Horseradish peroxidase (HRP), goat anti-human IgG, human IgG, anti-human IgG-peroxidase, bovine serum albumin and o-phenylenediamine dihydrochloride were purchased from Sigma, USA. Photoreactive-BSA is used for simultaneous binding of biomolecule and the matrix irrespective of functional groups either on the matrix or the biomolecules. Here, we report a novel proteinaceous photolinker prepared by a simple reaction of BSA and FNAB both of which are easily available. Hence, there is a need to prepare photolinker polymer by a cheap and simple method. However, this method is not popularized due to tedious and time consuming multistep procedure for preparation of such linking agent. Sigrist and co-workers has immobilized biomolecules on inert surfaces in presence of light using such type of linker prepared by the reaction of BSA and 3-(trifluoro methyl)-3-(m-isothiocyanophenyl) diazirine. Advantage of using a protein molecule as a base includes a large number of functional groups available in a single molecule for formation of covalent linkages to incorporate several molecules of a photolinker. Protein-based photolinker is a new class of photolinker having several photoactivable groups, some of which binds to the biomolecule and some to the matrix when exposed to light. However, in all these cases prior to covalent immobilization a matrix or a ligand has to be activated to get active functional group to facilitate covalent linking. Thermoreactive group of the photolinker on the other hand binds to a molecule having an active functional group. Photoreactive group yields highly activated species (nitrene, carbene or oxygen radical) on exposure to light and binds to any molecule irrespective of its functional group. Photolinker can be hetero-bifunctional having a photoreactive and a thermoreactive groups. Among covalent methods, photolinker-mediated immobilization of biomolecule is fast becoming popular due to its simple, non-invasive and mild procedure, ,, ,,. It leads to analytical advances in that the signal to noise ratio can be increased by thorough washing of the microplates after binding. Covalent method of immobilization is now gaining attention even in diagnostics involving ELISA, ,. In recent years, covalent immobilization of a biomolecule on a solid matrix has become the subject of intense research as it is rapid, stable and efficient. Thus the method is potentially useful for rapid ELISA or covalent immobilization of ligands onto an inert surface without prior activation. ELISA carried out in less than 3 h using photoreactive-BSA showed comparable results with that of conventional ELISA carried out in 18 h. The method is further exemplified by performing ELISA by covalent binding of antigen or antibody on a polystyrene microtiter plate in just 30 min using photoreactive-BSA. When an enzyme is placed on an inert polystyrene matrix in presence of photoreactive-BSA and exposed to light the later forms highly reactive nitrenes some of which bind to the matrix and the rest to the ligand resulting simultaneous formation of covalent bonds with the matrix and the enzyme. We have made photoreactive-BSA – a proteinaceous photolinker by the reaction of bovine serum albumin (BSA) with excess of 1-fluoro-2-nitro-4-azidobenzene (FNAB). Prerequisite of the method is a novel proteinaceous photolinker having multiple light-activable functional groups. A simple and versatile method is developed for covalently binding a protein ligand onto a matrix irrespective of functional groups either on the ligand or the matrix.
0 kommentar(er)
