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Updated: Aug 8, 2026
Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins
Published on: March 3, 2016
Anticentromere antibody specific to human cells directed against the CENP-B autoantigen
L A Bejarano1, J Bolivar, M M Valdivia
1Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Cádiz, Puerto Real, Spain.
Insights
Researchers developed a new antibody targeting human centromere protein B (CENP-B). This specific antibody recognizes the human CENP-B epitope but surprisingly does not bind to CENP-B in other mammalian species, indicating limited conservation.
Area of Science:
- Cell Biology
- Immunology
- Genetics
Background:
- Centromeres are crucial for chromosome segregation during cell division.
- Centromere protein B (CENP-B) is a key component of the centromere in human cells.
- Understanding CENP-B's structure and conservation is vital for cell biology research.
Purpose of the Study:
- To generate a specific antipeptide antibody against human CENP-B.
- To investigate the cross-reactivity and conservation of the human CENP-B epitope across different mammalian species.
Main Methods:
- Raised an antibody against a synthetic peptide of human CENP-B (amino acids 481-493).
- Utilized immunofluorescence and immunoblotting techniques.
- Tested antibody reactivity against centromeric proteins from human, mouse, hamster, and Indian muntjac cells.
Main Results:
- The generated antibody specifically binds to human CENP-B, showing patterns similar to CREST anticentromere antibodies.
- The antibody reacts with the 80-kDa human CENP-B autoantigen.
- Surprisingly, the human anti-CENP-B antibody did not react with centromeric proteins in mouse, hamster, or Indian muntjac cells.
Conclusions:
- The generated antipeptide antibody is specific to the human CENP-B epitope.
- The studied CENP-B epitope is not ubiquitously conserved across all mammalian species.
- This suggests potential species-specific variations in CENP-B structure or epitope presentation.
Abstract:
We describe the generation of a new antipeptide antibody that binds to the centromeric region of human mitotic chromosomes. This antibody was raised against a synthetic peptide corresponding to the 481-493 amino acid sequence of the human CENP-B autoantigen. Immunofluorescence analysis revealed that this anti-CENP-B serum showed an identical pattern to the human CREST anticentromere autoantibody in both mitotic cells and interphase nuclei. Immunoblotting showed that this antibody reacts with the recombinant human CENP-B autoantigen, indicating that it is directed to the 80-kDa centromere polypeptide. We have used this serum to determine, by indirect immunofluorescence, whether CENP-B is conserved in different mammalian species. Surprisingly, the human antipeptide antibody does not react with the centromeric proteins of cultured mouse, hamster, or Indian muntjac cells. Because the CENP-B gene has been cloned in human and mouse, our results suggest that the CENP-B epitope used as an immunogen in this study is not ubiquitous in mammalian cells, and that we have most probably established a monospecific antibody to the human centromere.
