NEN4 Antibody

What is EN4 antibody and what epitope does it recognize?

EN4 is a monoclonal antibody originally described as reacting specifically with human endothelial cells. More detailed characterization has revealed that EN4 recognizes the CD31 antigen, a 130 kD molecular weight protein. Evidence supporting this identification includes strong staining of murine fibroblasts transfected with the human CD31 gene and SDS-PAGE analysis of immunoprecipitates from surface-iodinated Jurkart T cells, which demonstrated that EN4 and reference CD31 monoclonal antibodies recognize the same antigen .

How does EN4 antibody compare to other CD31 antibodies in performance?

EN4 has been demonstrated to be consistently more efficient than reference anti-CD31 monoclonal antibodies, as evidenced by both the intensity of fluorescence in flow cytometry analysis and tissue staining in immunohistochemistry. This superior performance characteristic has enabled researchers to achieve better characterization of the tissue and cellular distribution of CD31 antigen . The enhanced sensitivity makes EN4 particularly valuable for applications requiring detection of low CD31 expression levels.

What is Nectin-4 antibody and what applications is it suitable for?

Nectin-4 antibody (such as AF2659) is a polyclonal antibody that recognizes human Nectin-4, specifically the region from Gly27 to Val351 (Accession # Q96NY8). This antibody has been validated for applications including immunohistochemistry on paraffin-embedded tissues and flow cytometry . It demonstrates specific binding to Nectin-4 in human placenta tissue samples and in cell lines such as MCF-7 human breast adenocarcinoma cells .

What is the recommended protocol for immunohistochemical detection using Nectin-4 antibody?

For optimal immunohistochemical detection of Nectin-4 in paraffin-embedded tissues, the following protocol has been validated: First, perform heat-induced epitope retrieval using an Antigen Retrieval Reagent-Basic. Incubate tissue sections with the Nectin-4 antibody (10 μg/mL concentration) overnight at 4°C. For visualization, an HRP-DAB detection system can be used, followed by hematoxylin counterstaining . As with all antibodies, optimal dilutions should be determined by each laboratory for their specific application.

How does the prevalence of pre-existing neutralizing antibodies against AAV serotypes impact gene therapy applications?

Pre-existing neutralizing antibodies (NAbs) against adeno-associated virus (AAV) vectors present a significant challenge for gene therapy applications. Research indicates variation in NAb prevalence across different AAV serotypes, with AAV1 generally showing the highest prevalence (74.9% at 1:1 serum dilution in adults) and AAV5 the lowest (63.9% at the same dilution) . This serotype-specific immunity pattern is critical for vector selection in clinical applications, as pre-existing NAbs can limit viral gene transfer efficiency and durability of transgene expression . Researchers should consider screening patients for NAbs against the specific AAV serotype being used in their therapeutic approach.

What demographic and geographic factors influence AAV neutralizing antibody seroprevalence?

Several demographic and geographic factors significantly impact AAV neutralizing antibody prevalence:

• Age: NAb prevalence and titers increase gradually from childhood to late adulthood, with higher prevalence in adults aged 41-60 years compared to those aged 16-40 years .

• Gender: A numerically higher prevalence of NAb positivity has been observed in females compared to males, with specific differences for serotypes like AAV9 .

• Ethnicity: Higher prevalence of anti-AAV NAbs has been documented in Asian participants compared to White participants, particularly for AAV1 and AAV5 .

• Geographic region: Considerable variability exists across countries and geographical regions. In the Asia-Pacific region, Australia and Japan showed similarly low levels of NAb positivity, contrasting with high prevalence in South Korea. Within the US, regional differences were observed with lower prevalence in the Midwest and higher prevalence in the West .

This demographic and geographic variability requires careful consideration when designing global clinical trials involving AAV vectors.

What is the pattern of co-prevalence of neutralizing antibodies against different AAV serotypes?

Analysis of co-prevalence patterns reveals important immunological relationships between AAV serotypes. Co-prevalence is most frequently observed between AAV1 and AAV6, while less frequently observed between AAV5 and other AAVs . This pattern differs from traditional phylogenetic classifications based on linear capsid sequences. Machine learning analyses have revealed unique clustering of AAVs that provides new insights into the biological relationships between the immunogenicity of AAVs in humans . This information is valuable for designing sequential gene therapy approaches or for selecting alternative serotypes in patients with pre-existing immunity.

What methodological considerations are important when determining patient eligibility for AAV gene therapy trials?

Assays to determine neutralizing antibody levels are poorly standardized across studies, with variations in methodology, cutoffs for sample positivity, and thresholds used to determine patient eligibility . This lack of standardization creates a situation where the same patient may be deemed eligible for administration of AAV gene therapy in one trial but ineligible in another . Researchers should consider implementing robust, highly sensitive cell-based transduction inhibition assays with clearly defined cutoff values. The sensitivity of the assay is particularly important, as demonstrated by the different detection capabilities across various serum dilutions (1:1 through 1:50,000) .

What protocol is recommended for flow cytometric detection of Nectin-4 in cell lines?

For flow cytometric detection of Nectin-4 in cell lines such as MCF-7 (human breast adenocarcinoma), researchers should follow these methodological steps: First, harvest cells in appropriate buffer to maintain cell viability. Incubate cells with the primary Nectin-4 antibody at an optimized concentration (typically starting with manufacturer recommendations, e.g., 10 μg/mL). After washing to remove unbound primary antibody, use a fluorochrome-conjugated secondary antibody appropriate for the host species of the primary antibody. Include appropriate controls such as isotype controls and unstained cells to determine background fluorescence and set proper gates . For multicolor flow cytometry, ensure compensation is properly set if detecting multiple antigens simultaneously.

How should researchers approach epitope retrieval for EN4/CD31 detection in different tissue types?

Epitope retrieval methods for EN4/CD31 detection should be optimized based on tissue type and fixation method. For paraffin-embedded tissues, heat-induced epitope retrieval (HIER) using a basic buffer (pH 9.0) is often effective, while for frozen sections, fixation in cold acetone for 10 minutes may be sufficient. The superior sensitivity of EN4 compared to other CD31 antibodies may allow for less aggressive retrieval methods, potentially preserving tissue morphology . Researchers should perform titration experiments to determine optimal antibody concentration for each tissue type and fixation method, with positive control tissues such as tonsil or placenta included to verify staining effectiveness.

What approaches can minimize interference from pre-existing anti-AAV antibodies in gene therapy applications?

Several approaches can be implemented to minimize interference from pre-existing anti-AAV antibodies:

• Serotype selection: Choose AAV serotypes with lower seroprevalence in the target population (e.g., AAV5 shows lower prevalence than AAV1) .

• Patient screening: Implement sensitive screening assays to identify patients without pre-existing NAbs to the selected serotype.

• Vector engineering: Modify AAV capsid proteins to reduce recognition by pre-existing antibodies while maintaining transduction efficiency.

• Immunomodulation: Consider transient immunosuppression during vector administration to reduce neutralizing antibody impact.

• Alternative delivery routes: Utilize compartmentalized delivery (e.g., intrathecal administration) to bypass circulating antibodies.

The choice of approach should be based on the specific therapeutic application, target tissue, and patient population demographics.

How should researchers interpret discrepancies in AAV neutralizing antibody detection across different assay methods?

Discrepancies in NAb detection across different assays stem from methodological variations that affect sensitivity and specificity. When encountering contradictory results, researchers should consider:

• Assay principle: Transduction inhibition assays tend to be more sensitive than ELISA-based methods for detecting functionally relevant NAbs.

• Serum dilution thresholds: Different studies use various dilution cutoffs (1:1, 1:2, 1:5, etc.) that significantly impact reported seroprevalence rates .

• Cell lines used: Different reporter cell lines may have varying susceptibility to AAV transduction and inhibition.

• Vector preparation: Contaminants in vector preparations can influence assay results.

To resolve discrepancies, researchers should standardize methods within their laboratory and calibrate against reference standards when available. The field would benefit from international standardization efforts for AAV NAb detection methodologies.

What factors should be considered when troubleshooting inconsistent staining with EN4 or Nectin-4 antibodies?

When encountering inconsistent staining with EN4 or Nectin-4 antibodies, researchers should systematically evaluate:

• Antibody integrity: Check for proper storage conditions and expiration dates. Perform positive control staining using tissues or cells known to express the target.

• Epitope accessibility: Optimize epitope retrieval methods based on tissue type and fixation protocol. For Nectin-4, heat-induced epitope retrieval using basic buffer has been validated .

• Fixation issues: Overfixation can mask epitopes, while inadequate fixation may result in poor tissue morphology. Standardize fixation time and conditions.

• Detection system sensitivity: For low-abundance targets, consider signal amplification methods (e.g., tyramide signal amplification).

• Non-specific binding: Increase blocking stringency and optimize antibody dilution to improve signal-to-noise ratio.

Creating a standardized protocol with detailed documentation of each experimental parameter will facilitate troubleshooting and improve reproducibility.

How might understanding AAV serotype immunogenicity patterns inform next-generation vector design?

The observed patterns of AAV serotype immunogenicity, particularly the machine learning-derived clustering that differs from traditional phylogenetic classifications, provide valuable insights for future vector design . Researchers could:

• Identify immunodominant epitopes: Map the specific epitopes responsible for cross-reactivity between serotypes (e.g., AAV1 and AAV6).

• Rational capsid engineering: Modify these immunodominant regions while preserving transduction efficiency.

• Develop chimeric vectors: Create novel capsids combining low-immunogenicity regions from different serotypes.

• Utilize computational prediction: Apply machine learning approaches to predict immunogenicity of newly designed capsids before in vivo testing.

• Explore naturally occurring variants: Screen for rare natural variants with reduced recognition by human antibodies.

These approaches could lead to development of AAV vectors with improved immune evasion properties for clinical gene therapy applications.

What are the implications of age-dependent AAV neutralizing antibody prevalence for pediatric versus adult gene therapy applications?

The observed age-dependent difference in AAV neutralizing antibody prevalence has significant implications for gene therapy applications across different age groups: