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Alternative ELISA Using a RNA Aptamer against Calf Intestinal Alkaline Phosphatase
Author(s) -
Huynh Vincent,
Wei Eric,
Ellington Andrew,
Stovall Gwendolyn
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.562.6
Subject(s) - aptamer , systematic evolution of ligands by exponential enrichment , rna , chemistry , selex aptamer technique , conjugated system , microbiology and biotechnology , alkaline phosphatase , nucleic acid , reagent , biochemistry , combinatorial chemistry , enzyme , biology , gene , organic chemistry , polymer
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 conjugation to a primary antibody or perhaps another aptamer. Anti‐CIAP aptamers were isolated using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). A diversified pool of RNA (“R50” pool) was used and iterative rounds of selection were performed to wash away the non‐binding RNA species and enrich RNA species specifically bound to CIAP. After nine rounds of selection, a reoccurring 14‐mer RNA motif (named VDH2.14 and identified in 15 of 40 clones sequenced) presented itself in a similar loop structure for the majority of RNA sequences. Furthermore, aptamers 4‐3, 4‐9, and 3‐6 were bound to CIAP with Kd values of 5 nM, 9.4 nM, 10.8 nM, respectively. In addition, a minimized variant was designed and bound to CIAP with a Kd value of 6.8 nM. The development of anti‐CIAP aptamers will allow us to generate an inexpensive and robust CIAP‐conjugated reagent for ELISA work and additional molecular techniques that are AP‐dependent.