Vernon Marsh Jr.'s CIAP Aptamer Project

A huge field of research within the scientific community is the diagnosis of diseases. Over 17 million people perish annually due to some type of infectious disease or illness [6]. Every year scientists discover new diseases, and every day scientists continuously work to find new and improved ways to diagnose these diseases more quickly, economically, and accurately. One way scientists have done this is through the use of an enzyme-linked immunosorbent assay or ELISA. Generally in ELISA specified antigens are immobilized on a surface. The surface antigens are then exposed to specific antigen-binding antibodies. This antibody, which is bound to the surface antigen, is linked to an enzyme. In the final step a substrate is introduced that reacts with the enzyme and produces a color change [7].

The enzyme used in this research project that induces the color in an ELISA mechanism will be calf intestinal alkaline phosphatase, commonly referred to as CIAP. CIAP is naturally found in the bovine intestine. CIAP dephosphorylates the 5’ phosphate groups of DNA and RNA. In addition, CIAP may be used to dephosphorylate the 5’ termini of DNA or RNA to prevent self-ligation. [1] CIAP is a dimeric enzyme, and is most stable at a pH between 7.5 and 9.5; however, for enzymatic activity it has a pH range between 8 and 10 [3]. The optimal pH of CIAP changes depending on substrate, substrate concentration, and ionic concentration. [3] At a pH of 7.5 CIAP has a charge of -11 .5, an isoelectric point (pI) of 5.99, and an isotopic molecular average weight of about 59kDA. [2] CIAP is stored in buffer containing 50 mM KCl, 10 mM Tris-HCl (pH 8.2), 1 mM MgCl2, 0.1 mM ZnCl2 and 50% glycerol. [1] The storage buffer has salts similar to that of HEPES. It is for this reason that HEPES buffer will be used as the selection buffer for CIAP. Since CIAP acted as a Biotin fuctionalized protein in this research project, the beads used in the selction process were tagged with streptavadin because biotin and streptavadin have high binding affinities for each other.

However, there are a few drawbacks in using antibodies in an ELISA mechanism. Antibodies used in ELISA do not have a very much stability and are easily denatured resulting in a limited shelf life. Most antibodies must be produced in vivo, meaning that a live animal is used in the production of antibodies. The production of antibodies is very long, laborious, and expensive. Antibodies are also typically recognized by the body’s immune system as foreign and dangerous, which in some cases evokes an undesired immune response. Performance of these antibodies varies, and is also subject to contamination. The target potential for antibodies is limited to only targets that cause a strong immune response. Lastly, antibodies cannot be modified without some form of negative consequence such as reduced activity.

For these reasons, this laboratory research will be centered on finding an aptamer against CIAP. In this application the aptamer will by used as a diagnostic tool to detect the presence of possible diseases within test subjects. The aptamer against CIAP (along with its antigen binding congregate) will be used to replace antibodies in the ELISA. There are many benefits to using an aptamer in this application. Aptamers are more stable and can withstand being denatured/re-natured repeatedly. Aptamer selection is in vitro meaning that animals are not used in the productions process. The in vitro selection process also allows aptamers to be easily modified to adapt to different conditions and targets. Since aptamers are made of nucleic acids, they would not not recognized to the body as foreign, and therefore would not evoke a negative immune response. The production of aptamers is also more economical, and has less of a risk of variation and contamination. Aptamers against CIAP have actually been developed before some of which have been in this lab (The Aptamer Research Stream at the University of Texas at Austin).

In this ELISA application an indirect ELISA will be used. In an indirect ELISA test, the CIAP aptamer species is isolated in-between the antibody and the CIAP enzyme. This can be seen more clearly in Figure 6. The addition of the CIAP enzyme will cause color to develop. The color change is directly proportional to the amount of bound sample antigen species. The more antigen species present in the sample, the more prevalent the color change. This format of indirect ELISA is suitable for determining the antibody level in samples [7]. The purpose of this lab is to isolate RNA sequences that have the highest binding affinity to the target biotinylated CIAP. After these species are found they are purified and concentrated, and then reverse transcribed to yield ssDNA. This ssDNA is then amplified in large scale by the polymerase chain reaction (PCR) and yields dsDNA. This dsDNA is then purified and concentrated and transcribed to yield ssRNA. The ssRNA is then purified by running it through a PAGE gel. The ssRNA found in the PAGE gel is then dissolved out and further purified and concentrated. The purified RNA is now what is known as an aptamer. This aptamer will then undergo further rounds of selection to increase selectivity and binding affinity. Future steps also include a binding assay to test the binding affinity of the aptamer to the CIAP target. Once an aptamer against CIAP is found it will then be sent for sequencing. This aptamer would then be used to replace the secondary antibody of an indirect ELISA mechanism. The use of this aptamer in an indirect ELISA would allow scientist to diagnose infectious diseases more quickly, economically, and accurately.

Click here for the Final Report

Citations

New England BioLabs Inc. | Alkaline Phosphatase, Calf Intestinal (CIP). Retrieved April 11, 2015 from

https://www.neb.com/products/m0290-alkaline-phosphatase-calf-intestinal-cip

Millan, J.L., (1998) Recombinant calf intestinal alkaline phosphatase. United States Patent. https://www.neb.com/products/m0290-alkaline-phosphatase-calf-intestinal-cip

SIGMA | Phosphatase, Alkaline from Bovine Intestinal Mucosa. Retrieved April 11, 2015 from https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/p5521pis.pdf

Hall, Brad., Aptamer Stream Website. Retrieved April 12, 2015 from https://sites.google.com/site/friaptamerstream/

Fosset, M., Chappelet-Tordo, D., Lazdunski, M., (1974) Intestinal Alkaline Phosphatase. Physical Properties and Quaternary Structure. Biochemistry. 13(9), 1783-1792.

World Health Organization | World Health Report. Retrieved September 12, 2015 from http://www.who.int/whr/1996/media_centre/press_release/en/

Sino Biological Inc. Biological Solution Specialist | Indirect ELISA, Conventional but Efficient. Retrieved September 13, 2015 from http://www.elisa-antibody.com/ELISA-Introduction/ELISA-types/indirect-elisa

Zhou J, Bobbin ML, Burnett JC and Rossi JJ (2012) Current progress of RNA aptamer-based therapeutics. Front. Gene. 3:234. doi: 10.3389/fgene.2012.00234

Stoltenburg, Regina, Christine Reinemann, and Beate Strehlitz. In Vitro Selection of Target-specific Aptamers Using SELEX Technology. Digital image. ScienceDirect. N.p., Oct. 2007. Web. 19 Sept. 2012. http://www.sciencedirect.com/science/article/pii/S1389034407000664