The immunochemistry of sandwich elisas—III. The stoichiometry and efficacy of the protein-avidin-biotin capture (PABC) system

Abstract

The protein-avidin-biotin capture (PABC) system was developed to decrease the adsorptioninduced loss of antigen capture capacity (AgCC) of capture antibodies (CAb) used in sandwich ELISAs. This system involves immobilization of biotinylated CAbs through linkage by streptavidin (SA) to biotinylated carrier proteins adsorbed on polystyrene. Studies reported here describe the stoichiometry of the system and the influence of biotinylation of different carrier proteins and CAbs on the reaction stoichiometry and the AgCC of CAbs. Because of the widespread use of sandwich ELISAs to measure the concn of multivalent protein antigens, the AgCCs of monoclonal and polyclonal CAbs to pig IgG in the PABC system were compared with the AgCCs of these Abs immobilized on the plastic by direct adsorption. Optimal assay conditions for the carrier were obtained when 1 μg/ml of the biotinylated protein was added to the polystyrene solid phase. An increasing degree of biotin substitution in three carrier proteins was paralleled by an increasing AgCC until a constant maximum was reached. Under conditions of maximal AgCC, 120ng of the carrier rabbit gamma globulin (RGG; i.e. RGG 25biot) was bound to polystyrene, which in turn yielded the maximum amount (i.e. 100 ng) of bound streptavidin (SA; Bd ng max) when 20 μg/ml of SA was added. Under conditions giving the Bd ng mas for SA, CAb 12biot yielded a higher Bd ng max than did CAb 25biot or CAb 2biot. When the AgCC of equal amounts of differentially biotinylated CAbs were compared, the following order of AgCC was observed: CAb 2biot > CAb 12biot > CAb 25biot. Hence, while the maximal amount of CAb is immobilized on SA when CAb 12biot is used, optimal AgCC is achieved with CAb 2biot. The carrier: SA: CAb 2biot ratio was 1:2:1 while that for carrier: SA: CAb 12biot was 1:2:2. The same ratio was obtained using IgG 2biot, from four different species. Monoclonal antibodies to swine IgG showed a 5–6-fold increase in Bd % max when immobilized as CAbs using the PABC system versus when adsorbed on polystyrene. Plots of these data suggest that the differences result from a loss of functional affinity. On the contrary, no significant differences in Bd % max and hence functional affinity were observed when a polyclonal antibody to pig IgG was compared using the two assay configurations. Furthermore, when the globulin fraction of the anti-pig polyclonal was adsorbed on plastic, it behaved nearly as well as its affinity-purified counterpart immobilized by the PABC system. The PABC system appears to offer significant advantages for sandwich ELISAs utilizing monoclonal antibodies as the CAb, and may offer some advantages in other systems.