Gliomas are the most frequent type of malignant brain tumor in adults, occurring in over 80% of cases.1 The enzyme isocitrate dehydrogenase, (IDH) plays a crucial role in regular metabolic processes and maintaining homeostasis. IDH mutations however, are characteristic of a specific group of diffuse gliomas.2 In particular, mutation of IDH1 is a key indicator for patient survival. Patients with secondary glioblastomas containing the IDH1(R132H) mutation have more favorable survival outcomes compared to those with primary glioblastomas carrying the wild- type(WT) IDH1 gene.3

Therefore, differentiation between WT and mutant IDH1(R132H) is critically important in proper diagnosis and treatment of glioblastomas. Compared to sequencing, the use of immunohistochemistry to identify IDH1(R132H) is faster, more cost effective and results in greater accuracy.4 Hence, validation of IHC antibodies specific for the mutant IDH1(R132H) is critically important, both for ongoing research and the development of diagnostic tests.

Identification of an IDH1(R132H) – specific antibody

OriGene has developed a tool for antibody validation called CytoSections. CytoSections are FFPE sections of transiently transfected cDNA specific gene targets over-expressed in mammalian cells. They are a cost-effective, easily renewable and unlimited source of controls for validating mutant-specific antibodies. Below, we describe how OriGene used CytoSections to validate an antibody specific to the key IDH1(R132H) mutation, for use in IHC assays.

CytoSections either over-expressing IDH1 or mutant IDH1( R132H), in addition to untransfected CytoSections, were used to screen two different mutant-specific antibodies against IDH1(R132H). Results are shown below.

CytoSections

Use of the DDK antibody (TA592569), resulted in positive (brown) staining, in WT (IDH1) and mutant (IDH1(R132H) CytoSections (1A and 1B) respectively. This demonstrates that the CytoSections are expressing either the DDK-tagged IDH1 WT construct, or the mutant construct, as expected. There is no positive staining in the untransfected negative control (1C). WT IDH1 antibody (TA800406) detected both WT (2A) and mutant IDH1(R132H) (2B) while the untransfected control (2C) is negative.

IDH1(R132H) Ab_1, (TA190113) results in positive staining using the CytoSection expressing IDH1(R132H) (3B). No positive staining is detected in the CytoSection expressing WT IDH1 (3A). This result shows that IDH1(R132H) Ab_1, (TA190113), is specific for the IDH1(R132H) mutation, since WT IDH1 is not detected.

A second antibody against mutant IDH1(R132H) Ab_2 (TA190164) was weakly positive using the mutant CytoSection. Untransfected CytoSection controls (3C and 4C) are negative.

Summary

A mutant-specific antibody (TA190113), was identified, which shows a strong signal and no cross-reactivity with WT IDH1. This antibody is a candidate for specific detection of IDH1(R132H) in glioma tissue samples. This study demonstrates the use of CytoSections as a tool to validate mutant-specific antibodies for IHC applications. Contact us to learn how OriGene can assist with antibody validation for your IHC assays.

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References

1. Dekker LJM, Verheul C, Wensveen N, Leenders W, Lamfers MLM, Leenstra S, Luider Effects of the IDH1 R132H Mutation on the Energy Metabolism: A Comparison between Tissue and Corresponding Primary Glioma Cell Cultures. ACS Omega. 2022 Jan 19;7(4):3568-3578. doi: 10.1021/acsomega.1c06121. PMID: 35128264; PMCID: PMC8811756.

2. Sharma N, Mallela AN, Shi DD, Tang LW, Abou-Al-Shaar H, Gersey ZC, Zhang X, McBrayer SK, Abdullah Isocitrate dehydrogenase mutations in gliomas: A review of current understanding and trials. Neurooncol Adv. 2023 May 10;5(1):vdad053. doi: 10.1093/noajnl/vdad053. PMID: 37287696; PMCID: PMC10243983.

3. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic- Haberle I, Jones S, Riggins GJ, Friedman H, Friedman A, Reardon D, Herndon J, Kinzler KW, Velculescu VE, Vogelstein B, Bigner IDH1 and IDH2 mutations in gliomas. N Engl J Med. 2009 Feb 19;360(8):765-73. doi: 10.1056/NEJMoa0808710. PMID: 19228619; PMCID: PMC2820383.

4. IDH1/2 Mutations in Glioma: ESMO Biomarker European Society for Medical Oncology. Updated Nov 23 2016. Available at: https://oncologypro.esmo.org/education-library/factsheets-on- biomarkers/idh1-2-mutations-in-glioma.