Enhanced Antibody Immobilization Through a Site-Oriented Process Integrated with Surface Modification

A novel antibody immobilization method was developed for enzyme-linked immunosorbent assay (ELISA). The proposed method utilizes amine-bearing polymer macromolecules, namely, polyamidoamine (PAMAM) or polyethylenimine (PEI) to enhance antibody binding on polystyrene (PS) microwells, as well as staphylococcal protein A (SPA) to realize orientation immobilization. After carboxylating the PS microwells with the solution of potassium permanganate (KMnO4) in diluted sulfuric acid (H2SO4), the PS surface was activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide and then grafted with PAMAM or PEI to introduce free amino groups. Glutaraldehyde was subsequently added to conjugate the amino groups and SPA prior to antibody adsorption. X-ray photoelectron spectroscopy and atomic force microscopy were performed to investigate the morphology of the PS surfaces before and after different treatments. The immunoglobulin binding sites were confirmed through anti-Fab and anti-Fc ELISA kits. Results from the alpha fetoprotein ELISA kit showed that the use of both PAMAM and SPA is the optimal method for antibody immobilization. In this treatment, absorbance exhibited an evident gradient with antigen concentrations and reached higher values over 200 ng/mL antigen compared with the ones of other methods.