Tae Ho Kim's CIAP Aptamer Project

ELISA (Enzyme Linked ImmunoSorbent Assay) is a well-known procedure used to detect the presence of small molecules such as proteins and hormones [1] (Figure 1 shows the ELISA procedure). Calf Intestine Alkaline Phosphatase (CIAP) is a dimeric enzyme consisting of two identical subunits with a molecular weight of about 69 kDA [3] [4]. CIAP is found naturally in bovine intestines and as well as in the outer membrane of microvilli in the small intestine with importance in the duodendum [3] and assists ELISA in detection as a reporter molecule [2]. With its complementing substrate, p-Nitrophenyl phosphate (pNPP), it binds to an antibody complex and the interaction verifies whether a particular antigen is present via development of a soluble end product [2]. This end product is usually yellow, as shown in Figure 2. Despite the record of success, this procedure has several drawbacks. ELISA, particularly the "direct ELISA" (Fig 1), require the labeling of all primary antibodies, which result in its high cost [1]. Another conflict is that when antibodies that are regularly used in ELISA conjugate with alkaline phosphatase, it has reduced immunoreactivity [2]. An aptamer for this target can also be used as a biosensor molecules such as thrombin using p-aminophenylphosphate (p-APP) as the substrate instead of the pNPP [11]. Currently, Jose L. Millan has been credited with a patent for producing a recombinant calf intestinal alkaline. [5]

Aptamers are single-stranded DNA or RNA molecules that can bind to a selected target(s) with a very high affinity. The stronger and cheaper aptamer for CIAP, that will be a part of ELISA, is used for diagnostic purposes as it aims to replace the fragile and expensive antibodies that interact with alkaline phosphatase. Aptamers can differ in shape they can have a wide range of targets with their tendency to form helices and single-stranded loops [8].Aptamers also have a variety in their applications to match. For example, in the field of medicine, Aptamers have been used as a therapeutic for conditions such as macular degeneration [9] and as a diagnostic tool for determining whether a growth hormone is either natural or recombinant [10].

Alkaline phosphatase works very well with the Diethanolamine (DEA) buffer as it is more insensitive to variations such as pH, buffer concentrations and incubation time [13]. However, the focus here is on ELISA and the assay buffer for ELISA is the PBS buffer. For this target, an aptamer has been found by Vincent Huynh, Eric Wei, Andrew Ellington, and Gwendolyn Stovall for an alternative ELISA [14].

The project has gone to the final stages in the first round before though it had to be backtracked due to problems during the binding and selection of the second round. In this selection, the objectives are to complete as many possible rounds of selection as possible (the target number is 2-3 rounds) in order to get closer to finding an aptamer that substitutes for the antibodies that are involved in the ELISA procedure. The procedure will then become more cost-efficient as well as avoiding the reduced immunoreactivity that happens when antibodies that are regularly used in ELISA conjugate with CIAP [2].

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Citations

[1] Wild, D. (2013). The Immunoassay Handbook: Theory and applications of ligand binding, ELISA and related techniques (Newnes).

[2]Seitz, H., and Schumacher, S. (2014). Molecular Diagnostics (Springer)

[3] Fosset, M., Chappelet-Tordo, D., and Lazdunski, M. (1974). Intestinal alkaline phosphatase. Physical properties and quaternary structure. Biochemistry 13, 1783–1788.

[4] Alkaline Phosphatase, Calf Intestinal (CIP) - NEB.

[5] Millan, J.L. (1998). Recombinant calf intestinal alkaline phosphatase

[6] I-detect.eu (2008) About i-Detect. Available at: http://www.i- detect.eu/en_produ.php

[7] Sigmaaldrich (2015) Phosphatase substrate 5 mg tablets | Sigma-Aldrich.