Abstract
Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.
Highlights
Characteristics of desired analyte and ligand molecules, which include enzymes, proteins, antibodies, nucleic acids, and glycans, are the primary criteria to be considered when designing sensing mechanisms
Biotinylated secondary antibody and streptavidin-Horseradish peroxide (HRP) are commonly used in the Enzyme Linked Immunosorbent Assay (ELISA) to improve the limit of detection (LOD)
We found that free biotin pre-mixed with streptavidin-HRP improved the detection of ESAT-6
Summary
Characteristics of desired analyte and ligand molecules, which include enzymes, proteins, antibodies, nucleic acids, and glycans, are the primary criteria to be considered when designing sensing mechanisms. Measurement of the signal generated upon analyte and ligand interaction is the basis of such sensing devices [1,2,3,4,5,6,7]. This approach has been applied to disease diagnosis, environmental monitoring, drug discovery, drug screening, therapeutics, and extension of the human life span [8,9,10,11,12,13,14,15]. A good biosensing system must have both high sensitivity and selectivity.
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