A simple general method of exactly comparing different binding activities and antigenic properties of antibodies by obtaining duplicate copies from a single isoelectric focusing gel.

We report a simple general method of exactly comparing different binding activities and/or antigenic properties of antibodies in ascites fluid after fractionation on a single isoelectric focusing gel. Two copies of the gel are made on nitrocellulose: 1 copy is overlaid with the radioiodinated form of one overlay protein and the other copy is overlaid with the radioiodinated form of another. By superimposing X-ray films obtained after radioautography, any 2 binding activities or antigenic properties of antibodies can be compared exactly and in detail. The procedure is simple, highly sensitive, and independent of the molecular weight of the overlay protein. This is accomplished without requiring isolation of the antibodies, denaturing agents, precipitation, chemical fixation or other modification reactions.