THE USE OF RECOMBINANT VACCINIA VIRUS TO GENERATE MONOCLONAL-ANTIBODIES AGAINST THE CELL-SURFACE GLYCOPROTEIN ENDOGLIN

Characterization of novel cell-surface protein molecules, initially id entified by cDNA cloning techniques, usually requires the generation o f specific antibodies to further analyze their biochemical and/or func tional properties. Here we report a simple method, using recombinant v accinia virus, for the generation of monoclonal antibodies (mAb) to th e cell-surface antigen endoglin, A recombinant vaccinia virus carrying a cDNA encoding human endoglin was inserted into the thymidine kinase locus under the control of the 7.5k vaccinia virus promoter, Infectio n of Balb/c mice with this recombinant virus led to the generation of specific polyclonal antibodies, as demonstrated by the antisera reacti vity against human endoglin transfectants, The spleen cells of these i nfected animals were fused to myeloma cells, allowing efficient genera tion of several hybridomas which secrete mAbs to human endoglin, its e videnced by their reactivity with purified endoglin as web as with end oglin transfectants, Some of the mAbs selected seem to be specific for regions of endoglin conserved among different species as evidenced by their cross-reactivity with chicken endoglin, These results underline the utility of recombinant vaccinia virus to generate antibodies with novel properties to new cell surface proteins such as endoglin. (C) 1 997 Federation of European Biochemical Societies.