(cloning in agarose/nitrocellulose filters/monoclonal antibodies/erythrocyte adherence assay/autoradiography)

A sensitive and rapid method for the detection of monoclonal antibodies secreted by hybridomas is described. Mouse myeloma cells are fused with spleen cells from immu- nized mice and directly cloned in soft agarose containing se- lective medium; hybrid clones can be seen after a week. Nitro- cellulose filters that have been coated with a specific protein antigen, with antigen-coupled erythrocyte ghosts, or with other cells used as antigens are then placed on the agarose surface. After incubation to allow immunoadsorption of any secreted antibodies specific for the filter-bound antigen, the filter is re- moved and overlaid with a suspension of antigen-coupled erythrocytes that react with the adsorbed antibodies; after un- bound erythrocytes are allowed to fall off the filter, red spots delineate the sites at which antibody-forming clones are present in the agarose. Alternatively, the filter may be treated with ra- diolabeled antigen followed by autoradiography. The reliability and sensitivity of the method are demonstrated with a-l-.3) specific antidextran myeloma J558, and the method's applica- bility is established by. detecting hybridomas with specificities for sheep erythrocytes and for a-(13) dextran.