ACA8 Antibody

What is the Annexin A8 (ANXA8) protein and what are its primary functions?

Annexin A8 (ANXA8, also known as Annexin VIII, ANX8, or Vascular anticoagulant-beta/VAC-beta) functions as an anticoagulant protein in the human body. Its primary role is acting as an indirect inhibitor of the thromboplastin-specific complex, which plays a crucial role in the blood coagulation cascade . This protein belongs to the larger annexin family, characterized by calcium-dependent phospholipid binding proteins that participate in various cellular processes including membrane organization, trafficking, and signal transduction.

What applications are ANXA8 antibodies suitable for in research?

ANXA8 antibodies, particularly the rabbit polyclonal variants, demonstrate versatility across multiple research applications. They have been validated for Western Blotting (WB), Immunohistochemistry on paraffin-embedded tissues (IHC-P), and Immunocytochemistry/Immunofluorescence (ICC/IF) applications . These antibodies primarily react with human samples and are generated using immunogens corresponding to recombinant fragment proteins within Human ANXA8 amino acids 50-300 . When selecting an ANXA8 antibody, researchers should verify the specific applications for which the antibody has been validated by the manufacturer to ensure optimal experimental results.

What is Carbonic Anhydrase VIII (CA8) and where is it predominantly expressed?

Carbonic Anhydrase VIII (CA8) is a protein that, despite structural similarities to other carbonic anhydrases, lacks the typical enzymatic activity of converting carbon dioxide to bicarbonate. CA8 is predominantly expressed in the central nervous system, with particularly high expression in the cerebellum . When detected via Western blot analysis using specific antibodies, CA8 appears as a band at approximately 36 kDa in both human and mouse brain (cerebellum) tissue lysates . This protein plays important roles in neurological function, and mutations in the CA8 gene have been associated with cerebellar ataxia and mild mental retardation.

What are the challenges associated with adeno-associated virus 8 (AAV8) antibodies in gene therapy research?

A significant challenge in AAV8-based gene therapy research is the presence of pre-existing anti-AAV8 antibodies in human populations. These antibodies can substantially impair the efficacy of AAV8 vector-mediated gene delivery by preventing vectors from transducing target tissues . Research has shown that approximately 40% of normal subjects possess AAV8 total antibodies (TAb), with 24% being neutralizing antibody (NAb) positive . This high prevalence necessitates careful screening of potential gene therapy recipients and may require the development of strategies to overcome neutralizing antibody responses, such as using alternative AAV serotypes or implementing immunomodulation approaches.

How do researchers differentiate between non-neutralizing and neutralizing anti-AAV8 antibodies in patient samples?

Differentiating between non-neutralizing and neutralizing anti-AAV8 antibodies requires a tiered testing approach. Initially, researchers employ a total antibody (TAb) screening assay, typically a chemiluminescence-based enzyme-linked immunosorbent assay (ELISA), to detect all anti-AAV8 antibodies in human serum . This is followed by a confirmatory assay to determine specificity, where a cut point factor of 2.65 for screening and 57.1% for confirmation has been established .

For definitive identification of neutralizing antibodies, researchers implement a cell-based neutralizing antibody (NAb) assay, often using COS-7 cells . The relationship between these assays shows high concordance—all NAb-positive subjects are confirmed TAb-positive and pass the confirmatory cut point (CCP) criteria, while NAb-negative subjects typically fail the CCP criterion . For gene therapy applications, implementing this tiered approach during pre-enrollment screening can effectively identify patients likely to have poor therapeutic responses due to pre-existing immunity.

What factors influence the cross-reactivity of AAV8 antibodies with other AAV serotypes?

The cross-reactivity of AAV8 antibodies with other AAV serotypes is determined by capsid protein homology and structural epitope conservation. For example, the human chimeric AAV8 antibody clone ADK8-h1 demonstrates significant cross-reactivity with AAV3, AAV7, AAVrh10, and AAVrh74, but shows no reactivity with AAV1, AAV2, AAV4, AAV5, AAV6, AAV9, or AAVDJ .

This pattern of cross-reactivity is directly related to the conservation of specific epitopes on the viral capsid surface across serotypes. Antibodies targeting highly conserved regions typically exhibit broader cross-reactivity, while those recognizing variable regions show greater serotype specificity. The binding affinity measurements reveal exceptionally high affinities (KD values <1.0E-12 M) for ADK8-h1 binding to AAV8, AAVrh10, and AAVrh74 , suggesting recognition of a highly conserved epitope among these serotypes. These cross-reactivity profiles are crucial considerations when selecting antibodies for specific research applications or when designing gene therapy vectors to evade pre-existing immunity.

How can structural modifications of antibodies enhance their research and therapeutic applications?

Structural modifications of antibodies can dramatically enhance their utility in both research and therapeutic applications. A remarkable example is the development of Ab8, which represents the variable, heavy chain (VH) domain of an immunoglobulin fused to part of the immunoglobulin tail region . This engineering approach creates a molecule that is approximately 10 times smaller than a full-sized antibody while retaining key immune functions .

The reduced size provides several advantages: enhanced tissue penetration, increased potential for diffusion, and compatibility with alternative administration routes such as inhalation . Additionally, engineered antibodies can be designed to lack binding to human cells, reducing the risk of off-target effects . The success of this approach was demonstrated with Ab8, which was highly effective against SARS-CoV-2 in animal models .

For researchers, similar engineering approaches can be applied to antibodies against target proteins like ANXA8 or CA8, potentially creating research reagents with enhanced properties for applications requiring tissue penetration or specialized delivery methods.

What are the optimal conditions for using ANXA8 antibodies in Western blot and immunohistochemistry applications?

For optimal Western blot applications with ANXA8 antibodies:

• Sample preparation: Extract proteins from human tissue or cell lines using a buffer containing protease inhibitors to prevent degradation.

• Protein loading: Load 20-30 μg of total protein per lane for cell lysates.

• Antibody dilution: Begin with 1:1000 dilution of the primary ANXA8 antibody . Optimize as needed.

• Incubation conditions: Incubate with primary antibody overnight at 4°C.

• Detection system: Use an appropriate HRP-conjugated secondary anti-rabbit antibody.

For immunohistochemistry on paraffin-embedded tissues (IHC-P):

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) is typically effective.

• Blocking: Block with 5% normal serum in PBS for 1 hour at room temperature.

• Antibody dilution: Start with 1:100-1:200 dilution of the ANXA8 antibody .

• Incubation time: Incubate overnight at 4°C in a humidified chamber.

• Visualization: Use an appropriate detection system compatible with rabbit primary antibodies.

For both applications, optimization of specific conditions may be required depending on sample type and experimental goals.

What methods are recommended for detection of human and mouse Carbonic Anhydrase VIII (CA8) by Western blot?

For optimal detection of human and mouse CA8 by Western blot, the following methodological approach is recommended:

• Tissue selection: Use cerebellum tissue as CA8 is highly expressed in this brain region .

• Membrane selection: Use PVDF membrane for protein transfer.

• Antibody concentration: Apply 1 μg/mL of Mouse Anti-Human/Mouse CA8 Monoclonal Antibody .

• Secondary antibody: Use HRP-conjugated Anti-Mouse IgG Secondary Antibody.

• Running conditions: Conduct the experiment under reducing conditions.

• Buffer system: Utilize Immunoblot Buffer Group 1 for optimal results.

• Expected results: A specific band for CA8 should be detected at approximately 36 kDa .

This methodology has been validated for both human and mouse cerebellum tissue lysates, making it valuable for comparative studies across these species .

How should researchers design and validate a total antibody (TAb) assay for screening anti-AAV8 antibodies?

Designing and validating a robust total antibody (TAb) assay for anti-AAV8 antibodies requires a systematic approach:

Assay Design:

• Platform selection: Develop a chemiluminescence-based enzyme-linked immunosorbent assay (ELISA) for high sensitivity .

• Antigen coating: Coat plates with purified AAV8 capsid proteins at optimized concentration.

• Blocking and detection: Use appropriate blocking agents to minimize background and sensitive detection systems for reliable signal measurement.

Validation Parameters:

• Cut point determination: Establish a screening cut point factor (2.65 has been validated) .

• Confirmatory cut point: Set a confirmatory cut point (57.1% has been effective) .

• Specificity analysis: Conduct competition studies with excess AAV8 antigen to confirm binding specificity.

• Cross-reactivity assessment: Test against other AAV serotypes to determine serotype specificity.

Verification Testing:

• Concordance testing: Compare results with cell-based neutralizing antibody (NAb) assays on the same samples .

• Population screening: Evaluate prevalence in normal subject populations (40% prevalence has been observed) .

• Clinical correlation: Correlate antibody levels with clinical outcomes in gene therapy applications.

This tiered approach ensures a reliable assay for patient screening prior to AAV8-mediated gene therapy, potentially replacing more complex cell-based NAb assays in clinical settings .

What experimental conditions are optimal for dot blot analysis of AAV8 using chimeric antibodies?

For optimal dot blot analysis of AAV8 using chimeric antibodies such as ADK8-h1, the following experimental conditions have been validated:

Sample Preparation:

• Native capsids: Use 1E+09-1E+10 capsids per dot .

• Denatured capsids: Use 1E+10 capsids denatured at 95°C for 10 minutes in sample buffer .

Blocking and Antibody Incubation:

• Blocking: Use 5% milk in PBST for 45 minutes at room temperature .

• Primary antibody: Dilute anti-AAV8 human chimeric antibody (ADK8-h1) in blocking buffer to 100 ng/ml concentration and incubate for 1.5 hours at room temperature .

• Secondary antibody: Use anti-human IgG goat polyclonal, HRP conjugate diluted in blocking buffer to 200 ng/ml and incubate for 1.5 hours at room temperature .

Detection:

• Visualization method: Use chemiluminescent detection with ECL Western Blotting Substrate .

• Expected results: Strong signals should be observed for AAV8, with cross-reactivity to AAV3, AAV7, AAVrh10, and AAVrh74, but not to AAV1, AAV2, AAV4, AAV5, AAV6, AAV9, or AAVDJ .

This optimized protocol enables sensitive detection of AAV8 and related serotypes, making it valuable for serotype identification and antibody characterization studies.

How might engineered antibody fragments like Ab8 influence future antibody development against ANXA8 or CA8?

The engineering approach used to develop Ab8, which is approximately 10 times smaller than a conventional antibody yet retains high specificity and neutralizing capacity , presents a compelling direction for future antibody development against targets like ANXA8 and CA8. This miniaturization strategy, which involves isolating the variable heavy chain (VH) domain and fusing it with part of the immunoglobulin tail region , could be applied to create minimized versions of existing ANXA8 and CA8 antibodies.

The potential advantages include:

• Enhanced tissue penetration: Smaller antibody fragments would likely penetrate tissues more effectively, particularly important for reaching CA8 in the cerebellum.

• Alternative administration routes: As demonstrated with Ab8, which can be administered via inhalation , miniaturized antibodies against ANXA8 or CA8 could potentially be delivered through novel routes.

• Improved binding kinetics: Smaller fragments often exhibit different binding kinetics that may be advantageous for certain research applications.

• Reduced immunogenicity: Engineered fragments can be designed to minimize interactions with human cells, potentially reducing off-target effects .

The methodology used to identify Ab8, which involved "fishing" in a pool of over 100 billion potential candidates using a target protein as bait , could be adapted to identify highly specific VH domains against ANXA8 or CA8, potentially leading to next-generation research tools with enhanced properties.

What implications do the prevalence rates of anti-AAV8 antibodies have for gene therapy research and clinical trials?

The observed prevalence of anti-AAV8 total antibodies in 40% of the normal population, with 24% being neutralizing antibody positive , has profound implications for AAV8-based gene therapy research and clinical trials:

Patient Selection Challenges:

• Screening necessity: Pre-screening of all potential gene therapy recipients becomes essential, requiring robust, standardized assays.

• Population variation: Research must account for potential variations in prevalence across different demographic groups.

• Reduced eligible population: The high prevalence rate substantially reduces the pool of eligible patients for AAV8-based therapies.

Research and Development Implications:

• Alternative serotype development: The significant prevalence of anti-AAV8 antibodies drives research toward developing alternative AAV serotypes with lower pre-existing immunity.

• Capsid engineering: Motivates engineering of AAV8 capsids to evade neutralizing antibodies while maintaining tropism for target tissues.

• Immunomodulation strategies: Encourages development of protocols to temporarily suppress immune responses during gene therapy administration.

Clinical Trial Design Considerations:

• Tiered testing approach: Implementation of screening followed by confirmatory assays before patient enrollment .

• Dose adjustment studies: Research into whether higher vector doses can overcome low-titer neutralizing antibodies.

• Re-administration challenges: Investigation of strategies to enable re-administration of AAV8 vectors despite development of treatment-induced immunity.

These implications underscore the importance of continued research into immunological aspects of gene therapy and the development of standardized assays for patient stratification.

How can researchers address contradictory results when using different anti-AAV8 antibody assays?

When researchers encounter contradictory results using different anti-AAV8 antibody assays, a systematic troubleshooting approach is essential:

Analysis of Assay Fundamentals:

• Assay principles: Understand the differences between total antibody (TAb) assays and neutralizing antibody (NAb) assays. TAb assays detect all antibodies that bind AAV8, while NAb assays specifically identify those that impair AAV8 function .

• Cut point variations: Different assays employ distinct cut points. For example, the TAb screening cut point factor of 2.65 and confirmatory cut point of 57.1% must be considered when comparing results across platforms.

Methodological Resolution Strategies:

• Implement a tiered approach: Begin with TAb screening, followed by confirmatory specificity testing, and finally NAb assays for comprehensive characterization .

• Standardize positive controls: Use identical positive control samples across all assay platforms to enable direct comparison of results.

• Cross-platform validation: Test a subset of samples on multiple platforms to establish correlation factors between assays.

Data Reconciliation Techniques:

• Concordance analysis: Systematically analyze concordance between assays. Research has shown that while all NAb-positive samples are TAb-positive, not all TAb-positive samples are NAb-positive (16% were NAb-negative in one study) .

• Threshold adjustment: Consider adjusting thresholds based on clinical outcomes data rather than purely statistical cut points.

• Report multiple metrics: When publishing results, provide data from multiple assay types to give a comprehensive view of anti-AAV8 immunity.

By implementing these strategies, researchers can reconcile contradictory results and develop a more complete understanding of anti-AAV8 immunity in their study populations.

Comparative Analysis of Anti-AAV8 Antibody Detection Methods

Note: This table synthesizes information from sources and to provide guidance on method selection.

Prevalence of Anti-AAV8 Antibodies in Normal Population

Note: Data derived from a study of 84 normal subjects as reported in source .

Cross-Reactivity Profile of Human Chimeric AAV8 Antibody (ADK8-h1)

Note: Data compiled from source showing the remarkable specificity pattern of the ADK8-h1 antibody.