Determining the Class and Subclass of a Monoclonal Antibody by Ouchterlony Double-Diffusion Assays

Originally, the Ouchterlony double-diffusion assays were the most common method for determining the class and subclass of a monoclonal antibody, and they still are useful, particularly when only a few assays will be performed. A sample of hybridoma tissue culture supernatant is placed in a well in a bed of agar, and class- and subclass-specific antisera are placed in other wells in a ring surrounding the test antibody. As the antibodies diffuse into the agar, they meet and multimeric immune complexes precipitate to form a visible "precipitin line."