Detection of specific hybridoma clones by replica immunoadsorption of their secreted antibodies.

Abstract

A sensitive and rapid method for the detection of monoclonal antibodies [Ab] secreted by hybridomas is described. Mouse myeloma cells are fused with spleen cells from immunized mice and directly cloned in soft agarose containing selective medium; hybrid clones can be seen after 1 wk. Nitrocellulose filters that were coated with a specific protein antigen [Ag], with Ag-coupled erythrocyte ghosts, or with other cells used as Ag are then placed on the agarose surface. After incubation to allow immunoadsorption of any secreted Ab specific for the filter-bound Ag, the filter is removed and overlaid with a suspension of Ag-coupled erythrocytes that react with the adsorbed Ab; after unbound erythrocytes are allowed to fall off the filter, red spots delineate the sites at which Ab-forming clones are present in the agarose. The filter may be treated with radiolabeled Ag followed by autoradiography. The reliability and sensitivity of the method are demonstrated with .alpha.-(1 .fwdarw. 3)-specific antidextran myeloma J558, and the method''s applicability is established by detecting hybridomas with specificities for sheep erythrocytes and for .alpha.-(1 .fwdarw. 3) dextran.