Preparation and characterization of a human aurora-A kinase monoclonal antibody.
We have developed monoclonal antibodies against the human aurora-A serine/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for monoclonals was an ELISA (Enzyme Linked Immunosorbant Assay) assay performed in 96-well plates against the purified recombinant histidine-tagged aurora-A. The second was a positive Western blot against the same recombinant protein. The third criteria was a positive western blot against an HeLa cell extract, the selected monoclonal should detect only one protein migrating at 46 kDa (kiloDalton) on SDS (Sodium Dodecyl Sulfate)-polyacrylamide gel electrophoresis. Finally, the monoclonal had to bind to duplicated centrosomes and spindle poles in human MCF7 cultured cells by indirect immunofluorescence. At this stage several monoclonals were still positive. We then increased the selectivity by searching for antibodies that were able to cross-react with the mouse aurora-A kinase both by western blot and indirect immunofluorescence. We selected and cloned the 35C1 hybridoma to produce the antibody. Further characterization of the 35C1 antibody revealed that it was able to immunoprecipitate the kinase, that it did not inhibit the aurora-A kinase activity and consequently could be used to measure the aurora-A kinase activity in vivo after immunoprecipitation.
Cremet
Jean Yves
JY
Groupe Cycle Cellulaire, UMR 6061 Génétique et Développement, CNRS-Université de Rennes 1, IFR 97 Génomique et Santé, Faculté de médecine, Rennes cedex, France.
Descamps
Simon
S
Vérite
Franck
F
Martin
Anne
A
Prigent
Claude
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D064096:Aurora Kinase A; D064127:Aurora Kinases; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D011994:Recombinant Proteins; C576918:Aurka protein, mouse; D064096:Aurora Kinase A; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases