title: Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay.
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.
authors.affiliation: Advanced Medical & Dental Institute (AMDI), Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia.
authors.forename: Thangavel
authors.initials: T
authors.lastname: Lakshmipriya
authors.affiliation: Institute of Nano Electronic Engineering (INEE), Universiti Malaysia Perlis, Kangar, Perlis, Malaysia.|School of Bioprocess Engineering, Universiti Malaysia Perlis, Arau, Perlis, Malaysia.
authors.forename: Subash C B
authors.initials: SC
authors.lastname: Gopinath
authors.affiliation: Advanced Medical & Dental Institute (AMDI), Universiti Sains Malaysia, Kepala Batas, Penang, Malaysia.
authors.forename: Thean-Hock
authors.initials: TH
authors.lastname: Tang
mesh_terms: D000906:Antibodies; D000942:Antigens, Bacterial / Q000276:immunology; D001710:Biotin / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D006801:Humans; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000906:Antibodies; D000942:Antigens, Bacterial; C525005:ESAT-6 antigen, Mycobacterium leprae; D001710:Biotin; D019809:Streptavidin