Alternative ELISA Using an RNA Aptamer Against Calf Intestinal Alkaline Phosphatase

Alkaline Phosphatase (AP) is extensively used in enzyme-linked immunosorbent assays (ELISA), most requiring expensive and time-intensive reagents to generate (such as AP-conjugated antibodies). In addition, these antibodies may have reduced immunoreactivity when conjugated with AP. We seek to address these problems by developing an alternative AP ligand in the form of an aptamer (nucleic acid binding species) against Calf Intestinal AP (CIAP). This CIAP-aptamer will lead to the development of an alternative reagent that is readily amendable to conjugation, such as to a primary antibody or perhaps another aptamer.

Anti-CIAP aptamers were isolated using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). After nine rounds of selection, we found a reoccurring 14-mer RNA motif that presented itself in a loop structure for the majority of RNA sequences. Aptamers 4-3, 4-9, 3-6, and minimized variant 3.1 bound to CIAP with Kd values of 5 nM, 9.4 nM, 10.8 nM, and 6.8 nM, respectively.

We designed a nucleic acid construct consisting of our CIAP aptamer along with a linker and extension, which hybridizes with an anti-PDGF-AB DNA aptamer. Preliminary results show an expected linear correlation between PDGF-AB concentration and absorbance at 405 nm, though absorbance readings are low. Future experiments include increasing the sensitivity and limit of detection of the assay, and improving the versatility of our reagent by swapping the PDGF-AB aptamer with a 2â??-fluoropyrimidine anti-FC aptamer.