Use of protein-bearing nitrocellulose as immunogen for in vitro production of monoclonal antibodies: application to myelin basic protein electrophoretically separated from a complex brain protein mixture

Abstract

A procedure for producing monoclonal antibody to myelin basic protein (MBP) using in vitro immunization with MBP transferred to nitrocellulose is described. Following the separation of brain proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic transfer of the electrophoretogram onto nitrocellulose, the MBP band located by immunodetection was excised from the nitrocellulose, ground, and used as immunogen for in vitro stimulation of unprimed mouse spleen cells. While in vitro immunization with soluble MBP was able to generate many hybrids, all the wells in the fusions carried out with the immobilized MBP contained hybrids, 33 to 42% of which were positive to MBP. Among these, six were further characterized; all were IgM and all bound to epitopes common to the 18.5K and 21.5K MBP forms of several species. In view of its simplicity, this technique should have a wide application for the rapid production of monoclonal antibodies to selected proteins or their fragments present in small quantity or difficult to purify on a large scale.