ECH1 Antibody

What is ECH1 and what is its biological significance?

ECH1, or Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, is a mitochondrial enzyme that functions in the auxiliary step of the fatty acid β-oxidation pathway . Specifically, it catalyzes the isomerization of 3-trans,5-cis-dienoyl-CoA to 2-trans,4-trans-dienoyl-CoA . This process is critical for the metabolism of unsaturated fatty acids that contain odd-numbered double bonds. ECH1 is widely expressed across multiple tissue types with particularly high expression in metabolically active tissues such as liver, heart, and muscle . Recent research has indicated ECH1's potential role in cancer development, particularly in lung cancer, where it can elicit autoimmune responses that may serve as diagnostic biomarkers .

What applications are ECH1 antibodies commonly used for in research?

ECH1 antibodies are employed in multiple experimental techniques, primarily:

• Western Blotting (WB): For detecting ECH1 protein expression levels in cell and tissue lysates, with expected band sizes of approximately 35-36 kDa

• Immunocytochemistry/Immunofluorescence (ICC/IF): For visualizing the subcellular localization of ECH1, predominantly in mitochondria

• ELISA: For detection of autoantibodies against ECH1 in serum samples, particularly in cancer research

Researchers should select antibodies validated for their specific application and species of interest. For instance, certain commercially available antibodies (e.g., ab153720) have been validated for human and mouse samples in WB and ICC/IF applications .

How should researchers optimize Western blot protocols for ECH1 detection?

When optimizing Western blot protocols for ECH1 detection, researchers should consider:

• Sample preparation: Use appropriate lysis buffers that effectively solubilize mitochondrial proteins

• Protein loading: 30 μg of total protein is typically sufficient for detection in cell extracts

• Gel percentage: 10% SDS-PAGE provides optimal separation for the 35-36 kDa ECH1 protein

• Antibody dilution: Start with manufacturer recommendations (e.g., 1:1000 to 1:2000 dilution for certain antibodies)

• Controls: Include both positive controls (tissues known to express ECH1) and negative controls (ECH1 knockout samples when available)

Western blot example data:

What validation steps should be taken to confirm ECH1 antibody specificity?

Antibody specificity validation is critical for reliable results. Researchers should:

• Compare results using multiple antibodies targeting different epitopes of ECH1

• Include knockout or knockdown controls when possible (e.g., ECH1 knockout 293T cell extracts)

• Perform peptide competition assays to confirm binding specificity

• Check for cross-reactivity with related proteins, particularly other hydratase/isomerase family members

• Validate antibody performance in the specific experimental system and conditions being used

How can ECH1 autoantibodies be utilized as biomarkers for early lung cancer detection?

ECH1 autoantibodies have shown promising potential as biomarkers for early lung cancer detection. In serological proteome analysis (SERPA) studies, autoantibodies against ECH1 have demonstrated significant diagnostic value:

• Discriminating lung cancer from normal individuals: AUC of 0.799 with sensitivity of 62.2% and specificity of 95.5%

• Distinguishing early-stage lung cancer from matched normal controls: AUC of 0.763 with sensitivity of 60.0% and specificity of 89.3%

• Early detection potential: Elevated autoantibody levels could be detected more than 2 years before clinical diagnosis of lung cancer

Researchers investigating this application should consider that ECH1 autoantibodies show a negative correlation with tumor size (rs = −0.256, p = 0.023), suggesting their potential utility for detection of smaller tumors in early stages . This makes them particularly valuable for screening high-risk populations before conventional imaging methods can detect tumors.

What methodological considerations should be addressed when using ELISA to detect ECH1 autoantibodies?

When developing ELISA protocols for ECH1 autoantibody detection, researchers should consider:

• Antigen preparation:

  • Use high-quality recombinant ECH1 protein (commercially available or lab-produced)

  • Optimal coating concentration: 0.5 μg/mL in PBS

  • Coating volume and time: Typically 100 μL per well, overnight at 4°C

• Sample handling:

  • Optimal serum dilution: 1:200 in blocking buffer

  • Include appropriate controls (healthy individuals, other disease states)

  • Consider using longitudinal samples if studying temporal changes

• Detection system:

  • Secondary antibody: Horseradish peroxidase-conjugated anti-human IgG at 1:4,000 dilution

  • Substrate: 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) with absorbance measured at 405 nm

  • Run samples in duplicate and use average OD values for analysis

• Data analysis:

  • Establish appropriate cutoff values using ROC analysis

  • Consider combining with other autoantibody markers for improved sensitivity

What is the relationship between ECH1 autoantibody levels and clinical parameters in cancer patients?

Research has revealed several significant correlations between ECH1 autoantibody levels and clinical parameters in lung cancer:

These correlations suggest ECH1 autoantibodies may be particularly valuable for early detection rather than monitoring disease progression or treatment response.

How does ECH1 contribute to cancer development and progression?

While the complete mechanistic role of ECH1 in cancer is still being elucidated, several findings provide insights:

• Metabolic reprogramming:

  • As an enzyme involved in fatty acid β-oxidation, ECH1 may contribute to altered energy metabolism in cancer cells

  • Changes in fatty acid oxidation can affect cancer cell survival and proliferation

• Tissue-specific effects:

  • In gastric cancer, higher ECH1 expression has been associated with lymph node metastasis, suggesting it may be a critical factor in metastatic development

  • In B-cell chronic lymphoid leukemia, downregulation of ECH1 has been linked to DNA damage-induced apoptosis resistance

  • Abnormal ECH1 expression has been associated with hepatocellular carcinoma secondary to hepatitis C virus infection

• Autoimmune response:

  • The presence of autoantibodies suggests ECH1 may be aberrantly expressed, modified, or localized in cancer cells

  • This altered presentation likely triggers immune recognition and autoantibody production

Understanding these mechanisms can help researchers develop more targeted approaches for using ECH1 in cancer diagnostics and potentially therapeutics.

What are the current limitations and challenges in ECH1 autoantibody research?

Researchers working with ECH1 autoantibodies should be aware of several limitations:

• Temporal variability:

  • Autoantibody positivity can change over time in the pre-diagnostic phase

  • Understanding the dynamics of these changes requires longitudinal sample analysis

• Specificity challenges:

  • While specificity for lung cancer vs. normal controls is high (95.5%), further research is needed to establish specificity against other cancer types and inflammatory conditions

• Combination approaches:

  • Single autoantibody markers may have limited sensitivity

  • Research suggests combining ECH1 with other autoantibody markers (like HNRNPA2B1) and conventional screening methods (low-dose CT) may provide optimal detection approaches

• Standardization issues:

  • Variability in ELISA protocols, recombinant protein quality, and cutoff determinations can affect reproducibility

  • Standardized methodologies are needed for clinical application

How might ECH1 antibodies contribute to emerging cancer detection technologies?

Several promising research directions for ECH1 antibodies in cancer diagnostics include:

• Multiplex autoantibody panels:

  • Combining ECH1 with other autoantibody markers (e.g., HNRNPA2B1, which showed an AUC of 0.874 with sensitivity of 72.2% and specificity of 95.5%)

  • Integration with existing panels like the early CDT-lung assay, which includes six different autoantibodies

• Point-of-care testing development:

  • Adaptation of laboratory ELISA protocols to rapid testing formats

  • Development of microfluidic or lateral flow assays for clinical implementation

• Liquid biopsy integration:

  • Combining autoantibody detection with circulating tumor DNA and other blood-based biomarkers

  • Creating comprehensive multi-analyte screening approaches

What research gaps remain in understanding ECH1's role in normal physiology and disease?

Despite progress in ECH1 research, several knowledge gaps remain:

• Regulatory mechanisms:

  • How ECH1 expression is regulated in different tissues and disease states

  • Post-translational modifications that might affect function or immunogenicity

• Structure-function relationships:

  • Detailed understanding of how ECH1 structure relates to its enzymatic activity

  • Identification of critical epitopes recognized by autoantibodies

• Therapeutic potential:

  • Whether ECH1 could serve as a therapeutic target in cancers where it plays a functional role

  • Development of methods to modulate ECH1 activity or expression

• Population differences:

  • Variations in ECH1 autoantibody prevalence across different ethnic groups

  • Genetic factors that might influence autoimmune responses to ECH1