The immunochemistry of sandwich ELISAs. II. A novel system prevents the denaturation of capture antibodies.

The studies reported here describe a model system by which the effect of surface adsorption on the antigen capture capacity (AgCC) of monoclonal antibodies (MoAbs) can be quantitated. Three mouse anti-fluorescein (FLU) MoAbs were biotinylated and iodinated with [125I]Na. The AgCC/ng of these MoAbs was compared when they were adsorbed directly on plastic or immobilized via a Protein Avidin Biotin Capture (PABC) system. In this system, biotinylated MoAbs were allowed to adsorb on biotinylated carrier proteins through a "bridging" process using Streptavidin. This permits MoAbs to complex with antigen at some distance from the plastic surface. The AgCCs of the MoAbs immobilized using the PABC system were 5-400-fold higher than when they were directly adsorbed on plastic.