Efficient generation of monoclonal antibodies against surface-expressed proteins by hyperexpression in rodent cells.
The generation of antibodies is one of the first requirements in the characterisation of a newly cloned protein. However, this requires expression and purification of the protein in sufficient yield and purity for immunisation of animals and screening of fusion wells. Even with the development of highly efficient protocols based upon incorporation of specific peptide tags, this can be a tedious and time-consuming process. In an effort to improve the speed and efficiency of obtaining antibodies reactive with newly cloned proteins we have developed an approach based upon the expression of the protein at high level in cell lines originating from the species to be used for immunisation. To illustrate this approach we describe the generation of antibodies against two recently cloned proteins that are normally expressed at the membrane, the rat and mouse analogues of human decay accelerating factor (DAF; CD55). However, the strategy is applicable not only to membrane proteins but also to other proteins which can be expressed on the cell membrane by incorporating at the carboxy terminus the signal sequence for glycosyl phosphatidylinositol (GPI) anchor addition derived from DAF or another GPI-anchored protein. The strategy also permits rapid and efficient screening using flow cytometry on expressing cells.
Spiller
O B
OB
Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, UK. spillerb@cardiff.ac.uk
Harris
C L
CL
Morgan
B P
BP
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D018958:CD55 Antigens / Q000235:genetics / Q000276:immunology*; D016466:CHO Cells; D002462:Cell Membrane / Q000276:immunology; D006224:Cricetinae; D005057:Eukaryotic Cells; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger; D051381:Rats; D014162:Transfection
D000911:Antibodies, Monoclonal; D018958:CD55 Antigens; D012333:RNA, Messenger