. Obrenovich,Tianbing Liu,Hsien Chan,George Perry,Mark A. Smith,Ralph N. Martins, (2003), Amyloid-β: a chameleon walking in two worlds: a review of the trophic and toxic properties of amyloid-β

Huang, Yadong et al., (2011) Cell, Alzheimer Mechanisms and Therapeutic Strategies Volume 148 , Issue 6 , 1204 - 1222

Lequin, M., Rudolf (2005) Enzyme Immunoassay (EIAI/Enzyme-Linked Immunosorbent Assay (ELISA), Clinical Chemistry 51:12, 2415-2418

Patel, S., Shah, R. J., Coleman, P., & Sabbagh, M. (2011). Potential Peripheral Biomarkers for the Diagnosis of Alzheimer’s Disease. International Journal of Alzheimer ’s disease, 2011, 572495. http://doi.org/10.4061/2011/572495

Veitch, N., (2004) Horseradish Peroxidase: a modern view of a classic enzyme, Phytochemistry, 2004, Volume 65, Issue 3, 249-259.