DEGP4 Antibody

What are the binding properties of monoclonal antibodies against DENV4?

Monoclonal antibodies against DENV4 exhibit diverse binding properties that critically impact their function. Research indicates that some DENV4 mAbs bind specifically to soluble E protein, while others preferentially recognize epitopes displayed only on intact virions. For instance, in characterization studies, antibodies like L8, M1, B10, and I7 demonstrated binding to both DENV2 soluble E protein and reporter virus particles, while others such as J9 and C4 bound exclusively to virus particles, suggesting epitopes preferentially displayed on the intact virion . When designing binding assays, researchers should evaluate antibody binding using both ELISA with purified proteins and assays with intact viral particles to comprehensively profile binding characteristics.

How are neutralizing versus non-neutralizing DENV4 antibodies differentiated experimentally?

Differentiation between neutralizing and non-neutralizing antibodies requires systematic evaluation through plaque reduction neutralization tests (PRNT). The methodology involves:

• Serial dilution of purified antibodies (typically from 10 μg/ml downward)

• Pre-incubation with standardized viral inocula

• Infection of susceptible cells (e.g., BHK-21, Vero)

• Quantification of plaque formation compared to control

Neutralizing antibodies demonstrate dose-dependent reduction in plaque formation, often achieving complete neutralization. In contrast, non-neutralizing antibodies show minimal impact on viral infection rates even at high concentrations. For example, research has identified antibodies like J9 that completely neutralize DENV1-4 with IC₅₀ values ranging from 6-39 ng/ml, while others like B10 and M1 demonstrate incomplete neutralization with 10-50% infectivity remaining at antibody concentrations as high as 10 μg/ml .

How do researchers identify broadly neutralizing antibody (bNAb) epitopes against multiple dengue serotypes?

Identification of bNAb epitopes against multiple dengue serotypes requires a multi-faceted experimental approach:

• Antibody Isolation and Characterization:

  • Single-cell sorting of plasmablasts from DENV-infected individuals

  • Transcriptomic analysis of antibody sequences

  • Cloning and expression of monoclonal antibodies

• Cross-Reactivity and Neutralization Assessment:

  • Binding assays against all four DENV serotypes

  • Neutralization assays against reporter flaviviruses (DENV1-4, ZIKV, WNV)

  • Quantification of neutralization potency (IC₅₀ values)

• Epitope Mapping:

  • Mutagenesis studies on E protein domains

  • Competition binding assays with known antibodies

  • Structural analysis (cryo-EM or crystallography)

This approach has successfully identified antibodies like J9 and J8, which target determinants in E protein domain I (DI) and potently neutralize all four DENV serotypes with IC₅₀ values in the low picomolar range . The identification of these distinct epitopes from previously characterized bNAbs provides valuable targets for rational vaccine design efforts.

What methodologies are most effective for studying antibody-dependent enhancement (ADE) of dengue infection?

ADE studies require careful experimental design, incorporating:

In Vitro Methodology:

• Cell Selection: Use Fc receptor-bearing cells such as K562 (human myeloid) or P-388D1 (mouse macrophage-like) cells

• Antibody Preparation: Dilute antibodies serially to sub-neutralizing concentrations

• Infection Protocol:

  • Pre-incubate virus with diluted antibodies

  • Infect cells at multiplicity of infection (MOI) 0.1-1

  • Measure enhancement using:

    • Flow cytometry (% infected cells)

    • Viral RNA quantification (RT-qPCR)

    • Viral plaque assays (from supernatants)

In Vivo Methodology:

• Animal Model Selection: AG129 mice (lacking IFN-α/β and -γ receptors)

• Protocol:

  • Administer antibodies at defined concentrations

  • Challenge with sub-lethal DENV dose

  • Monitor:

    • Viremia levels

    • Clinical symptoms

    • Survival rates

Studies have identified that cross-reactive, poorly neutralizing mAbs like DD11-4 and DD18-5 strongly enhance DENV1-4 infection in K562 cells and increase mortality in AG129 mice . The epitope residues for these enhancing antibodies were identified as W212 and E26 on the E protein using virus-like particle (VLP) mutants .

How can researchers track the evolutionary pathways of B cell lineages producing broadly neutralizing antibodies against DENV?

Tracking B cell evolutionary pathways involves sophisticated immunological and computational techniques:

• Sample Collection Timeline:

  • Acute phase (1-7 days post-symptom onset)

  • Early convalescent phase (15-30 days)

  • Late convalescent phase (3-6 months)

• B Cell Repertoire Analysis:

  • Single-cell RNA sequencing of plasmablasts

  • Bulk B cell receptor (BCR) sequencing

  • Germline gene assignment and clonal family grouping

• Somatic Hypermutation (SHM) Analysis:

  • Construction of phylogenetic trees for antibody lineages

  • Reversion mutations to germline sequences

  • Expression of ancestral and intermediate antibodies

• Functional Assessment:

  • Binding affinity measurements (SPR/BLI)

  • Neutralization potency against multiple serotypes

  • Epitope mapping at each developmental stage

This approach has revealed that clonally related bNAbs like J9 and J8 can follow divergent somatic hypermutation pathways, suggesting multiple evolutionary routes to generate broadly neutralizing activity within a single lineage . Analysis has shown that a limited number of key mutations may be sufficient for neutralizing activity, providing insights for immunogen design to elicit similar antibodies through vaccination.

What are the optimal conditions for using DENV4 antibodies in multiplex detection assays?

When incorporating DENV4 antibodies into multiplex detection platforms, researchers should consider:

Antibody Panel Design:

• Group antibodies based on epitope abundance (ranging from 0.05 to 10 μg/mL)

• Conduct titration experiments to determine optimal concentration

• Balance between signal strength and background

Concentration Optimization:
Research demonstrates that antibody dilution affects signal differently based on target abundance. A four-fold dilution of antibody mixtures results in only 38-51% reduction in UMI counts, rather than the expected 75% reduction . This suggests antibodies reach saturation plateaus between 0.62-2.5 μg/mL for many epitopes, and higher concentrations only increase background signal.

Staining Volume Optimization:
Reducing staining volume from 50μl to 25μl (effectively halving antibody amount while doubling cell density) produces different effects based on epitope expression levels:

• Highly expressed markers show most significant signal increases

• Markers with low positive signal benefit from increased cell:antibody ratio

• Background signal percentage can be dramatically reduced (e.g., CD86 background reduced from 76.5% to 12.6%)

How should researchers interpret heterogeneous epitope distribution in DENV4 antibody cross-reactivity studies?

The heterogeneous distribution of epitopes across dengue serotypes creates interpretive challenges. When analyzing cross-reactivity:

• Strain Variation Analysis:

  • Test antibodies against multiple strains within each serotype

  • Group viral strains into subgroups based on reactivity patterns

  • Document epitope conservation across evolutionary distances

• Epitope Mapping Approach:

  • Use virus-like particle (VLP) mutants to identify key residues

  • Compare reactivity patterns across serotype representatives

  • Create epitope maps based on neutralization fingerprints

• Data Interpretation Framework:

  • Distinguish between serotype-specific and cross-reactive epitopes

  • Identify strain-specific variations within serotypes

  • Correlate epitope recognition with functional properties

What quality control measures ensure reproducible results when working with DENV4 antibodies?

Implementing rigorous quality control is essential for reproducible antibody-based experiments:

Pre-Experimental QC:

• Antibody Validation:

  • Confirm specificity via Western blot against recombinant E protein

  • Verify binding to infected cells by immunofluorescence

  • Test for cross-reactivity against all four DENV serotypes

• Standardized Titration:

  • Perform binding curves using ELISA (0.01-10 μg/mL range)

  • Determine EC₅₀ values for each lot of antibody

  • Establish working concentration at 2-5× EC₅₀

Experimental Controls:

• Positive Controls:

  • Include well-characterized reference antibodies (e.g., EDE antibodies C10 and B7)

  • Use serotype-specific and pan-DENV reactive controls

• Negative Controls:

  • Isotype-matched non-specific antibodies

  • Uninfected cell lysates or particles

• Technical Replicates:

  • Minimum of three technical replicates per condition

  • Inter-assay controls for day-to-day variability

Post-Experimental Validation:

• Signal-to-Noise Ratio Analysis:

  • Calculate S/N ratio for each antibody application

  • Establish minimum acceptable S/N thresholds

• Reproducibility Assessment:

  • Compare results across multiple experiments

  • Document lot-to-lot variation in antibody performance

Implementing these measures ensures that variations in experimental outcomes reflect biological differences rather than technical inconsistencies in antibody performance or assay conditions.

How can DENV4 antibody studies inform vaccine design strategies?

DENV4 antibody studies provide critical insights for vaccine development through:

Epitope-Focused Vaccine Design:

• Identification of bNAb Targets:

  • E protein domain I (DI) epitopes targeted by antibodies like J9 and J8

  • Novel epitopes distinct from previously characterized bNAbs

• Multiple Specificity Integration:

  • Include multiple distinct epitopes in vaccine formulations

  • Target conserved regions across serotypes

  • Minimize exposure of enhancing epitopes (like W212 and E26)

B Cell Lineage-Based Approaches:

• Germline-Targeting Strategies:

  • Design immunogens targeting germline precursors of bNAb lineages

  • Create sequential immunization protocols to guide affinity maturation

• Minimal Mutation Pathways:

  • Focus on key mutations sufficient for neutralizing activity

  • Design immunogens that specifically elicit these critical mutations

ADE Risk Mitigation:

• Epitope Engineering:

  • Modify immunogens to reduce presentation of enhancing epitopes

  • Engineer out epitopes recognized by antibodies like DD11-4 and DD18-5

• Balanced Immune Response:

  • Ensure balanced response against all four serotypes

  • Promote high-quality neutralizing antibodies over enhancing antibodies

These approaches address the fundamental challenge in dengue vaccine development: eliciting protective immunity against all serotypes while avoiding enhancement of infection upon subsequent exposure to heterologous serotypes.

What methodological approaches best characterize the function of DBF4 antibodies in cellular research?

When working with antibodies against DBF4 (also known as ASK):

Antibody Selection Criteria:

• Specificity Verification:

  • Western blot confirmation against recombinant DBF4 protein

  • Immunoprecipitation followed by mass spectrometry

  • Knockdown/knockout validation in relevant cell lines

• Application-Specific Validation:

  • Western blot (WB) for protein expression analysis

  • Immunocytochemistry (ICC) for subcellular localization

  • Immunohistochemistry (IHC) for tissue expression patterns

  • Immunofluorescence (IF) for co-localization studies

Experimental Protocols:

• Nuclear Protein Detection:

  • Optimize nuclear extraction protocols

  • Use appropriate nuclear markers for co-localization

  • Apply subcellular fractionation techniques

• Functional Studies:

  • Chromatin immunoprecipitation (ChIP) for DNA binding analysis

  • Co-immunoprecipitation for protein interaction studies

  • Combine with cell cycle synchronization techniques

The DBF4 protein is a 674-amino acid nuclear protein with a mass of approximately 76,858 daltons . When selecting anti-DBF4/ASK antibodies, researchers should consider the specific application requirements and validate antibody performance in their particular experimental system.

How might single-cell technologies advance our understanding of DENV4 antibody responses?

Single-cell technologies offer unprecedented opportunities to dissect DENV4 antibody responses:

Advanced Methodological Approaches:

• Single-Cell Antibody Repertoire Sequencing:

  • Paired heavy/light chain sequencing from individual B cells

  • Linkage of transcriptional state with antibody sequences

  • Correlation of clonal expansion with neutralization breadth

• Spatial Transcriptomics of Germinal Centers:

  • Mapping of B cell maturation in lymphoid tissues

  • Spatial relationships between T follicular helper cells and maturing B cells

  • Tracking of clonal selection in germinal center reactions

• Multi-modal Single-Cell Analysis:

  • Integration of protein expression with transcriptional profiles

  • Optimization of oligo-conjugated antibody signal in multimodal assays

  • Balancing antibody concentrations for effective signal detection

These technologies will provide deeper insights into:

• Developmental trajectories of broadly neutralizing antibody lineages

• Molecular determinants of cross-reactivity versus serotype-specificity

• Factors influencing the balance between protective and enhancing antibodies

By applying these advanced approaches, researchers can develop more effective vaccination strategies and therapeutic antibodies targeting DENV4 and other dengue serotypes.