ENHO Antibody

What is the ENHO gene and its encoded protein adropin?

ENHO (Energy Homeostasis Associated) is a gene that encodes adropin, a protein implicated in the maintenance of energy homeostasis and insulin resistance. Adropin expression is regulated by energy status and dietary nutrient content and is altered in obesity. The protein regulates the expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor gamma (PPAR-gamma). Research has shown that adropin levels increase with dietary fat content. Additionally, adropin has been proposed to play a role in the regulation of endothelial function .

What applications are ENHO antibodies validated for?

ENHO antibodies are validated for multiple applications depending on the specific antibody. Common applications include:

Most commercially available ENHO antibodies are rabbit polyclonal antibodies with reactivity against human, mouse, and/or rat proteins .

What is the difference between polyclonal and monoclonal ENHO antibodies?

Polyclonal ENHO antibodies:

• Recognize multiple epitopes on the adropin protein

• Typically derived from rabbit serum after immunization with ENHO peptides

• Offer higher sensitivity due to binding multiple epitopes

• Show greater batch-to-batch variation

• Most commercially available ENHO antibodies are polyclonal

Monoclonal ENHO antibodies:

• Recognize a single epitope

• Produced from a single B-cell clone

• Provide higher specificity and reproducibility

• May have lower sensitivity than polyclonal antibodies

• Less commonly available for ENHO compared to polyclonal options

The choice between polyclonal and monoclonal depends on the experimental requirements, with polyclonals offering higher sensitivity and monoclonals providing better specificity.

How should I design experiments to validate ENHO antibody specificity?

A robust experimental design for validating ENHO antibody specificity should include:

• Positive and negative controls:

  • Positive: Tissues or cells known to express ENHO (e.g., astrocytes, which show high ENHO expression)

  • Negative: ENHO knockout tissues/cells or tissues with known low expression

• Multiple techniques validation:

  • Confirm specificity across at least two different techniques (e.g., WB and IHC)

  • Compare results with alternative antibodies targeting different epitopes

• Peptide competition assay:

  • Pre-incubate antibody with the immunizing peptide

  • Loss of signal confirms epitope-specific binding

• Cross-reactivity testing:

  • Test across intended species (human, mouse, rat as applicable)

  • Validate against related proteins to confirm specificity

• Quantitative validation:

  • Compare staining/signal intensity with known ENHO expression levels

  • Use siRNA knockdown to confirm signal reduction correlates with expression

This multi-technique approach is essential as studies have found varying sensitivities and specificities for antibodies, including recently developed rabbit monoclonal antibodies .

What experimental controls should be included when using ENHO antibodies in multicolor flow cytometry?

When designing multicolor flow cytometry experiments with ENHO antibodies, proper controls are essential for accurate interpretation:

• Single-color compensation controls:

  • Run single-color BD compensation control tubes for each antibody

  • Use compensation beads for antibody fluorophores

• Fluorescence Minus One (FMO) controls:

  • Include tubes containing all fluorochromes except the one conjugated to ENHO antibody

  • Essential for accurate gating and identifying true positive populations

• Isotype controls:

  • Include matching isotype control antibodies with the same fluorophore

  • Ensure the fluorophore-to-protein (F/P) ratio matches the test antibody

  • Purchase from the same company as the ENHO antibody for consistency

• Blocking controls:

  • Pre-incubate cells with unconjugated blocking antibody to prevent non-specific binding

  • Important when measuring activation markers

• Live/dead discrimination:

  • Include viability dye to exclude dead cells that may bind antibodies non-specifically

Example of FMO control design for a 4-color panel including ENHO:

• Tube 1: All antibodies except ENHO

• Tube 2: Complete panel with ENHO

• Tube 3: Complete panel with isotype control replacing ENHO

This approach allows for proper compensation and accurate identification of positive populations .

How can ENHO antibodies be used to study the relationship between adropin and neurological conditions?

ENHO/adropin's expression in the nervous system has significant implications for neurological research. When designing studies to investigate this relationship:

• Cell type-specific expression analysis:

  • Single-nucleus RNA sequencing (snRNA-seq) has revealed that astrocytes are a major site of ENHO expression in the human nervous system

  • Oligodendrocyte precursor cells (OPCs) also exhibit comparable levels of ENHO expression

  • Certain neuronal populations (e.g., Pax6+ve and Vip+ve neurons) show ENHO expression but at lower levels than astrocytes

• Age-related expression patterns:

  • ENHO expression appears to decline with aging in the brain

  • In astrocytes isolated from human donors, ENHO expression levels decrease with donor age

  • This decline may reflect a general reduction of metabolic processes and cellular activity in astrocytes

• Experimental approaches:

  • Use ENHO antibodies for immunohistochemistry to map expression in different brain regions

  • Combine with other cell-type markers to confirm cell-specific expression

  • Correlate ENHO protein levels with cognitive assessments in aging studies

  • Investigate the potential role of inflammation on ENHO expression, as adropin expression is suppressed by inflammatory factors like TNFα and Poly I:C

• Methodological considerations:

  • When comparing young vs. aged samples, consistent antibody batches and standardized protocols are essential

  • Quantitative analysis should include normalization to appropriate housekeeping proteins

  • Consider the influence of post-translational modifications on antibody detection

What methodological approaches should be used when employing ENHO antibodies in biomarker studies?

Recent research has identified ENHO as a potential biomarker in systemic sclerosis (SSc) . When designing biomarker studies:

• Diagnostic model development:

  • SSc research identified ENHO and NOX4 as novel biomarkers using machine learning approaches (LASSO regression and SVM)

  • ENHO was found to be down-regulated in skin of SSc patients

  • Expression differences were more pronounced in patients with diffuse cutaneous SSc than in those with limited cutaneous SSc

• Cohort validation strategy:

  • Perform initial discovery in one dataset (e.g., GSE130955)

  • Validate in multiple independent cohorts

  • Confirm with your own study cohort using ENHO antibodies

  • Quantify expression differences between disease and control samples

• Antibody selection considerations:

  • Choose antibodies validated for the specific application (IHC for tissue studies, ELISA for serum studies)

  • Consider epitope location relative to known functional domains of adropin

  • Select antibodies with demonstrated specificity in the tissue of interest

• Integration with functional studies:

  • Correlate ENHO expression with functional parameters

  • In SSc studies, negative correlations were observed between ENHO levels and Macrophages M1 and M2

  • Explore connections between ENHO expression and disease-related pathways

How can I optimize Design of Experiments (DoE) for ENHO antibody validation and use?

Design of Experiments (DoE) is a powerful methodology for systematically optimizing conditions when working with antibodies like those targeting ENHO:

• DoE workflow for antibody optimization:

  • Step 1: Define objectives, factors, and ranges

    • Objectives: Screening, optimization, or robustness testing

    • Factors: Primary antibody concentration, incubation time, temperature, buffer composition

    • Ranges: Determine upper and lower values for each factor

  • Step 2: Define responses and measurement systems

    • Responses: Signal-to-noise ratio, background staining, specific signal intensity

    • Ensure measurement system requirements are compatible with expected outcomes

  • Step 3: Create the experimental design

    • Determine design resolution and number of experiments needed

    • Address systematic bias through randomization

    • Consider replication of experiments for statistical power

• Practical implementation:

  • Use statistical software to create factorial designs

  • Perform experiments in a randomized order

  • Analyze results using response surface methodology

  • Generate mathematical models to predict optimal conditions

• Considerations specific to ENHO antibodies:

  • Begin with manufacturer-recommended dilutions and conditions

  • Include known positive controls (tissues with high ENHO expression, such as astrocytes)

  • Consider epitope accessibility in fixed tissues or denatured proteins

  • Optimize antigen retrieval methods for IHC applications

Using DoE approaches rather than one-factor-at-a-time methods provides more comprehensive understanding of factor interactions and more efficient optimization .

What strategies can resolve contradictory results when using different ENHO antibodies?

When different ENHO antibodies yield contradictory results, a systematic troubleshooting approach is necessary:

• Epitope differences analysis:

  • Map the epitopes recognized by each antibody (e.g., ABIN7139711 targets AA 37-54 of human adropin)

  • Different epitopes may be differentially accessible in various applications

  • Post-translational modifications may affect epitope recognition

• Validation comparison:

  • Review validation data for each antibody (Western blot, IHC images)

  • Check species reactivity claims and cross-reference with your samples

  • Examine antibody production methods (immunogen used, purification approach)

• Standardized comparison experiment:

  • Design side-by-side testing under identical conditions

  • Include positive and negative controls

  • Quantify results using digital image analysis for objective comparison

• Performance analysis by application:

  • Some antibodies perform better in specific applications (WB vs. IHC)

  • For example, in HER2/neu testing, studies found that rabbit monoclonal antibody SP3 showed about a 50% reduction in equivocal scores compared to rabbit polyclonal antibody A0485

• Resolution strategy:

  • Use multiple antibodies targeting different epitopes

  • Correlate antibody results with mRNA expression data

  • Consider alternative detection methods (e.g., mass spectrometry) for confirmation

How can binding strength of anti-ENHO antibodies be assessed beyond traditional affinity measurements?

Traditional affinity measurements may not fully characterize antibody-antigen interactions. Advanced methods to assess binding strength include:

• Chaotrope-based assays:

  • Measure resistance of antibody-antigen complexes to chaotropic agents

  • Calculate avidity index as the reciprocal titer after chaotrope exposure divided by the reciprocal titer without chaotrope

  • Alternative measures include functional affinity index (FAI) and Bmax index (BMI)

• Limitations of chaotrope-based methods:

  • Antibody binding to continuous epitopes resists chaotrope-treatment more than binding to discontinuous epitopes

  • Higher valency interactions show greater resistance to chaotropes

  • The chaotrope may perturb antigen conformation, affecting binding independently of affinity

• Alternative binding kinetics approaches:

  • Surface Plasmon Resonance (SPR) to measure real-time binding kinetics

  • Bio-Layer Interferometry (BLI) for label-free kinetic analysis

  • Isothermal Titration Calorimetry (ITC) to measure thermodynamic parameters

• Considerations for polyclonal responses:

  • Computational neutralization fingerprinting can predict epitope specificities

  • Next-generation fingerprinting algorithms improve prediction accuracy

  • These methods can detect multiple specificities in a sample

When working with ENHO antibodies, these advanced methods provide more comprehensive characterization of binding properties than traditional ELISA or immunoprecipitation assays alone.

How can ENHO antibodies contribute to HIV-1 vaccine research methodologies?

While ENHO itself is not directly related to HIV-1, research methodologies using antibodies in HIV vaccine development can inform approaches with ENHO antibodies:

• Structure-based design principles:

  • HIV-1 research uses structural biology to guide antibody development against the Envelope glycoprotein (Env)

  • Similar approaches could target specific structural domains of adropin

  • AlphaFold2 modeling could be used to design optimized epitopes for ENHO antibody generation

• Consensus sequence approaches:

  • HIV vaccine research uses consensus sequences to overcome diversity

  • For ENHO research across species, consensus epitope design could improve cross-reactivity

  • Chemical modifications and redesigned loops can enhance antibody binding

• Experimental medicine study design:

  • Sequential immunization protocols can be adapted for raising high-affinity anti-ENHO antibodies

  • Monitoring seroconversion rates and antibody persistence over time

  • Correlating binding to specific epitopes with functional outcomes

• Analytical methodologies:

  • Next-generation neutralization fingerprinting algorithms can map polyclonal antibody responses

  • Machine learning approaches to select optimized panels for analysis

  • Confidence estimation for antibody predictions

These advanced methodologies from HIV-1 vaccine research represent cutting-edge approaches that could be applied to enhance ENHO antibody development and characterization for research applications.

What are the optimal protocols for using ENHO antibodies in different applications?

Based on validated research applications, here are optimized protocols for different techniques:

Immunohistochemistry (IHC) Protocol for ENHO:

• Tissue preparation:

  • For paraffin sections: Use heat-induced epitope retrieval with Antigen Retrieval Reagent-Basic

  • For frozen sections: Fix with 4% paraformaldehyde for 10 minutes

• Blocking and antibody incubation:

  • Block with 5% normal serum (matching secondary antibody species) for 1 hour

  • Incubate with anti-ENHO antibody at 5-15 μg/mL for 1 hour at room temperature

  • For rabbit polyclonal antibodies (e.g., ABIN7139711), use at 1:100-1:200 dilution

• Detection system:

  • Use Anti-Mouse/Rabbit IgG HRP Polymer Antibody

  • Develop with DAB and counterstain with hematoxylin

  • Expected pattern: Cytoplasmic staining in positive cells

Immunocytochemistry (ICC) Protocol:

• Cell preparation:

  • Fix cells in 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

• Antibody incubation:

  • Block with 1% BSA in PBS for 30 minutes

  • Incubate with anti-ENHO antibody at 25 μg/mL for 3 hours at room temperature

  • Wash 3 times with PBS

• Detection and visualization:

  • Incubate with fluorophore-conjugated secondary antibody (e.g., NorthernLights 557-conjugated Anti-Mouse/Rabbit IgG)

  • Counterstain nuclei with DAPI

  • Expected pattern: Cytoplasmic localization

These protocols should be optimized for specific antibodies and experimental conditions using the DoE approach described in section 4.1.

How can I interpret ENHO expression patterns in different tissues and cell types?

Interpreting ENHO expression requires understanding typical expression patterns across tissues and cell types:

• Nervous system expression:

  • Highest in astrocytes and oligodendrocyte precursor cells (OPCs)

  • Some neuronal populations (Pax6+ve and Vip+ve neurons) show expression but at lower levels

  • Expression appears to decline with age in the brain

  • In human brain cortex, adropin localizes to neuronal cell bodies and processes

• Kidney expression:

  • Cytoplasmic staining in glomeruli

  • Use appropriate kidney-specific markers for co-localization studies

• Expression changes in disease states:

  • Downregulated in systemic sclerosis (SSc) skin samples

  • More pronounced decrease in diffuse cutaneous SSc compared to limited cutaneous SSc

• Regulatory factors affecting expression:

  • Suppressed by inflammatory factors (TNFα, Poly I:C)

  • Not regulated by hypoxia in astrocytes

  • Regulated by energy status and dietary nutrient content

  • Expression levels increase with dietary fat content

• Correlated gene expression patterns:

  • ENHO expression correlates with genes involved in mitochondrial oxidoreductase reactions

  • Also correlates with genes involved in energy-demanding synthesis of micro- and macromolecules

  • Inverse correlation with genes that increase with age

When interpreting ENHO staining patterns, consider both the expected cellular localization (primarily cytoplasmic) and the relative intensity across different cell types within the same tissue section.

What are the emerging applications of ENHO antibodies in metabolic and neurological research?

Recent advances have expanded the potential applications of ENHO antibodies in several research areas:

• Metabolic research applications:

  • Investigation of adropin's role in regulation of glucose homeostasis and lipid metabolism

  • Exploring connections between ENHO expression and obesity/insulin resistance

  • Studying the relationship between adropin and PPAR-gamma signaling

• Neurological research developments:

  • Brain aging studies: ENHO expression declines with age in human brain tissue

  • Correlation between adropin levels and cognitive function

  • Potential connection to neurodegenerative diseases through energy metabolism dysfunction

  • Investigation of astrocyte-specific ENHO function and its impact on neuronal health

• Systemic sclerosis biomarker research:

  • ENHO identified as a potential diagnostic biomarker for early detection of SSc

  • Combination with NOX4 provides a diagnostic prediction model

  • Negative correlation observed between ENHO levels and macrophage populations (M1/M2)

• Methodological advances:

  • Development of more specific monoclonal antibodies targeting functional domains

  • Custom antibodies against different species-specific ENHO variants

  • Antibodies specifically designed for live-cell applications

As research into adropin's functions continues to expand, antibodies against ENHO will become increasingly important tools for understanding its diverse roles in multiple physiological systems.

How will advances in antibody technology impact future ENHO research?

The next generation of antibody technologies will significantly enhance ENHO research capabilities:

• Recombinant antibody production:

  • Greater batch-to-batch consistency compared to traditional polyclonal antibodies

  • Engineered antibody fragments (Fab, scFv) for improved tissue penetration

  • Humanized antibodies for therapeutic development targeting adropin pathways

• Multiplexed detection systems:

  • Simultaneous detection of ENHO with multiple related proteins

  • Spatial transcriptomics combined with antibody-based protein detection

  • Mass cytometry (CyTOF) for high-dimensional analysis of ENHO in relation to dozens of other markers

• Advanced imaging applications:

  • Super-resolution microscopy for precise subcellular localization

  • Intravital imaging with labeled anti-ENHO antibodies

  • Correlative light and electron microscopy to link ENHO localization with ultrastructure

• Computational approaches:

  • Machine learning algorithms for automated quantification of ENHO expression patterns

  • Bioinformatic integration of antibody-based detection with multi-omics data

  • Next-generation fingerprinting algorithms to map polyclonal antibody responses

• Structure-guided antibody development:

  • AlphaFold2 and other AI tools to model ENHO structure for optimal epitope selection

  • Antibodies designed to distinguish between different conformational states of adropin

  • Chemical modifications to enhance binding properties

These technological advances will enable more precise and comprehensive investigations into adropin's functions across different tissues, cell types, and disease states.