Ace Antibody, HRP conjugated

What is ACE-2 and why are antibodies against it important in research?

ACE-2 is a type I transmembrane zinc protease that cleaves angiotensins I and II to produce vasodilatory and anti-proliferative peptides. The balance between ACE-1 and ACE-2 activity is critical for maintaining cardiovascular, renal, and pulmonary function. Additionally, ACE-2 functions as the cellular uptake receptor for the SARS coronavirus, making it a key target in COVID-19 research . Antibodies against ACE-2 allow researchers to detect and study this protein in various experimental settings, characterize its expression across tissues, and investigate its role in disease pathogenesis.

How do HRP-conjugated ACE-2 antibodies differ from fluorophore-conjugated versions?

HRP-conjugated ACE-2 antibodies utilize enzymatic signal amplification, where the HRP enzyme catalyzes the oxidation of a substrate to produce a detectable signal. This differs from fluorophore conjugates such as PE (Phycoerythrin) or Alexa Fluor that directly emit fluorescent signals without requiring substrate addition . HRP conjugation offers several advantages: higher sensitivity through signal amplification, compatibility with permanent archival samples, and versatility across multiple detection platforms. In contrast, fluorophore conjugates excel in applications requiring multiplexing and direct visualization, such as flow cytometry and fluorescence microscopy .

What major applications use HRP-conjugated ACE-2 antibodies?

HRP-conjugated ACE-2 antibodies are particularly valuable for:

• Western blot analysis for protein expression quantification

• Enzyme-linked immunosorbent assays (ELISA) for sensitive detection

• Immunohistochemistry (IHC) for tissue localization studies

• Chromogenic in situ hybridization

• Proximity ligation assays for protein-protein interaction studies

These applications benefit from the signal amplification provided by the HRP enzyme, allowing for detection of even low-abundance ACE-2 expression in various tissues and experimental contexts .

How should researchers validate ACE-2 antibody specificity?

Validation of ACE-2 antibodies should follow the recommendations of the International Working Group for Antibody Validation (IWGAV). The most effective approach includes:

• Orthogonal validation: Correlating antibody detection with mRNA expression data from sources like HPA, GTEx, and FANTOM5 .

• Independent antibody strategy: Using multiple antibodies targeting different epitopes of ACE-2 to confirm consistent expression patterns .

• Genetic controls: Testing in systems with ACE-2 overexpression (like transfected HEK293 cells) and comparing with negative controls .

• Tissue panel validation: Testing across tissues with known differential expression (high in intestine and kidney, low/variable in lung) .

Research shows that antibodies like MAB933 from R&D Systems have been validated through multiple approaches, including comparison with transfected cell lines and correlation with transcriptomic data .

What positive and negative control tissues are recommended for ACE-2 antibody validation?

Based on comprehensive tissue expression profiling:

Recommended positive controls:

• Small intestine (duodenum): Shows consistently high ACE-2 expression

• Kidney (proximal tubular cells): Demonstrates reliable ACE-2 expression

• Testis: Exhibits strong and consistent expression

Recommended negative controls:

• Lung tissue: Majority of samples (357/360 in one study) show negative staining

• Brain tissue: Consistently negative for ACE-2 expression

• Skeletal muscle: Shows minimal ACE-2 expression

Researchers should note that expression in respiratory epithelia (nasal mucosa, bronchus) is rare and heterogeneous, with only a small subset of ciliated cells in a minority of individuals showing positivity .

What are the optimal storage conditions for maintaining ACE-2 HRP-conjugated antibody activity?

For optimal stability and performance:

• Store at 2-8°C (not frozen) for up to 12 months from receipt date

• Protect from light to prevent photodegradation of the conjugate

• Do not freeze as this can damage the HRP enzyme activity

• Avoid repeated temperature fluctuations

• Use sterile technique when handling to prevent contamination

• Consider adding carrier protein (0.1-1% BSA) if diluting for storage

Following these guidelines will help maintain antibody performance and extend shelf life for research applications .

How can researchers optimize detection of low ACE-2 expression in respiratory tissues?

Detecting ACE-2 in respiratory tissues presents significant challenges due to its sparse and heterogeneous expression. To optimize detection:

• Use large tissue sections rather than tissue microarrays (TMAs) to increase the probability of finding rare positive cells .

• Employ enhanced antigen retrieval protocols optimized for membrane proteins.

• Utilize signal amplification systems like tyramide signal amplification for HRP-conjugated antibodies.

• Screen multiple individuals, as expression varies significantly between subjects (in one study, only 6/12 nasal mucosa and 2/8 bronchus samples showed any positivity) .

• Focus on specific cell types where expression is more likely (ciliated cells in nasal mucosa and bronchus, AT2 cells in lung) .

• Compare with known positive control tissues (intestine, kidney) within the same experiment.

The comprehensive study by Uhlén et al. demonstrated that only 2/360 lung samples showed ACE-2 positivity in structures likely representing AT2 cells, emphasizing the importance of thorough sampling .

What approaches can resolve discrepancies between different ACE-2 detection methods?

When facing discrepancies between different detection methods:

• Cross-validate with multiple techniques:

  • Compare IHC results with Western blotting from the same tissues

  • Correlate protein detection with mRNA analysis (RT-PCR or RNA-seq)

  • Use mass spectrometry-based proteomics as an antibody-independent method

• Employ multiple antibodies:

  • Use independently developed antibodies targeting different epitopes

  • Compare commercial antibodies that meet IWGAV validation criteria

  • Include both monoclonal and polyclonal antibodies in parallel

• Control for technical variables:

  • Standardize fixation and processing protocols

  • Optimize antigen retrieval methods

  • Ensure proper antibody concentration and incubation times

• Quantitative assessment:

  • Develop a scoring system with defined criteria for positivity

  • Use digital image analysis for objective quantification

  • Include internal calibration standards

The study by Uhlén et al. demonstrated how this approach successfully reconciled conflicting reports about ACE-2 expression in respiratory tissues .

How can dual immunostaining be performed with ACE-2 HRP-conjugated antibodies?

For dual immunostaining with ACE-2 HRP-conjugated antibodies:

• Sequential Double Staining Protocol:

  • Complete the first staining with HRP-conjugated ACE-2 antibody

  • Develop with a substrate (e.g., DAB for brown color)

  • Denature residual HRP activity (using 3% H₂O₂ or microwave treatment)

  • Apply the second primary antibody

  • Use a different enzyme system (e.g., alkaline phosphatase) with the second antibody

  • Develop with a contrasting substrate (e.g., Fast Red)

• Considerations for Successful Dual Staining:

  • Optimize antibody concentrations individually before combining

  • Test cross-reactivity between detection systems

  • Use antibodies from different host species when possible

  • Include appropriate single-stained and negative controls

  • Consider the order of staining (typically start with the less abundant target)

This approach is particularly valuable for co-localization studies, such as examining ACE-2 expression in specific cell types identified by lineage markers .

What approaches enable quantitative analysis of ACE-2 expression using HRP-conjugated antibodies?

For quantitative analysis of ACE-2 expression using HRP-conjugated antibodies:

• Immunohistochemistry Quantification:

  • Use digital pathology software for image analysis

  • Quantify parameters including:

    • Staining intensity (0-3+ scale)

    • Percentage of positive cells

    • H-score calculation (intensity × percentage, range 0-300)

  • Include calibration standards with known expression levels

  • Compare results across different tissues using the same methodology

• Western Blot Densitometry:

  • Include recombinant ACE-2 standards for calibration

  • Use appropriate loading controls (β-actin, GAPDH)

  • Ensure detection is within the linear range

  • Utilize image analysis software for band intensity quantification

  • Normalize to loading controls to account for sample variation

• ELISA-Based Quantification:

  • Develop standard curves using recombinant ACE-2

  • Optimize sample preparation to maintain protein integrity

  • Include appropriate quality controls

  • Calculate concentrations based on standard curves

These approaches enable objective comparison of ACE-2 expression across different experimental conditions, tissues, and disease states .

What is the tissue distribution pattern of ACE-2 protein and how does it affect experimental design?

ACE-2 protein shows a distinct tissue distribution pattern that significantly impacts experimental design:

High Expression Tissues:

• Intestinal tract (especially duodenum and small intestine)

• Kidney (proximal tubular cells)

• Testis

• Gallbladder

• Eye (conjunctiva and cornea)

Moderate Expression Tissues:

• Heart (cardiomyocytes) - with some antibody variability

• Thyroid gland

• Pancreas

• Placenta (syncytiotrophoblasts)

Low/Variable Expression Tissues:

• Respiratory tract (rare expression in ciliated cells)

• Lung (very rare expression in AT2 cells)

• Vascular endothelium (tissue-specific patterns)

This distribution has important implications for experimental design:

• Positive control selection should prioritize high-expression tissues

• Detection methods for low-expression tissues require increased sensitivity

• Sampling strategies must account for heterogeneous expression

• Inter-individual variation must be considered, especially in respiratory tissues

According to comprehensive studies, expression in respiratory tissues is found in only a small minority of samples, with just 6/12 nasal mucosa and 2/8 bronchus samples showing any positivity in ciliated cells .

How does ACE-2 expression in the vascular system impact detection strategies?

ACE-2 expression in the vascular system shows unique patterns that require specialized detection strategies:

• Tissue-Specific Vascular Expression:

  • Small capillaries show consistent ACE-2 staining only in specific organs: heart, pancreas, thyroid gland, parathyroid gland, and adrenal gland

  • Other vascular beds show minimal or undetectable expression

• Cell Type Considerations:

  • Expression may be localized to specific vascular cell populations (endothelial cells vs. pericytes)

  • The exact cellular localization requires high-resolution microscopy techniques

• Optimized Detection Approaches:

  • Use thin sections (3-5μm) to better visualize capillary structures

  • Employ dual staining with endothelial markers (CD31, vWF) to confirm vascular localization

  • Apply tyramide signal amplification to enhance detection sensitivity

  • Consider regional heterogeneity within vascular beds

• Experimental Implications:

  • The restricted vascular expression pattern suggests functional specialization

  • Studies examining ACE-2 in vascular biology should focus on organs with confirmed expression

  • Systemic vascular studies must account for organ-specific differences

This tissue-specific vascular expression pattern has important implications for understanding ACE-2 biology and its role in cardiovascular physiology and pathology .

How do monoclonal and polyclonal ACE-2 antibodies compare in research applications?

Monoclonal and polyclonal ACE-2 antibodies offer distinct advantages and limitations across research applications:

Application-specific performance:

• Flow cytometry: Both perform well, with monoclonals often preferred for cleaner background

• Western blot: Polyclonals often provide stronger signal, especially for denatured proteins

• IHC/ICC: Each type has advantages; polyclonals may detect partially degraded antigens better

• ELISA: Monoclonals typically preferred for capture, polyclonals for detection

The optimal choice depends on the specific application, required sensitivity, and target accessibility .

What are the relative advantages of different conjugates for ACE-2 antibody detection?

Different conjugates offer distinct advantages for ACE-2 detection:

Key considerations when selecting conjugates:

• For tissue detection: HRP conjugates provide superior sensitivity and permanent signal

• For flow cytometry: PE and Alexa Fluor conjugates allow direct detection without substrate

• For multiplexed detection: Fluorescent conjugates enable simultaneous multi-target analysis

• For quantitative analysis: Each conjugate system requires specific standardization

• For long-term storage: HRP-DAB produces permanent signals for archival samples

The research context and specific experimental requirements should determine the optimal conjugate selection .

What are common pitfalls in ACE-2 antibody experiments and how can they be avoided?

Common pitfalls in ACE-2 antibody experiments include:

• False Negative Results:

  • Cause: Insufficient antigen retrieval, epitope masking, low expression levels

  • Solution: Optimize antigen retrieval protocols, use multiple antibodies targeting different epitopes, include positive control tissues (intestine, kidney)

• False Positive Results:

  • Cause: Cross-reactivity, excessive antibody concentration, endogenous peroxidase activity

  • Solution: Validate antibody specificity, optimize dilutions, include appropriate blocking steps, use isotype controls

• Inconsistent Detection in Respiratory Tissues:

  • Cause: Extremely rare and heterogeneous expression, individual variation

  • Solution: Examine larger tissue sections, increase sample size, focus on specific cell types

• Discordance Between Protein and mRNA Data:

  • Cause: Post-transcriptional regulation, antibody specificity issues

  • Solution: Validate with multiple antibodies, correlate with orthogonal methods

• Batch-to-Batch Variation:

  • Cause: Manufacturing differences, storage conditions

  • Solution: Include consistent positive controls, document lot numbers, standardize protocols

Research has shown that even well-validated antibodies may detect ACE-2 differently across tissues, with respiratory tissues being particularly challenging. In one comprehensive study, only 2/360 lung samples showed positivity in AT2 cells .

What quality control measures ensure reliable results with ACE-2 HRP-conjugated antibodies?

Essential quality control measures include:

• Antibody Validation Controls:

  • Positive tissue controls with known ACE-2 expression (intestine, kidney)

  • Negative tissue controls with minimal expression (brain, skeletal muscle)

  • Isotype-matched control antibodies at equivalent concentrations

  • Absorption controls using recombinant ACE-2 protein

• Technical Quality Controls:

  • Endogenous peroxidase blocking verification steps

  • Background assessment in antibody-omitted sections

  • Inclusion of standardized positive samples across experiments

  • Documentation of lot numbers and storage conditions

• Signal Verification:

  • Correlation with orthogonal methods (Western blot, RNA-seq)

  • Verification with multiple antibodies targeting different epitopes

  • Concentration-dependent signal demonstration

  • Appropriate subcellular localization assessment

• Quantification Controls:

  • Standard curves with recombinant protein (for quantitative applications)

  • Internal reference standards for normalization

  • Replicates to assess technical variation

  • Statistical validation of quantitative results

Implementation of these comprehensive quality control measures aligns with best practices recommended by the IWGAV and ensures reliable, reproducible results in ACE-2 research .

How can ACE-2 antibodies contribute to SARS-CoV-2 research?

ACE-2 antibodies provide critical tools for SARS-CoV-2 research in several areas:

• Receptor Expression Mapping:

  • Characterizing ACE-2 distribution across tissues to identify potential sites of viral entry

  • Quantifying expression levels in susceptible cell populations

  • Examining expression in special populations (children vs. adults, healthy vs. diseased)

• Viral Entry Mechanisms:

  • Visualizing ACE-2/SARS-CoV-2 spike protein interactions

  • Monitoring receptor internalization following viral binding

  • Studying co-localization with other entry factors (TMPRSS2, furin)

• Therapeutic Development:

  • Screening for antibodies that block viral binding without affecting physiological function

  • Identifying compounds that modulate ACE-2 expression or accessibility

  • Evaluating receptor occupancy during recombinant ACE-2 therapy

• Pathogenesis Studies:

  • Examining ACE-2 downregulation after viral infection

  • Correlating expression patterns with disease severity

  • Investigating tissue-specific consequences of ACE-2 dysregulation

Recent research has revealed significant heterogeneity in ACE-2 expression across tissues, with implications for understanding viral tropism and developing targeted interventions .

What novel methodological approaches are being developed for ACE-2 detection?

Innovative approaches for ACE-2 detection include:

• Single-Cell Analysis:

  • Integration of ACE-2 protein detection with single-cell RNA sequencing

  • Mass cytometry (CyTOF) for high-dimensional analysis of ACE-2 expression

  • Single-cell proteomics to correlate ACE-2 with broader protein networks

• Advanced Imaging Techniques:

  • Super-resolution microscopy to visualize ACE-2 distribution in membrane microdomains

  • Live-cell imaging to track receptor dynamics in real-time

  • Expansion microscopy for enhanced visualization of subcellular localization

• Functional Detection Systems:

  • FRET-based biosensors to monitor ACE-2 conformational changes

  • Split-reporter systems to detect protein-protein interactions in live cells

  • Activity-based probes to distinguish functionally active ACE-2

• Multiplexed Detection:

  • Cyclic immunofluorescence to analyze dozens of proteins on the same tissue section

  • Spatial transcriptomics combined with protein detection

  • Digital spatial profiling for quantitative spatial analysis

These emerging methodologies allow researchers to move beyond simple presence/absence detection toward functional, quantitative, and spatially resolved analysis of ACE-2 in complex biological systems .