IDH1 Antibody

What is IDH1 and why is it significant in glioma diagnosis?

Isocitrate dehydrogenase 1 (IDH1) is a cytosolic enzyme that has become central to the integrated diagnosis and classification of diffuse gliomas. The significance of IDH1 emerged over the past decade when mutations in the IDH1 gene were established as key molecular markers in gliomagenesis. Approximately 90% of IDH mutant gliomas carry a specific G-to-A mutation at position 395 of the IDH1 gene, resulting in an R132H mutant protein where arginine is replaced by histidine . This mutation has profound diagnostic, prognostic, and therapeutic implications in neuro-oncology.

The current WHO classification system for central nervous system tumors incorporates IDH mutation status as a fundamental parameter in an integrated diagnostic approach that includes: (1) traditional histopathology to determine cell lineage and grade, (2) IDH gene status, and (3) additional molecular features such as 1p/19q codeletion, ATRX loss, and TP53 mutations . The integration of IDH1 status has significantly improved diagnostic reproducibility and provided more accurate prognostic information for patients with gliomas.

How do IDH1 antibodies distinguish between wild-type and R132H mutant proteins?

IDH1 antibodies used in research and diagnostics fall into two categories: those that detect all forms of IDH1 (wild-type and mutant) and those specifically designed to recognize only the R132H mutant protein.

Mutant-specific antibodies such as MRQ-67 and H09 are engineered to recognize the conformational change resulting from the single amino acid substitution at position 132. These antibodies bind selectively to the R132H mutant epitope with minimal cross-reactivity to wild-type IDH1. The specificity of these antibodies can be demonstrated through various validation approaches:

• Enzyme-linked immunosorbent assays (ELISA) confirm selective binding to the R132H mutant protein

• Western blot analyses show specific detection of the mutant protein in IDH1 R132H-expressing cell lines but not in wild-type IDH1 cells

• Dot immunoassays demonstrate higher binding capacity to the mutant protein compared to wild-type

For detecting total IDH1 expression (both wild-type and mutant forms), non-specific IDH1 antibodies like the rabbit polyclonal IDH1 antibody or mouse monoclonal antibody 66197-1-Ig are employed .

What are the primary applications of IDH1 antibodies in research laboratories?

IDH1 antibodies have multiple research applications, with specific protocols optimized for different experimental purposes:

IDH1 R132H antibodies are particularly valuable for:

• Screening glioma samples for the presence of the R132H mutation

• Distinguishing diffuse gliomas from histological mimics like reactive gliosis and pilocytic astrocytoma

• Identifying infiltrating tumor cells at surgical margins

• Distinguishing between primary and secondary glioblastomas

What cell lines are typically used for validating IDH1 antibody specificity?

Several established cell lines are used for validating IDH1 antibody specificity, each serving different validation purposes:

For wild-type IDH1 detection:

• HepG2 human hepatocellular carcinoma cell line: Expresses wild-type IDH1 and has been well-characterized for IDH1 protein expression

• HeLa human cervical epithelial carcinoma cell line: Used as a standard control for wild-type IDH1 expression

• NIH-3T3 mouse embryonic fibroblast cell line: Validates cross-reactivity with mouse IDH1

• Rat-2 rat embryonic fibroblast cell line: Tests cross-reactivity with rat IDH1

For IDH1 R132H mutant detection:

• Genetically modified cell lines expressing the R132H mutant protein

• Patient-derived glioma cell lines harboring the naturally occurring R132H mutation

• HeLa IDH1 knockout cell lines (negative control): Used to confirm antibody specificity by showing absence of staining

The Western blot detection of IDH1 typically shows a specific band at approximately 46 kDa, which is absent in knockout cell lines, confirming antibody specificity .

What tissues typically express IDH1, and how is this relevant for experimental controls?

IDH1 expression patterns in normal tissues provide important reference points for experimental controls:

Human tissues with documented IDH1 expression:

• Brain (cortex): Particularly in astrocytes, as demonstrated by IHC staining

• Liver: Shows consistent IDH1 expression, making liver cancer tissues useful positive controls

• SK-BR-3 human breast cancer cell line: Shows cytoplasmic localization of IDH1

Tissue-specific localization:

• In brain tissue, IDH1 is primarily localized in astrocytes and glial cells

• Subcellular localization is predominantly cytoplasmic, consistent with IDH1's role in cellular metabolism

• In rat brain, IDH1 staining is specifically localized to glial cell cytoplasm

When designing experiments, appropriate positive and negative tissue controls should be included to validate staining patterns and antibody specificity.

How do different IDH1 R132H mutant-specific antibody clones compare in terms of performance?

Several monoclonal antibodies have been developed for specific detection of the IDH1 R132H mutant, with varying performance characteristics:

Comparison of widely used IDH1 R132H antibody clones:

Other less frequently used clones include IMab-1, HMab-1, HMab-2, and IHC132, each with specific performance characteristics that may be advantageous for particular experimental designs .

What are the optimal protocols for IDH1 R132H immunohistochemistry in FFPE glioma samples?

Successful immunohistochemical detection of IDH1 R132H in FFPE glioma samples requires careful attention to technical details:

Recommended IHC protocol for IDH1 R132H detection:

• Tissue preparation:

  • 4% formalin fixation followed by paraffin embedding

  • 4-5 μm section thickness is optimal

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) is essential

  • Options include:

    • TE buffer pH 9.0 (preferred for 66197-1-Ig)

    • Citrate buffer pH 6.0 (alternative)

    • Antigen Retrieval Reagent-Basic (as used with MAB7049)

• Primary antibody incubation:

  • MRQ-67 or H09: 1-2 μg/mL concentration

  • Incubation overnight at 4°C yields optimal results

  • For MAB7049: 15 μg/mL overnight at 4°C

• Detection systems:

  • For brightfield microscopy: HRP-DAB detection systems (e.g., Anti-Mouse HRP-DAB Cell & Tissue Staining Kit)

  • For fluorescence detection: Fluorophore-conjugated secondary antibodies (e.g., NorthernLights™ 557-conjugated Anti-Mouse/Rabbit IgG)

• Counterstaining:

  • Hematoxylin for brightfield microscopy

  • DAPI for fluorescence microscopy

This protocol has demonstrated reliable performance in detecting IDH1 R132H in diffuse astrocytomas, oligodendrogliomas, and secondary glioblastomas, while consistently showing negative results in primary glioblastomas .

How can researchers troubleshoot false negative or high background staining issues with IDH1 R132H antibodies?

Several factors can contribute to false negative results or high background staining when using IDH1 R132H antibodies:

Common causes of false negative results:

• Inadequate antigen retrieval: Insufficient heat-induced epitope retrieval can mask the R132H epitope

• Prolonged fixation: Over-fixation with formalin can cross-link proteins and obscure antibody binding sites

• Tissue processing artifacts: Freezing/thawing procedures can affect antibody sensitivity, particularly with the H09 clone

• Low mutant protein expression: Some low-grade gliomas may express low levels of the mutant protein

• Alternative IDH mutations: R132H-specific antibodies will not detect other IDH1 mutations (R132C, R132G, R132S, R132L) or IDH2 mutations

Strategies for resolving false negatives:

• Optimize antigen retrieval conditions (temperature, duration, pH)

• Test alternative IDH1 R132H antibody clones

• Perform DNA sequencing to confirm mutation status

• Consider using a more sensitive detection system

Causes of high background staining:

• Insufficient blocking

• High antibody concentration

• Cross-reactivity with other proteins

• Endogenous peroxidase activity

• Necrotic tissue areas

Approaches to reduce background:

• Increase blocking time/concentration

• Optimize antibody dilution

• Try alternative antibody clones (e.g., MRQ-67 shows less background than H09)

• Include proper quenching of endogenous peroxidase activity

• Implement more stringent washing protocols

When troubleshooting, it's advisable to include known positive and negative controls to differentiate between technical issues and true biological findings.

What is the correlation between IDH1 R132H immunohistochemistry and molecular sequencing results?

The correlation between IDH1 R132H immunohistochemistry and DNA sequencing is an essential consideration for diagnostic accuracy:

Correlation findings:

• Studies comparing IHC with sequencing show high concordance for R132H detection

• In the validation study for MRQ-67, all IHC-positive cases (5/5) were confirmed by sequencing to harbor the R132H mutation

• All IHC-negative cases (13/13) were confirmed by sequencing to have wild-type IDH1

Important considerations:

• IHC cannot detect non-R132H mutations in IDH1 or any IDH2 mutations

• False negatives can occur due to technical factors or low expression levels

• Rare instances of false positives have been reported, particularly with older antibody clones

Recommended diagnostic algorithm:

• Initial screening with IDH1 R132H IHC

• Sequencing for IHC-negative cases in clinical/radiological contexts suggestive of diffuse glioma

• Consider targeted sequencing panels that include both IDH1 and IDH2 for comprehensive mutation detection

This stepwise approach is cost-effective while maintaining diagnostic accuracy, as approximately 90% of IDH-mutant gliomas harbor the R132H mutation detectable by IHC .

How can IDH1 antibodies be utilized to identify infiltrating tumor cells at surgical margins?

The detection of infiltrating tumor cells at surgical margins represents a significant challenge in neuro-oncology and a valuable application of IDH1 R132H immunohistochemistry:

Methodological approach:

• Sample collection: Obtain tissue samples from apparent tumor margins and adjacent "normal" brain tissue

• IHC protocol: Apply standard IDH1 R132H IHC protocol with particular attention to:

  • Optimal fixation and processing

  • Enhanced antigen retrieval

  • Sensitive detection systems

• Microscopic evaluation: Systematic examination at low and high magnification

• Interpretation: Single R132H-positive cells in apparently normal brain tissue represent infiltrating tumor cells

Clinical significance:

• IDH1 R132H IHC can detect cryptically infiltrating tumor cells in apparently uninvolved brain tissues at surgical margins

• This application is particularly valuable for low-grade diffuse gliomas, where infiltrating cells may be morphologically indistinguishable from normal brain cells

• Positive staining of isolated cells at margins has prognostic implications and may influence post-surgical treatment decisions

Technical considerations:

• Use of highly sensitive detection systems is crucial for identifying isolated positive cells

• Double immunostaining with glial markers can further characterize infiltrating cells

• Background staining must be minimized to avoid false-positive interpretations

• Comparison with intraoperative frozen sections can provide valuable correlation

This application of IDH1 R132H IHC represents an important advancement in the surgical management of diffuse gliomas by providing more accurate assessment of tumor extent beyond what is visible by conventional histopathology.

How can IDH1 antibodies be applied in differentiating glioma from non-neoplastic conditions?

Distinguishing low-grade diffuse gliomas from reactive gliosis represents a significant diagnostic challenge that can be addressed using IDH1 R132H immunohistochemistry:

Methodological approach:

• Perform IDH1 R132H IHC on questionable lesions using standardized protocols

• Compare with appropriate positive and negative controls

• Interpret in conjunction with conventional histology and additional immunomarkers

Interpretative guidelines:

• Positive R132H staining strongly supports a neoplastic diagnosis of diffuse glioma

• Staining pattern helps distinguish between:

  • Diffuse infiltrative IDH1-mutant gliomas (positive)

  • Reactive gliosis (negative)

  • Pilocytic astrocytoma (negative)

  • Primary glioblastoma (generally negative)

This application helps resolve diagnostically challenging cases where morphological features are ambiguous or sample size is limited. The presence of IDH1 R132H mutation is highly specific for diffuse glioma and can be a decisive diagnostic marker in appropriate clinical contexts .

What are the comparative performance characteristics of different detection systems for IDH1 R132H immunohistochemistry?

The choice of detection system significantly impacts the sensitivity and specificity of IDH1 R132H immunohistochemistry:

Comparison of detection methods:

Specific systems validated with IDH1 R132H antibodies:

• Anti-Mouse HRP-DAB Cell & Tissue Staining Kit: Validated with MAB7049 (15 μg/mL)

• NorthernLights™ 557-conjugated secondary antibodies: Effective for fluorescence detection with MAB7049

The selection of detection system should be guided by the specific research question, with amplification-based systems preferred for applications requiring the highest sensitivity, such as detecting isolated infiltrating tumor cells.

How do pre-analytical variables affect IDH1 R132H immunohistochemistry results?

Pre-analytical variables significantly impact the quality and reliability of IDH1 R132H immunohistochemistry:

Critical pre-analytical factors:

• Fixation variables:

  • Type of fixative: 10% neutral-buffered formalin is optimal

  • Fixation duration: 24-48 hours recommended; overfixation reduces sensitivity

  • Fixation delay: Immediate fixation preserves antigenicity

• Tissue processing:

  • Paraffin type and embedding temperature affect epitope preservation

  • Section thickness (4-5 μm optimal)

  • Section storage conditions and age

• Decalcification:

  • Necessary for bone-infiltrating tumors

  • EDTA-based decalcification preferred over acid-based methods

• Frozen tissue considerations:

  • Freezing/thawing can reduce immunoreactivity with some antibody clones, particularly H09

  • Flash freezing in liquid nitrogen preferable to slow freezing

To minimize pre-analytical variability, laboratories should establish standardized protocols for tissue handling, fixation, and processing, with particular attention to fixation time and conditions for optimal IDH1 R132H detection.

What are the optimal dilution ranges for different IDH1 antibodies across various applications?

Proper antibody dilution is critical for achieving optimal signal-to-noise ratio in different applications:

Recommended dilutions for IDH1 antibody (66197-1-Ig):

Recommendations for MAB7049 (anti-IDH1):

• Western blot: 0.25 μg/mL under reducing conditions

• ICC: 10 μg/mL for 3 hours at room temperature

• IHC (FFPE): 15 μg/mL overnight at 4°C

• IHC (frozen): 15 μg/mL overnight at 4°C

It is critical to optimize antibody concentration for each specific application and sample type. Titration experiments should be performed to determine the optimal dilution that provides specific staining with minimal background.

What validation studies confirm the specificity of IDH1 R132H antibodies?

Rigorous validation studies are essential to confirm antibody specificity for IDH1 R132H:

Validated specificity tests for IDH1 R132H antibodies:

• Enzyme-linked immunosorbent assay (ELISA):

  • Demonstrates selective binding to R132H mutant peptide

  • MRQ-67 shows higher affinity than H09

• Western blot validation:

  • Testing against cell lines with known IDH1 status

  • The use of IDH1 knockout cell lines as negative controls

  • For MAB7049: Testing against HeLa parental and IDH1 knockout lines shows specific band at 46 kDa in parental line only

• Dot immunoassay:

  • MRQ-67 binds specifically to IDH1 R132H with higher capacity than H09

• DNA sequencing correlation:

  • All IHC-positive cases (5/5) confirmed by sequencing to have R132H mutation

  • All IHC-negative cases (13/13) confirmed to have wild-type IDH1

• Tissue microarray testing:

  • Systematic evaluation across multiple glioma subtypes

  • Positive in diffuse astrocytomas (16/22), oligodendrogliomas (9/15), and secondary glioblastomas (3/3)

  • Negative in primary glioblastomas (0/24)

These validation approaches collectively confirm the specificity of IDH1 R132H antibodies and establish their reliability for diagnostic and research applications.

How can IDH1 antibodies contribute to research on treatment response monitoring?

IDH1 antibodies offer promising applications in monitoring treatment responses and disease progression:

Research applications in treatment monitoring:

• Residual disease detection:

  • IDH1 R132H IHC can detect minimal residual disease not visible by conventional imaging

  • Application in post-treatment biopsy specimens to assess treatment efficacy

• Liquid biopsy correlation:

  • Potential integration with circulating tumor DNA (ctDNA) detection of IDH1 mutations

  • Correlation between tissue IHC and liquid biopsy findings may enhance monitoring precision

• Therapy-induced changes:

  • Monitoring changes in IDH1 R132H expression patterns following targeted therapies

  • Evaluation of potential mechanisms of treatment resistance

• Experimental therapeutic contexts:

  • IDH1 inhibitors are being developed as targeted therapeutics

  • IDH1 R132H IHC may serve as a companion diagnostic for patient selection

  • Potential for monitoring changes in mutant protein expression with treatment

As targeted therapies for IDH-mutant gliomas continue to develop, IDH1 R132H antibodies will likely play an increasingly important role in treatment response assessment and patient monitoring.

What are the technical considerations for multiplexed immunofluorescence including IDH1 antibodies?

Multiplexed immunofluorescence incorporating IDH1 antibodies enables simultaneous analysis of multiple biomarkers:

Technical considerations for multiplexed approaches:

• Antibody compatibility:

  • Selection of primary antibodies from different host species

  • For example, mouse anti-IDH1 (MAB7049) can be combined with rabbit antibodies against other markers

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • NorthernLights™ 557-conjugated secondary antibodies have been validated with IDH1 antibodies

• Sequential staining approaches:

  • For same-species antibodies, sequential staining with intermediate blocking steps

  • Tyramide signal amplification (TSA) enables use of same-species antibodies

• Counterstaining:

  • DAPI nuclear counterstain provides cellular context

  • Autofluorescence quenching may be necessary, especially in brain tissue

• Validated marker combinations:

  • IDH1 R132H + ATRX: Helps distinguish astrocytomas from oligodendrogliomas

  • IDH1 R132H + Ki-67: Assesses proliferation in mutant cells

  • IDH1 R132H + GFAP/Olig2: Confirms glial lineage of mutant cells

Multiplexed approaches provide richer biological context and enable more sophisticated analyses of tumor heterogeneity, microenvironment interactions, and cellular phenotypes in IDH1-mutant gliomas.