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Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells
Published on: October 22, 2012
A monoclonal antibody raised against an undecapeptide sequence from human transforming growth factor alpha recognizes
K A Ellem1, M S Wilson, C Fardoulys
1Queensland Institute of Medical Research, Bramston Terrace, Brisbane, Australia.
Insights
A novel monoclonal antibody targets a specific epitope on human transforming growth factor alpha. This antibody unexpectedly binds to centrosomes, identifying a new protein involved in mitotic spindle organization.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Monoclonal antibody (mAbIIa138) was generated against a peptide from human transforming growth factor alpha (TGF-α).
- The antibody targets a hexapeptide epitope (RFLVQE) within the [22-27] residue range of TGF-α.
- TGF-α plays a role in cell receptor binding.
Purpose of the Study:
- To characterize the binding specificity of mAbIIa138.
- To investigate the potential of mAbIIa138 as a cellular marker.
- To identify novel centrosomal proteins involved in cell division.
Main Methods:
- Enzyme-linked immunosorbent assay (ELISA) to test antibody reactivity with native and reduced TGF-α.
- Immunostaining of mammalian cells to determine antibody binding sites.
- Analysis of epitope antigenic stringency using amino acid replacement sets.
- Bioinformatic searches for homologous proteins.
Main Results:
- mAbIIa138 did not react with native or reduced TGF-α, suggesting conformational interference.
- The antibody specifically bound to centrosomes in late-interphase and mitotic cells.
- Centrosome staining intensity correlated with mitotic spindle organization activity.
- The epitope demonstrated high tolerance to single amino acid substitutions (25 replacements).
Conclusions:
- mAbIIa138 recognizes an epitope on a novel centrosomal protein, designated CSP alpha.
- CSP alpha is involved in mitotic spindle organization or function.
- CSP alpha is an unsequenced protein, specific to the centrosome.
Abstract:
A monoclonal antibody (mAbIIa138) raised against the second-loop undecapeptide (residues [21-31]) of human transforming growth factor alpha, known to be involved in binding to its cell receptor, was found to define a hexapeptide epitope (RFLVQE, residues [22-27]). In enzyme-linked immunosorbent assay testing mAbIIa138 did not react with either native or reduced human transforming growth factor alpha, indicating that conformational restraints in this protein interfered with the antigenicity of the cognate sequence. Despite its failure to react with human transforming growth factor alpha, this monoclonal antibody proved very interesting when used to immunostain mammalian cells. mAbIIa138 was found to bind with great specificity to the centrosomes of late-interphase and mitotic cells. The staining of the centrosomes was most intense when the centrosomes were active in organizing the mitotic spindle and faded during telophase as the spindle was disaggregated. The epitope that mAbIIa138 recognizes is thus in a centrosomal protein, denoted CSP alpha, involved in some aspect of mitotic spindle organization or function. Analysis of the antigenic stringency of the epitope, using an amino acid replacement set, showed that 25 individual single amino acid replacements were possible without significant loss of antigenicity. Data bank searches for proteins of possible relevance were unsuccessful, so CSP alpha is an as yet unsequenced protein, specific to the centrosome.
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