Cloning and expression of the H chain V region of antibody OVB3 that reacts with human ovarian cancer.
Hybridoma OVB3 produces an antibody (IgG2b kappa) that reacts with an Ag present on the surface of almost all human ovarian carcinomas. An EcoRI fragment of genomic DNA containing the rearranged H chain V region of monoclonal OVB3 was cloned from a lambda gtWES library and then sub-cloned into a pGEM4 vector. To show that the cloned DNA sequence did encode the V region of a functional antibody, the DNA fragment was inserted into plasmid pSV-VNP gamma SNase in place of VNp to produce pSV-VOVB3 gamma SNase. This plasmid was then transfected into variant OVB3 hybridoma cells, which no longer produced the H chain of antibody OVB3, and functional antibody was secreted by the recipient cells. The recombinant chimeric antibody bound to ovarian cancer cells and contained Staphylococcus aureus nuclease activity, proving that a functional V region gene had been cloned.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D000937:Antigen-Antibody Reactions; D002469:Cell Separation; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D010051:Ovarian Neoplasms / Q000235:genetics / Q000276:immunology*; D010446:Peptide Fragments / Q000235:genetics
D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D004247:DNA