ERCC1 Monoclonal Antibody

What is ERCC1 and what role does it play in DNA repair?

ERCC1 (Excision Repair Cross-Complementing group 1) is a mammalian nucleotide excision repair (NER) enzyme encoded by the ERCC1 gene located on chromosome 19 . It plays a crucial role in repairing damaged DNA, particularly in the nucleotide excision repair pathway. ERCC1 is homologous to RAD10 in Saccharomyces cerevisiae and is required for mitotic intrachromosomal recombination and repair processes .

The protein functions by forming a heterodimer with XPF endonuclease, which is essential for the repair of DNA damage introduced by platinum compounds like cisplatin. ERCC1 specifically participates in the excision of damaged DNA segments during the repair process, making it particularly important in contexts where DNA damage occurs through crosslinking agents .

How does ERCC1 expression correlate with platinum-based chemotherapy resistance?

ERCC1 overexpression has been associated with poor prognosis after cisplatin (CDDP) treatment in various cancers, including gastric and non-small cell lung cancers . This correlation exists because high ERCC1 expression enhances the cell's ability to repair platinum-induced DNA damage, effectively neutralizing the cytotoxic effects of these chemotherapeutic agents.

Research has demonstrated that in advanced gastric cancer, elevated ERCC1 mRNA levels correlate with resistance to cisplatin and 5-fluorouracil-based therapy . Conversely, patients with tumors showing low ERCC1 expression typically demonstrate better disease control rates when treated with platinum-containing chemotherapy regimens . This relationship makes ERCC1 a valuable predictive biomarker for platinum-based chemotherapy efficacy across multiple cancer types.

What are the major ERCC1 isoforms and their functional differences?

The ERCC1 gene produces four main isoforms (201, 202, 203, and 204) through alternative splicing, with distinct functional characteristics:

• Isoform 202 has been specifically implicated in cisplatin resistance in xenograft models of lung cancer cells

• Isoform 204 notably lacks the exon 3 coding region, distinguishing it from the other major isoforms

• Isoforms 201, 202, and 203 contain exon 3 coding regions and appear to be the predominant isoforms expressed in various cancer cell lines, particularly gastric cancer cell lines

This isoform diversity is significant because different antibodies may detect specific isoforms but not others, which can affect research outcomes and clinical interpretations. For instance, the 9D11 antibody described in search result specifically detects isoforms 201, 202, and 203, but not 204.

What techniques are most effective for detecting ERCC1 expression in clinical specimens?

Multiple techniques can be employed for ERCC1 detection in clinical specimens, each with specific advantages:

• Immunohistochemistry (IHC): Most commonly used in clinical settings for its ability to directly visualize protein expression in tissue sections. The 9D11 antibody demonstrated clear nuclear staining patterns in clinical specimens of gastric cancers . Other antibodies like EP219 are also suitable for both paraffin-embedded and frozen tissues .

• Western Blotting: Provides quantitative assessment of protein expression and can distinguish between different isoforms. In the study referenced, Western blotting analysis confirmed that all seventeen gastric cancer cell lines expressed either 201, 202, and/or 203 as major isoforms of ERCC1 .

• qRT-PCR: Enables quantification of ERCC1 mRNA levels, which has shown correlation with platinum resistance in some studies. This technique was used to select stable cell lines expressing human ERCC1 in the referenced research .

• ELISA: Useful for screening of antibody specificity and sensitivity, as demonstrated in the hybridoma screening process for the 9D11 antibody .

For clinical specimens, IHC remains the most practical approach, though researchers should be aware of potential variability in antibody performance over time, as noted in the 2021 study of NSCLC patients .

How can researchers validate the specificity of ERCC1 antibodies?

Validating antibody specificity is crucial for reliable ERCC1 detection. Recommended validation approaches include:

• Expression of recombinant proteins: Test antibody specificity against various ERCC1 isoforms expressed in bacterial or human cell systems. In the referenced study, researchers used BL21 (DE3) to induce expression of ERCC1 isoforms and plasmids RC208787 and RC228204 to express the ERCC1 isoforms 201 and 203 with C-terminal Myc and FLAG tags in HeLa cells .

• Western blotting with isoform controls: Compare detection patterns against known isoform expression profiles. This allows confirmation of which isoforms the antibody can detect .

• Immunohistochemical controls: Include positive control tissues with known ERCC1 expression. Recommended control tissues include tonsil, testis, breast, prostate, fallopian tube, and various carcinomas .

• Comparison with established antibodies: Benchmark new antibodies against previously validated ones (with awareness of their limitations). The 9D11 antibody development included comparison with other ERCC1 antibodies like 4F9 and FL297 .

• Genetic knockdown/knockout validation: Confirm specificity by testing in ERCC1 knockdown or knockout models to verify signal reduction or elimination.

How do various ERCC1 monoclonal antibodies compare in terms of isoform specificity?

Different ERCC1 monoclonal antibodies exhibit varying isoform specificities, which can significantly impact experimental outcomes:

When selecting an antibody, researchers should consider which isoforms are relevant to their research question. For comprehensive detection of clinically relevant isoforms, antibodies that detect multiple isoforms containing exon 3 coding regions (like 9D11) may be preferable for most applications .

What challenges exist in standardizing ERCC1 expression evaluation for clinical use?

Several significant challenges have hindered the standardization of ERCC1 expression evaluation for clinical applications:

• Antibody specificity issues: Some antibodies, like 8F1, have demonstrated cross-reactivity with unrelated proteins, compromising result reliability .

• Changes in antibody performance over time: Research has shown that antibody performance can change, with testing done in 2012 and 2018 producing inconsistent results despite using the latest available antibodies at each time point .

• Isoform-specific functionality: Evidence suggests that only specific isoforms (particularly 202) contribute significantly to cisplatin resistance, complicating the interpretation of total ERCC1 expression data .

• Technical variability in detection methods: Different detection methodologies and scoring systems create difficulties in comparing results across studies.

• Tissue fixation and processing variables: Differences in sample preparation can affect antibody binding and signal intensity, particularly in FFPE samples.

These challenges underscore the need for standardized technologies to evaluate ERCC1 expression, as highlighted in the 2021 study of NSCLC patients . Without such standardization, the clinical utility of ERCC1 as a predictive biomarker remains limited despite its strong biological rationale.

How can researchers address contradictory findings in ERCC1 biomarker studies?

Contradictory findings regarding ERCC1 as a biomarker are not uncommon. To address these discrepancies, researchers should:

• Clearly specify antibody characteristics: Document the specific clone, manufacturer, and detection protocol used, as different antibodies may yield different results .

• Evaluate multiple isoforms: Since different isoforms may have distinct functions in platinum resistance, analyzing specific isoform expression rather than total ERCC1 may provide more consistent results .

• Implement rigorous validation: Include appropriate positive and negative controls, and validate antibody specificity through multiple methods .

• Consider mixed prognostic/predictive effects: Be aware that ERCC1 may have both prognostic value (independent of treatment) and predictive value (treatment-dependent). For example, in surgically removed NSCLC tumors that receive no further therapy, ERCC1-positive tumors have better survival than ERCC1-negative ones, despite ERCC1 positivity predicting poorer response to platinum therapy .

• Combine with other biomarkers: Integrating ERCC1 with other DNA repair markers may improve predictive accuracy compared to single-marker approaches.

• Re-validate antibodies periodically: Given evidence of changing antibody performance over time, regular re-validation is advisable for longitudinal studies .

What novel approaches show promise for improving ERCC1 detection reliability?

Emerging approaches that may enhance ERCC1 detection reliability include:

• Isoform-specific antibodies with improved validation: Development of highly specific monoclonal antibodies targeting individual ERCC1 isoforms, particularly isoform 202, which appears most relevant to platinum resistance .

• Multi-epitope detection strategies: Using antibody panels targeting different ERCC1 epitopes to improve detection accuracy and reduce false positives.

• Digital pathology and AI-assisted scoring: Automated quantification methods that could reduce inter-observer variability in IHC interpretation.

• Combination of protein and mRNA detection: Correlating protein expression with mRNA levels for more comprehensive assessment.

• Functional assays: Complementing expression analysis with functional readouts of ERCC1-dependent DNA repair capacity.

Recent efforts like the development of the 9D11 antibody demonstrate progress in creating more specific detection tools that recognize clinically relevant isoforms while maintaining clear staining patterns in clinical specimens .

How does ERCC1 expression pattern vary across different tumor types?

ERCC1 expression patterns show notable variation across cancer types, with important implications for its use as a biomarker:

• Non-small cell lung cancer (NSCLC): ERCC1 expression has been extensively studied in NSCLC, where it has demonstrated both predictive value for platinum therapy response and prognostic value independent of treatment .

• Gastric cancer: All seventeen human gastric cancer cell lines examined in one study expressed ERCC1 isoforms 201, 202, and/or 203, but not 204 . High expression has been associated with resistance to cisplatin and 5-fluorouracil-based therapy in advanced gastric cancer .

• Squamous cell carcinoma of the head: High ERCC1 expression has been linked to tumor progression in this cancer type .

• Ovarian and esophageal cancers: Both show associations between ERCC1 expression levels and tumor progression .

In clinical specimens, ERCC1 protein is typically exclusively detected in cell nuclei, with vascular endothelial cells often showing a moderate level of constant positivity that can serve as an internal control . This nuclear localization pattern is consistent across different tumor types when detected with validated antibodies.

What is the current consensus on optimal scoring methods for ERCC1 immunohistochemistry?

While no universally accepted scoring system exists for ERCC1 immunohistochemistry, several approaches have been employed:

• H-score method: Used in the 2021 NSCLC study, which revealed significantly higher ERCC1 H-scores in patients with disease progression compared to those without progression after platinum-containing chemotherapy .

• Nuclear staining assessment: Since ERCC1 is exclusively detected in nuclei of cells, scoring should focus on nuclear rather than cytoplasmic staining .

• Internal control normalization: Using the relatively constant ERCC1 expression in vascular endothelial cells as an internal reference point can help standardize scoring across specimens .

• Categorical scoring: Some studies use simpler positive/negative categorization based on percentage of stained tumor cells and staining intensity.

Researchers should select scoring methods based on their specific application while acknowledging the limitations of current approaches. The inconsistency in results between 2012 and 2018 evaluation in the NSCLC study highlights the need for standardized evaluation technology .

What controls should be included when using ERCC1 antibodies in experimental systems?

Robust control strategies for ERCC1 antibody experiments should include:

• Positive tissue controls: Include tissues known to express ERCC1, such as tonsil, testis, breast, prostate, and fallopian tube .

• Cellular internal controls: Vascular endothelial cells typically show moderate, constant ERCC1 positivity and can serve as internal controls in tissue sections .

• Isoform expression controls: When testing isoform specificity, include cells transfected with plasmids expressing specific ERCC1 isoforms, as demonstrated with plasmids RC208787 and RC228204 for isoforms 201 and 203 .

• Overexpression systems: Stable cell lines overexpressing ERCC1, such as the MKN45 cells transfected with pCDNA3 Hs-ERCC1 described in the literature, provide useful positive controls .

• Negative controls: Include antibody diluent without primary antibody to detect non-specific binding of secondary detection systems.

• Cross-reactivity controls: If available, include samples from ERCC1 knockout models to confirm antibody specificity.

Proper implementation of these controls helps ensure the reliability and reproducibility of ERCC1 detection across different experimental conditions.

How can researchers optimize ERCC1 immunohistochemistry protocols for different sample types?

Optimizing ERCC1 immunohistochemistry requires tailored approaches for different sample types:

• Formalin-fixed paraffin-embedded (FFPE) tissues:

  • Ensure optimal antigen retrieval, as ERCC1 epitopes may be masked during fixation

  • Test multiple antibody dilutions to determine optimal signal-to-noise ratio

  • Consider using amplification systems for low-expressing samples

  • Validate staining patterns against known positive controls

• Frozen tissues:

  • Adjust fixation protocols (typically briefer than for FFPE)

  • Optimize antibody concentration, which may differ from FFPE protocols

  • Control background staining, which can be more problematic in frozen sections

• Cell lines:

  • For immunocytochemistry, standardize fixation methods (typically paraformaldehyde-based)

  • Consider permeabilization requirements for nuclear antigen access

  • Include both high and low ERCC1-expressing cell lines as controls

• Tissue microarrays (TMAs):

  • Ensure inclusion of appropriate control tissues in each TMA

  • Consider potential heterogeneity in ERCC1 expression when interpreting TMA results

For all sample types, it's critical to validate that the selected antibody maintains its specificity and sensitivity in the specific application and fixation conditions being used.

These optimized protocols can significantly improve the reliability and reproducibility of ERCC1 detection across diverse research and clinical applications.