High-Performance Immunolatex Possessing a Mixed-PEG/Antibody Coimmobilized Surface: Highly Sensitive Ferritin Immunodiagnostics

To create a high-performance immunoassay system based on a nanosphere/antibody complex, pentaethylenehexamine-ended poly(ethylene glycol), N6-PEG comprising N6-PEG-5k (Mn = 6000 g/mol) and N6-PEG-2k (Mn = 2000 g/mol) was employed as a novel blocking agent to modify the surface of nanospheres. Both the antibody (antiferritin) and the N6-PEG were covalently bonded onto the nanospheres by the linkage of their amino groups with the activated carboxyl groups of those particles. The quantification of antiferritin and tethered N6-PEG polymer was carried out using the copper reduction/bicinchoninic acid reaction (the Micro BCA method). Dynamic-light-scattering (DLS) and electrophoretic mobility (μe) measurements were performed to characterize the nanosphere/antiferritin/N6-PEG complex, which was prepared under various conditions. Simultaneously, the immune response of the complex obtained in this manner was measured by the turbidimetric monitoring method in phosphate buffer (10 mM, pH = 7.4). On the basis of all the results, the optimum conditions for preparing an acceptable nanosphere/antiferritin/N6-PEG complex were determined. Interestingly, compared to the blocking treatment with bovine serum albumin (BSA), which is a well-known blocking agent, surface modification with N6-PEG, especially that using a mixture of N6-PEG-5k and N6-PEG-2k, improved the performance (increased immune response yield and decreased detection limit) of the nanosphere/antiferritin complex to a remarkable degree in both phosphate buffer and 100% fetal bovine serum (FBS), thus significantly demonstrating the potential of the nanosphere/antibody/mixed-PEG complex as central to a high-performance immunoassay system.