Reversed dot-blotting in hybridoma screening and epitope mapping. A model study with human alpha 2-macroglobulin to select complex-specific monoclonal antibodies

Abstract

In reversed dot-blotting monoclonal antibodies are immobilized on polyspecific anti-Ig antibodies bound to nitrocellulose paper. The paper is then challenged with the radiolabeled antigen and processed for autoradiography. We found this technique specific and useful in the screening of hybridomas prepared from mice immunized with human alpha 2-macroglobulin. Using 125I-labeled alpha 2M and 125I-labeled alpha 2M-trypsin complexes, antibodies to epitopes expressed only by either native alpha 2M or by alpha 2M-trypsin were detected. This procedure allowed us to map these epitopes directly in the first screening, thereby saving time and materials. The specificities of the selected hybridomas were then easily confirmed by rate electrophoresis.