An immunofiltration apparatus for accelerating the visualization of antigen on membrane supports

Although proteins can be transferred and bound to a membrane with several different methods such as capillary transfer, electroblotting, etc., only the "shaker/incubation" method is commonly used for visualization of the proteins. We have tested an apparatus for the immunofiltration of solutions through nitrocellulose membrane which greatly accelerates the kinetics for the visualization of proteins. As a model system, avidin, bound on nitrocellulose membrane, was detected with 5-min filtering steps of a 1:2000 dilution of ascites of murine monoclonal antibody against avidin followed by a 1:5000 dilution of goat anti-mouse IgG-horseradish peroxidase and 0.5 mg/ml chloronapthol in the presence of 0.01% peroxide. The same solutions used with 5-min incubation steps with the shaker/incubation method could not detect avidin at almost 10 times that amount. Further studies with monoclonal antibodies specific for native C-reactive protein and modified-CRP, 15.1D6 and 13.3H12 respectively, showed that immunofiltration did not result in altered specificity compared to the shaker/incubation method. Also, data are presented showing the advantages of a 10-slot top for the immunofiltration of solutions through distinct areas of a membrane.