In a collaboration with Professor Richard Grundy and Dr Farhana Haque at the Children’s Brain Tumour Research Centre (CBTRC), Layfield and co-workers employed novel mutation specific antibodies generated by Cambridge Research Biochemicals to discriminate missense (G34R and G34V) mutant histone H3 (H3.3) proteins from their wild type counterparts. The antibodies generated were shown to detect both exogenous and endogenous H3.3 G34R and H3.3 G34V mutant proteins by Western Blot and immunocytochemistry. Moreover, the H3.3 G34R antibody showed high specificity and selectivity to the G34R mutation in paediatric brain tumour sections by immunohistochemistry.
The research focus of the group, based at the University of Nottingham, includes a range of different brain tumours. The recent publication released by the group assesses the prevalence of somatic histone H3.1 and H3.3 mutations in paediatric diffuse intrinsic pontine gliomas (DIPG), non-brain stem paediatric high grade gliomas (pHGG) and adult glioblastoma multiforme (GBM).
To better understand the effect of these mutations on the molecular pathology of gliomagenesis the group enlisted the assistance of Cambridge Research Biochemicals to raise highly specific custom polyclonal antibodies to recognise only the G34R and G34V mutant histone H3 proteins. Peptide sequences were carefully designed to ensure specificity to the single mutated residues along with the incorporation of highly refined affinity chromatography processes to ensure effective removal of wild-type specific antibodies. ELISA analysis of the anti-sera generated throughout the course of the immunisation programmes demonstrated selectivity towards the mutant sequence when compared to the wild type analogue.
Cambridge Research Biochemicals are world leaders in peptide synthesis (antigen peptides and other variants/ modifications) and antibody generation, with over 37 years of industry expertise. Mutant specific anti-peptide antibodies are highly sought-after research tools for gaining knowledge and insight into target specific mutations. The screening and purification strategies refined throughout CRBs 37 years of successful antibody generation has helped to ensure and improve the specificity of the antibodies produced.
The figure below shows antibody staining of patient derived cell lines with histone mutations, detected by Immunofluorescence microscopy. The H3-G34R and H3-G34V antibodies were generated by Cambridge Research Biochemicals. The H3-K27M and H3 wild type antibodies are commercially available.
The Western blot below shows selective detection of purified recombinant GST-mutant specific histone proteins from the wild type sequences with antibodies, H3-G34R and H3-G34V, generated by Cambridge Research Biochemicals.
Brain tumours are a major cause of cancer related death in childhood and the anti-peptide antibodies generated by Cambridge Research Biochemicals may prove to be a useful diagnostic tool in the detection of such tumours. The mutant specific antibodies will be invaluable for furthering knowledge in the field of histone H3 mutant brain tumours. Further applications using these antibodies are underway by the Group to look at associated downstream targets and signalling pathways. For example the H3-G34R antibody has also been certified suitable for chromatin immunoprecipitation (ChIP) which will be invaluable for future investigations.
“We were delighted when Cambridge Research Biochemicals were able to help us generate and purify the mutant-specific antibodies – this was a risky project and downstream work was wholly dependent on this being successful. What really surprised us was that the production of such reagents is far from commonplace. For example hundreds of different phospho-specific antibodies have been developed, but only a handful of mutant-specific ones. The purified antibodies from Cambridge Research Biochemicals exceeded our expectations and have already provided new insights into tumour biology.” – Professor Robert Layfield