Establishment of a strategy for the rapid generation of a monoclonal antibody against the human protein SNEV (hNMP200) by flow-cytometric cell sorting.
The screening for antigen-specific hybridoma cells with adequate production rates is still a time-, labour- and money-consuming procedure. A reduction in cell culture testing by specifically selecting those fused cells that produce antibody could therefore make hybridoma technology more attractive, even for small research groups or for newly discovered proteins at an early stage of research. Additional problems, such as the requirement to produce sufficient amounts of the unknown protein at a purity that allows specific immunisation of mice and testing of the resulting hybridoma clones, also need to be overcome. Here we present a new strategy to isolate rapidly and efficiently monoclonal antibodies against new proteins, for which only sequence information at the DNA level is known. The strategy consists of fusion of the protein to a hexa-His-tag to allow easy purification, production in yeast and insect cells to reduce background immunisation with host cell proteins and the selection of IgG-producing hybridoma cells by flow-cytometric cell sorting using the affinity matrix secretion assay technique.
Böhm
Ernst
E
Institute of Applied Microbiology, Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Wien/Vienna, Austria.
Grillari
Johannes
J
Voglauer
Regina
R
Gross
Stefan
S
Ernst
Wolfgang
W
Ferko
Boris
B
Kunert
Renate
R
Katinger
Hermann
H
Borth
Nicole
N
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D001360:Avidin / Q000737:chemistry; D019921:Biotinylation; D015153:Blotting, Western; D003208:Concanavalin A / Q000737:chemistry; D045643:DNA Repair Enzymes; D042783:Endothelial Cells / Q000737:chemistry; D005347:Fibroblasts / Q000737:chemistry; D005434:Flow Cytometry / Q000379:methods*; D006639:Histidine / Q000235:genetics; D006801:Humans; D006825:Hybridomas / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology*; D000072260:RNA Splicing Factors; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000276:immunology*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D000072260:RNA Splicing Factors; D011993:Recombinant Fusion Proteins; C026887:biotinylconcanavalin A; D003208:Concanavalin A; D001360:Avidin; C033223:polyhistidine; D006639:Histidine; D044767:Ubiquitin-Protein Ligases; D045643:DNA Repair Enzymes; C417041:PRPF19 protein, human