Enzyme-labelled antibody-avidin conjugates: new flexible and sensitive immunochemical reagents

We have prepared avidin-labelled antibodies ('shuttles') with the aim of increasing the sensitivity of detecting mouse IgG and human complement factors in ELISA tests and of detecting monoclonal antibodies and digoxigenin haptens (DIG) in hybridization and immunoblot procedures. Avidin-D was conjugated to goat IgG anti-mouse IgG or to anti-digoxigenin antibodies by thiol/maleimide chemistry. Conjugates of different molecular weight were obtained by Superdex 200 gel filtration. The avidin-D-labelled antibodies were then incubated with biotinylated horseradish peroxidase or with biotinylated alkaline phosphatase. Such preformed enzyme-labelled complexes were subsequently used in the various assays. A 5-8-fold increase in sensitivity was found when the preformed enzyme-labelled antibody-avidin-D complexes were compared to directly enzyme-labelled antibodies or antibody fragments. Furthermore it was shown that ELISA procedures employing digoxigenin-labelled polyclonal antibodies detected by shuttle conjugates were approximately five times more sensitive than biotinylated antibodies detected by avidin-biotin complexes (ABC method). The greatest sensitivity was obtained using antibody-avidin complexes which consisted of two IgG molecules and 4-6 avidin-D molecules.