RESUMEN
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 µL 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.