methods a second mouse monoclonal antibody directed against a different epitope on rabbit igg should be conjugated with fad again using previously described methods the desired detection complex is somewhat like a sandwich with the bead capture antibody here the bead antibody is a first anti rabbit igg antibody forming the first layer the antigen here rabbit igg is used as the antigen forming the middle layer and the fad conjugated anti antigen antibody here a second anti rabbit igg antibody that binds to a different epitope on rabbit igg forming the top layer this structure is built up in stages and the beads are washed in between each stage first the middle layer is added to the first layer and then the top layer is added to the middle layer and lower layer purified rabbit igg should be suspended 0 1m phosphate buffered saline ph 7 0 at a concentration of 0 1 mg ml and incubated with a 1 solution of the mouse anti rabbit igg conjugated microbeads for 1 hour washed 3 u00d7 in the same buffer by repeated microcentrifugation and then resuspended to the original 1 concentration to add the top layer to the sandwich the fad conjugated monoclonal mouse anti rabbit igg should then be added to these washed microbeads to a final concentration of 0 2 mg ml of antibody and allowed to bind for 1 hour the beads should be washed 3 u00d7 in phosphate buffer and resuspended in the 1 5m trehalose buffer at a 1 suspension the net result is to create a sandwich structure of the type in which the fad conjugated anti rabbit igg monoclonal antibody binds to the rabbit igg which in turn binds to the anti rabbit igg coupled to the beads these beads should.