line b104 1 1 emulsified in freund 39 s adjuvant spleens from immune mice are fused with the aminopterin sensitive ns 1 myeloma line and hybridomas are selected in hypoxanthine aminopterin thymidine media culture supernatants from growing hybridomas are initially screened for the presence of antibody capable of binding b104 1 1 cells by indirect immunofluorescence using fluorescence activated cell sorting facs positive supernatants are then tested for specificity by determining whether they contain antibody capable of binding normal nih 3t3 cells or nih 3t3 cells transformed by transfection with harvey sarcoma virus proviral dna cell line xht 1 1a the heavy chain isotypes of the monoclonal antibodies characterized here are determined by double immunodiffusion in agar according to the method of ouchterlony in hudson l and f c hay eds practical immunology blackwell scientific publications london p 117 which is specifically incorporated herein hybridoma cells are washed several times in hbss and injected into pristine primed 400 rad irradiated c3d2f1 mice to induce ascites fluid production when the mice develop significant ascites the fluid is removed by aspiration with a 19 gauge needle and hybridoma cells and debris are removed by centrifugation at 1000 u00d7 g the clarified ascites fluid is then stored at 70 c prior to purification or is purified immediately purification is performed according to the method of drebin et al in immunology and cancer m l kripke and p frost eds university of texas press austin tex p 277 which is specifically incorporated herein cells are removed from dishes with buffered edta versene gibco and washed twice in facs medium hank 39 s balanced salt solution hbbs gibco supplemented with 2 fetal calf serum fcs 0 1 sodium azide and 10 mm hepes 1 u00d7 10 6 cells in 0 1.