Comparison of different blocking agents and nitrocelluloses in the solid phase detection of proteins by labelled antisera and protein A.

Five different brands of nitrocellulose (NC), each of pore size 0.45 micron and without adsorbed antigen, bound different amounts of two labelled antisera and labelled protein A. Experiments with some non-ionic surface active agents and proteins showed that milk powder and bovine serum albumin were the most effective agents for blocking non-specific binding of labelled protein to NC. With some of the NCs, Nonidet P-40 (NP-40) and Tween 20 were almost as effective as milk powder. The protein-binding capacity of unblocked NC and the level of protein binding after blocking were found to be inversely proportional to the pore size of the NC. A comparison of blocking agents in an immunoassay with pollen proteins adsorbed to NC discs revealed that the highest specific uptakes of antiserum occurred with NP-40 and Tween and not with any of the protein blocking agents such as milk powder. Hence, for the detection of proteins using NC-based assays (but not necessarily following electroblotting), the best choices would appear to be: NC of pore size 0.45 micron; a brand of NC that provides a suitable balance between protein binding capacity and non-specific uptake of protein after blocking; a non-ionic detergent such as NP-40 or Tween 20.