Leila Nyandusi's CIAP Aptamer Project (2017)
Optimized Human Immunodeficiency Virus Diagnostic Using an Anti-Calf Intestinal Alkaline Phosphatase Aptamer
Introduction and Background
ELISA, which stands for enzyme-linked immunosorbent assay, is a diagnostic tool which utilizes antibodies and color change to identify the presence of an antibody. There are several types of ELISA testing and the one in interest for this project is Indirect ELISA. In Indirect ELISA, a flat plate with multiple wells is incubated with an antigen, bovine serum albumin (BSA) is then spreaded across the plate, and the samples with their corresponding antibodies are added (Sino Biological Inc.). Following this, a substrate is added and the enzymes on the antibodies detect the presence of the substrate and elicit a fluorescent or color-change signal (Sino Biological Inc.) ELISA is important as it is used to diagnose communicable diseases such as HIV, a life-threatening disease which has infected 70 million people and killed 35 million people since the beginning of the epidemic in the 1980s (World Health Organization). Using aptamers, quick and efficient diagnoses of such diseases can be made, preventing the loss of millions of lives.
Aptamers are oligonucleotides (single strand of DNA, or RNA in this case) that bind very tightly and specifically to an unknown region of another strand. Aptamers have all the benefits of antibodies and more, such as low cost as they can be created synthetically utilizing a known and easily replicable process. Aptamers are created in vitro as opposed to antibodies which are created in vivo, making for an overall cheaper and more efficient option. To enrich an aptamer, an approach called SELEX (systematic evolution of ligands by exponential enrichment) is utilized. SELEX involves a selection step where a random DNA or RNA oligonucleotide library is exposed to the target, a washing step where the unbound template is removed, an elution that gathers the bound template, reverse transcription in which the template is converted to cDNA, PCR which amplifies the template, and transcription in which DNA is copied to RNA. Each step is pivotal in ensuring the discovery of an aptamer that binds tightly and is highly specific to the target. SELEX is repeated, each repetition constituting a round of selection wherein the pool of oligonucleotides becomes more specific and will bind tightly to the target. The downstream applications of an aptamers range from therapeutics to drug delivery, diagnostics, synthetic biology, and developmental biology (Stoltenburg).
An aptamer against calf intestinal alkaline phosphatase (CIAP) is being selected for with the purpose of reengineering the ELISA diagnostic tool. Calf Intestinal Alkaline Phosphatase is an enzyme found in bovine intestinal mucosa and is most stable in the pH range 7.5 - 9.5, and has a molecular weight of 69 kDa (New England Biolabs). Research being done on CIAP includes a study published in the journal of inflammation and immunopharmacology, it was observed that 80% of mice injected with CIAP survived a lethal escherichia coli infection as CIAP is able to detoxify lipopolysaccharide -mediated diseases (Beumer). An aptamer has been developed for this target by a student of the FRI Aptamers Stream at the University of Texas at Austin (Huynh).
The anti-CIAP aptamer will be serving a diagnostic function by making a more efficient and cost-effective ELISA, thereby quickening the diagnosis process for HIV.. Following the figures to the right, the top figure displays the mechanism of a standard Indirect ELISA. In the top figure an antigen binds to an anti-antigen antibody. A secondary antibody, which is bound to a reporter molecule, binds to the anti-antigen antibody. The reporter molecule elicits a measurable response. In the bottom figure, one sees that the primary antigen is the p24 antigen, a protein which composes the majority of HIV (National AIDS Manual) . An aptamer will bind to the p24 antigen and the Images: Standard (top) and Optimized (bottom) ELISAs (Bio-Rad) anti-CIAP aptamer will be affixed to the anti-p24 aptamer. CIAP serves as the reporter molecule which will elicit a response that will indicate the level of p24 antigen in the sample. The target, CIAP, would replace the antigen (seen in Figure 1) and the bound aptamer would replace the primary antibody and the enzyme-linked secondary antibody in the Indirect ELISA method.
Standard Indirect ELISAs are currently unable to discern trace levels of p24 antigen, resulting in a delay in diagnosis confirmation until the patient has accumulated a larger amount of viral p24 antigen in their body. In delaying diagnosis, a patient is unable to begin treatment at the time of infection and instead must wait until the disease has progressed further. The optimized Indirect ELISA which is not only economical, as aptamers are cheaper to produce than antibodies, but will be able to detect smaller quantities of p24 antigen in a sample because aptamers bind more specifically than antibodies. Patients can receive a diagnosis sooner utilizing the optimized ELISA. Currently, this project has progressed to the second round of selection and is on the cycle course PCR step.
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References
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