The use of recombinant vaccinia virus to generate monoclonal antibodies against the cell-surface glycoprotein endoglin.
Characterization of novel cell-surface protein molecules, initially identified by cDNA cloning techniques, usually requires the generation of specific antibodies to further analyze their biochemical and/or functional properties. Here we report a simple method, using recombinant vaccinia virus, for the generation of monoclonal antibodies (mAb) to the 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. Infection of Balb/c mice with this recombinant virus led to the generation of specific polyclonal antibodies, as demonstrated by the antisera reactivity against human endoglin transfectants. The spleen cells of these infected animals were fused to myeloma cells, allowing efficient generation of several hybridomas which secrete mAbs to human endoglin, as evidenced by their reactivity with purified endoglin as well as with endoglin 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.
Luque
A
A
Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, Universidad Complutense, Madrid, Spain.
Cabañas
C
C
Raab
U
U
Letamendia
A
A
Páez
E
E
Herreros
L
L
Sánchez-Madrid
F
F
Bernabeu
C
C
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D015703:Antigens, CD; D000071063:Endoglin; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D013937:Thymidine Kinase / Q000235:genetics; D014615:Vaccinia / Q000276:immunology*; D014616:Vaccinia virus / Q000235:genetics / Q000276:immunology*; D019010:Vascular Cell Adhesion Molecule-1 / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C064206:ENG protein, human; D000071063:Endoglin; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D019010:Vascular Cell Adhesion Molecule-1; D013937:Thymidine Kinase