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Updated: Jul 2, 2025
Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes
Published on: October 3, 2019
Biotin as a structural component in the detection of small model antigens in BLA-S-ELISA
Mladen Simonović1, Sanja Ostojić1, Darko Micić1
1Institute of General and Physical Chemistry, University of Belgrade, Belgrade, Serbia.
Abstract:
An interesting biotin-linked-antigen-Sandwich-ELISA was developed (BLA-S-ELISA), which based on the captured Trinitrophenol-Biotin (TNP-Biotin) molecule between the immobilised monovalent antibody and enzyme-conjugated streptavidin. Monoclonal anti-Trinitrotoluene single chain fragment antibody (anti-TNT-scFv) was cloned and expressed in E. coli cells, and then used as an immobilised component in an assay. Thereafter, the previously synthesised TNP-Biotin was added as antigen followed by the addition of streptavidin-horseradish peroxidase (streptavidin-HRP) conjugate which led finally to the formation of a three-component system (antibody/TNP-Biotin/streptavidin-HRP). The assay was performed with a range of different dilutions of TNP-Biotin to establish its minimal detectable concentration. The detection limit of TNP-Biotin was 4 ngmL-1 (i.e. 200 pg or 0.42 pmol antigen calculated on the basis of 50 microL sample or 8.4 nM expressed in concentration units). According to our best knowledge, this is the very first time for any model antigen to be detected with such a form of biotin-streptavidin sandwich-assay.
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