Antibody Immobilizing Through Polymer Macromolecular PAMAM/PEI

In immunoassay,the sensitivity,regenerability,and stability directly depend on antibodies or antigens that immobilized on the solid surface.The traditional binding method often suffers from small quantity,easy to denaturation and inactivation,wrong orientation.So,looking for a method that can immobilize antibodies or antigens with high quantity,remaining activity,and proper orientation is always the object.In this paper,antibodies were immobilized on polystyrene(PS)surface by six different methods.(A)Antibodies were directly adsorbed to the untreated PS microwells.(B)Polyethylenimine(PEI)was initially immobilized on the carboxylated PS via 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(EDC)and N-hydroxysuccinimide(NHS).Glutaraldehyde(GU)was then used to conjugate antibodies and PEI.(C)The same process as(B)was applied,but polyamidoamine(PAMAM),instead of PEI,was immobilized.(D)Immobilized antibodies with asstaphylococcal protein A(SPA)were adsorbed to the untreated PS microwells.(E)First,PEI was immobilized on the carboxylated PS via EDC and NHS.GU was then used to conjugate SPA and PEI.Finally,antibodies were absorbed by SPA.(F)The same process as(E)was applied,but PAMAM,instead of PEI,was immobilized.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.At the same time,X-ray photoelectron spectroscopy(XPS)was performed to investigate the morphology of the PS surfaces before and after amination.The result indicates nitrogen was not formed on the pristine PS surface,but after the treatment with PEI or PAMAM,nitrogen content significantly increased to 7.35% and 9.69% separately.This meant amine groups were doped.The roughnesses of the PS surfaces before and after the antibody immobilization were determined through atomic force microscopy(AFM),and the results show after antibody immobilization,granular structures were found on the surfaces.Also,few grains were formed on the PS surface with directly adsorbed antibodies than that on the other modified surfaces.This result implies that this treatment immobilized less antibodies.Anti-Fab-and anti-Fc-conjugated HRPs ELISA indicates when antibodies were binding by SPA,they showed almost the proper orientation,or else,there were almost the same Fab and Fc exposed,this means many active sites were hidden.