Colloidal Particles as Immunodiagnostics: Preparation and FFF Characterization
Teresa Basińska, Karin Dahlgren Caldwell · ACS symposium series · 1999
The preparation and characterization of two types of particles with covalently attached proteins is described. The first type is a Pluronic F108 coated polystyrene latex (PS) where the surfactant has been end-group activated with pyridyl disulfide groups (PS-F108-PDS) for the subsequent protein attachment, while the second type is a synthesized poly(styrene/acrolein) latex (PSA), whose surface aldehyde groups allow a direct immobilization of protein via Schiff's base formation. Human immunoglobulin (IgG) was first covalently bound to the surface of these particles and in a second step a polyclonal antibody directed against this protein, namely a rabbit anti human IgG (α-IgG), was specifically adsorbed. Determinations of the surface concentrations of protein adsorbed in the two steps were accomplished by the sedimentation field-flow fractionation (SdFFF) technique, as well as by the more conventional bicinchoninic acid (μBCA) and amino acid analysis (AAA) methods, whereby a reasonable agreement was found between the three. It is shown that the mode of linking antigen (IgG) to surface has a significant influence on the efficiency of its antibody binding, with the tethered attachment providing greater access to the antibody. This study represents the first example of SdFFF characterization of a multi-layered composite where the different layers consisted of different chemical species.