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Updated: Jul 2, 2026
Generation of Murine Monoclonal Antibodies by Hybridoma Technology
Published on: January 2, 2017
Generation and characterization of novel monoclonal antibodies against human aurora-A
Liming Wu1, Tom Beito, Junjie Chen
1Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Abstract:
The mitotic kinase Aurora-A is essential for mitotic progression, including centrosome maturation, mitotic spindle formation, and faithful segregation of chromosomes to daughter cells. Several lines of evidences also suggest that the mammalian aurora kinase family proteins play a role in tumorigenesis. We have previously shown that human Aurora-A was ubiquitinated and negatively regulated by an early mitotic checkpoint protein, Chfr (checkpoint protein with FHA and RING domain). Here, we established several mouse anti-Aurora-A monoclonal antibodies (MAb). GST-tagged human Aurora-A was expressed in BL21 and used as an antigen to immunize mice. Three different hybridomas were obtained and antibodies produced by these hybridomas were analyzed. The results reveal that these antibodies specifically recognize endogenous Aurora-A in both immunoblotting and immunofluroscence experiments. They are useful tools for further analysis of human Aurora-A.
Insights
Researchers developed new mouse monoclonal antibodies targeting Aurora-A, a key mitotic kinase involved in cell division and cancer. These antibodies specifically detect Aurora-A, aiding further study of this important protein.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Aurora-A is a crucial mitotic kinase regulating cell division processes like spindle formation and chromosome segregation.
- Dysregulation of Aurora-A is implicated in tumorigenesis.
- Previous work identified Chfr (checkpoint protein with FHA and RING domain) as a negative regulator of human Aurora-A via ubiquitination.
Purpose of the Study:
- To establish and characterize mouse monoclonal antibodies against human Aurora-A.
- To generate tools for investigating Aurora-A's role in cell biology and cancer.
Main Methods:
- Expression of GST-tagged human Aurora-A in BL21 bacteria for use as an antigen.
- Immunization of mice with the Aurora-A antigen.
- Development of hybridomas and production of monoclonal antibodies.
- Analysis of antibody specificity using immunoblotting and immunofluorescence.
Main Results:
- Three distinct hybridomas producing anti-Aurora-A antibodies were successfully generated.
- The developed antibodies demonstrated specific recognition of endogenous Aurora-A.
- Antibody efficacy was confirmed in both immunoblotting and immunofluorescence assays.
Conclusions:
- Novel mouse monoclonal antibodies against human Aurora-A have been successfully established.
- These antibodies are validated tools for detecting endogenous Aurora-A.
- The antibodies will facilitate further research into Aurora-A function and its role in tumorigenesis.
