title: Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays.
abstract: This protocol describes an improved and optimized approach to develop rapid and high-sensitivity ELISAs by covalently immobilizing antibody on chemically modified polymeric surfaces. The method involves initial surface activation with KOH and an O(2) plasma, and then amine functionalization with 3-aminopropyltriethoxysilane. The second step requires covalent antibody immobilization on the aminated surface, followed by ELISA. The ELISA procedure developed is 16-fold more sensitive than established methods. This protocol could be used generally as a quantitative analytical approach to perform high-sensitivity and rapid assays in clinical situations, and would provide a faster approach to screen phage-displayed libraries in antibody development facilities. The antibody immobilization procedure is of ∼3 h duration and facilitates rapid ELISAs. This method can be used to perform assays on a wide range of commercially relevant solid support matrices (including those that are chemically inert) with various biosensor formats.
authors.affiliation: Centre for Bioanalytical Sciences, National Centre for Sensor Research, Dublin City University, Dublin, Ireland.
authors.forename: Chandra Kumar
authors.initials: CK
authors.lastname: Dixit
authors.forename: Sandeep Kumar
authors.initials: SK
authors.lastname: Vashist
authors.forename: Brian D
authors.initials: BD
authors.lastname: MacCraith
authors.forename: Richard
authors.initials: R
authors.lastname: O'Kennedy
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006878:Hydroxides / Q000737:chemistry; D010084:Oxidation-Reduction; D017680:Potassium Compounds / Q000737:chemistry; D011437:Propylamines; D012680:Sensitivity and Specificity; D012821:Silanes / Q000737:chemistry; D013499:Surface Properties
chemical_list: D055910:Antibodies, Immobilized; D006878:Hydroxides; D017680:Potassium Compounds; D011437:Propylamines; D012821:Silanes; C477625:amino-propyl-triethoxysilane; C029943:potassium hydroxide