Zixin Yi's CIAP Aptamer Project (2018)

Detecting Gallbladder Diseases with Calf Intestinal Alkaline Phosphatase RNA Aptamers

Approximately 10% to 15% of adults are suffering from gallbladder diseases, a leading cause of gastrointestinal problems (Stinton 2012). Gallbladder diseases cause acute pain in the abdomen area with symptoms including nausea, vomiting, and complications. Serious gallbladder diseases may induce cholelithiasis, gallbladder cancer, and even require the surgery. The most well-known gallbladder disease is gallstone, where stones formed within the gallbladder out of bile components (Figure 1). However, since risk factors of gallbladder dieases include age increase, gender, liver fatty level, and so forth, they are mostly asymptomatic and discovered incidentally (Acalovschi 2014). Therefore, a more efficient gallbladder diseases diagnostic method can provide patients with faster and cheaper results in order to receive the treatment in time. Currently, the Alkaline Phosphatase test, an antibody-based ELISA kit, as known as enzyme-linked immunosorbent assay, is utilized to quantify the amount of alkaline phosphatase enzymes in the bloodstream that if ALP expressed over the normal range of 30-136 IU/L, gallbaldder problems could exist (Aslam 2013). Therefore, aptamer against calf intestinal alkaline phosphate (CIAP), can be involved to substitute the antibody against alkaline phosphate (ALP) in detection method and to diagnose gallbladder diseases more efficiently.

Aptamers are RNA or DNA oligonucleotides developed to bind the target of interest through Systematic Evolution of Ligands by Exponential Enrichment, the SELEX method, which selects best-binding oligonucleotide sequences from cycles of bead-based selection, reverse transcription, PCR amplification, transcription, and PAGE RNA purification, and repetitive selections can enrich binding species from a highly diverse population. In clinical diagnostic use, aptamers function similar to how the antibody bind the protein, but they make the binding process more efficient, cost-effective, and less time-consuming (Darmostuk 2015). Therefore, CIAP aptamers are substituting alkaline phosphatase enzyme antibodies in an ELISA assay to construct a sandwich ELONA assay, so that when detecting the presence of alkaline phosphatase, the substrate’s color changes to yellow (Figure 2). One CIAP aptamer was in the buffer plate already to bind and immobilize the CIAP target, and secondary aptamer, specific to the CIAP aptamer and conjugated with CIAP brought the color- changing labeling system in to detect the presence or concentration of the target.

In this research, the N71 pool will be used to offer RNA nucleotides to bind on CIAP, and find out the most rapidly and tightly bound aptamers to indicate the conditions of liver and gallbladder.

Calf Intestinal Alkaline Phosphate, CIAP target, found in the bovine intestine is a reporter molecule that detects the presence of another molecule by creating a colorimetric response. For this individual project finding aptamers against CIAP for gallbladder diseases, the first round of CIAP aptamer selection has proceeded to RNA Elution. A full round of CIAP diagnostic aptamer selection is expected to be finished in this fall semester. After ample rounds of aptamer selection, a CIAP aptamer is supposed to be found and sequenced in order to establish a sandwich ELONA essay for gallbladder diseases.

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References

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Aslam, H. M., Saleem, S., Edhi, M. M., Shaikh, H. A., khan, J. D., Hafiz, M., & Saleem, M.

(2013). Assessment of gallstone predictor: comparative analysis of ultrasonographic and

biochemical parameters. International Archives of Medicine, 6, 17. http://doi.org/10.1186/1755-

7682-6-17

Darmostuk, M., Rimpelova. S., Gbelcova. H., Ruml. T. (2015). Current approaches in SELEX:

An update to aptamer selection technology. Biotechnol Adv. 2015 Nov 1;33(6 Pt 2):1141-61. doi:

10.1016/j.biotechadv.2015.02.008. Epub 2015 Feb 20.

Mayo Foundation for Medical Education and Research. Gallstones. Digital Image. 2018.

ThermoFisher Scientific. CIAP (Calf Intestinal Alkaline Phosphate). 2018.

Stinton, L. M., & Shaffer, E. A. (2012). Epidemiology of Gallbladder Disease: Cholelithiasis and

Cancer. Gut and Liver, 6(2), 172–187. http://doi.org/10.5009/gnl.2012.6.2.172

Xue. Q., Cao. X., Zhang. C., Xian. Y. (2018). Polydopamine nanodots are viable probes for

fluorometric determination of the activity of alkaline phosphatase via the in situ regulation of a

redox reaction triggered by the enzyme. Mikrochim Acta. 2018 Mar 19;185(4):231. doi:

10.1007/s00604-018-2769-7.