Ba71V-126 Antibody Pair

What is the BA71V strain and how does it differ from virulent ASFV isolates?

BA71V is an attenuated tissue-culture adapted strain of African Swine Fever Virus that has been extensively used in laboratory research. Unlike virulent field isolates such as the Georgia 2007/1 (GRG) strain, BA71V can be grown in cell culture systems rather than requiring primary macrophages. The key differences between BA71V and virulent isolates lie in their gene complement, particularly in multigene family (MGF) members, which are often implicated in host immune response suppression .

Transcriptome analysis has revealed that despite these genomic differences, the expression patterns of conserved genes between BA71V and virulent isolates show surprising similarities in both expression levels and temporal regulation. This indicates that virulence determinants are primarily driven by virulent isolate-specific genes rather than differential regulation of shared genes .

What are the key antigenic targets for antibody development in ASFV research?

Several ASFV proteins serve as important antigenic targets for antibody development, including:

• CD2v protein (EP402R gene product) - A heavily glycosylated membrane protein that mediates hemadsorption and is implicated in virulence and host range

• p72 capsid protein (B646L) - The major structural protein and commonly used diagnostic target

• p30 (CP204L) - An early protein frequently used for diagnostic purposes

• p54 (E183L) - A membrane protein involved in virus attachment

The selection of appropriate antigens for antibody development depends on the research objectives. CD2v extracellular domain expressed in mammalian systems (like 293F cells) offers advantages for generating antibodies against conformational epitopes due to proper glycosylation .

How can I verify the specificity of antibodies against BA71V-126 proteins?

Verification of antibody specificity requires multiple complementary approaches:

For comprehensive validation, test antibodies against both recombinant proteins and virus-infected cells. Additionally, include appropriate controls such as uninfected cells and isotype control antibodies to ensure specificity .

What are the best expression systems for producing ASFV antigens for antibody development?

The choice of expression system significantly impacts the quality of antibodies generated against ASFV proteins:

For heavily glycosylated proteins like CD2v, mammalian expression systems are strongly recommended. Research has shown that the 293F expression system provides proper glycosylation of the CD2v extracellular region, which is critical for generating antibodies that recognize native viral proteins . For non-glycosylated structural proteins, bacterial expression systems may be sufficient.

How should I design experiments to compare gene expression between BA71V and virulent ASFV strains?

When designing transcriptome experiments comparing BA71V and virulent ASFV strains:

• Standardize infection conditions: Use a high multiplicity of infection (MOI of ~5) to ensure synchronized infection cycles and minimize the complication of variable proportions of uninfected cells .

• Select appropriate time points: Include both early (~5 hpi) and late (~16 hpi) time points to capture the temporal dynamics of gene expression .

• Choose appropriate sequencing methodology: Cap Analysis of Gene Expression sequencing (CAGE-seq) allows precise mapping of transcription start sites and quantification of transcript levels .

• Normalize data appropriately: For direct comparisons, use reads per million (RPM) to account for differences in sequencing depth across samples .

• Validate key findings: Confirm RNA-seq results using qRT-PCR or protein expression analysis.

Data analysis should include both differential expression analysis for individual genes and cluster analysis to identify co-regulated gene groups .

What are the critical considerations for developing monoclonal antibodies against BA71V proteins?

Developing effective monoclonal antibodies against BA71V proteins requires careful attention to:

• Immunogen design: Express the full extracellular domain or functional regions of membrane proteins in mammalian systems to ensure proper folding and glycosylation .

• Immunization protocol: Use a prime-boost strategy with Freund's complete adjuvant for primary immunization, followed by incomplete adjuvant for boosters at 21 and 42 days .

• Hybridoma screening strategy:

  • Initial screening by ELISA against recombinant protein

  • Secondary screening by Western blot

  • Confirmatory testing by IPMA or IFA with virus-infected cells

• Clone selection criteria: Select hybridomas based on:

  • Antibody titer and affinity

  • Specificity in multiple assay formats

  • Recognition of native viral protein in infected cells

  • Stability in culture

• Epitope mapping: Characterize the binding epitopes through truncation mutants or peptide arrays to understand antibody specificity .

How should I interpret changes in viral gene expression patterns during infection?

Interpretation of ASFV gene expression patterns requires understanding of both temporal regulation and relative abundance:

• Early vs. Late genes: Categorize genes based on expression patterns:

  • Early genes: High expression at early time points (5 hpi) that may persist or decline

  • Late genes: Low expression early, with significant upregulation at late time points (16 hpi)

• Expression clusters: More refined analysis reveals five distinct expression patterns:

  • Cluster 1 (H-H): High expression maintained throughout infection

  • Cluster 2 (L-H): Low early, high late expression

  • Cluster 3 (L-M): Low early, moderate late expression

  • Cluster 4 (M-M): Moderate expression maintained throughout

  • Cluster 5 (LM-LM): Low-moderate expression maintained throughout

• Functional correlation: Genes with similar functions often share expression patterns. For example:

  • RNA polymerase subunits typically belong to cluster 4 (M-M)

  • Structural proteins often belong to cluster 2 (L-H)

  • Transcription initiation factors may show different patterns depending on their roles in the infection cycle

• Strain comparisons: When comparing BA71V to virulent strains like GRG, focus on:

  • Expression differences in conserved genes (relatively rare)

  • Presence/absence and expression of strain-specific genes (particularly MGFs)

  • Differences in the timing or magnitude of expression changes

What approaches should be used to map epitopes recognized by antibodies against BA71V proteins?

For comprehensive epitope mapping:

• Initial mapping with truncated proteins:

  • Create a series of truncated protein constructs

  • Express constructs in mammalian cells

  • Test antibody binding by Western blot or ELISA

• Fine mapping with overlapping peptides:

  • Synthesize overlapping peptides (15-20 amino acids with 5-10 residue overlap)

  • Test binding using Dot-Blot or peptide ELISA

  • Identify minimal epitope sequence through peptide truncation

• Confirmation with point mutations:

  • Introduce alanine substitutions at key positions

  • Test impact on antibody binding

  • Identify critical contact residues

• Structural context analysis:

  • Map epitope onto protein structure (if available)

  • Evaluate surface exposure and accessibility

  • Assess conservation across ASFV strains

For example, in studies with CD2v protein, researchers identified a linear epitope (154SILE157) using a combination of truncation constructs followed by testing with overlapping peptides in Dot-Blot, ELISA, and IFA tests .

How can I interpret differences in promoter motifs between early and late ASFV genes?

Analysis of ASFV promoter architecture reveals distinct patterns that control gene expression timing:

• Early Promoter Motifs (EPM):

  • Contain conserved sequence signatures

  • Positioned approximately 9-10 nucleotides upstream of transcription start site (TSS)

  • Often include characteristic 'TA' motif at the Initiator (Inr) element

• Late Promoter Features:

  • Often lack strong conserved sequence motifs

  • May depend more on genome replication and changes in DNA accessibility

  • Correlate with increased pervasive transcription during late infection

• Contextual analysis:

  • Examine the relationship between promoter sequences and expression timing

  • Compare motifs across different ASFV strains (BA71V vs. virulent isolates)

  • Correlate with functional gene categories

• Biological implications:

  • Early genes often encode regulatory and immune evasion proteins

  • Late genes typically encode structural and virion assembly proteins

  • Temporal regulation ensures proper virus assembly sequence

Understanding these promoter elements is essential for predicting gene expression timing and designing experiments to target specific stages of the viral lifecycle.

How can I address low antibody specificity or cross-reactivity issues with BA71V proteins?

When troubleshooting specificity issues:

• Analyze potential cross-reactive targets:

  • Perform sequence homology searches

  • Test against related viral proteins

  • Include appropriate controls (uninfected cells, isotype controls)

• Optimize assay conditions:

  • Adjust antibody concentration

  • Modify blocking reagents (try different blockers like BSA, milk, or commercial blockers)

  • Increase washing stringency (higher salt, or addition of detergents)

• Epitope engineering approaches:

  • Select unique regions for immunization

  • Avoid conserved domains shared with host proteins

  • Consider affinity maturation techniques

• Purification strategies:

  • Perform negative selection against cross-reactive antigens

  • Use epitope-specific affinity purification

  • Consider subclass switching if isotype may contribute to non-specific binding

What approaches can be used to study the temporal dynamics of BA71V gene expression?

For detailed temporal analysis:

• High-resolution time course:

  • Sample at multiple time points (0, 2, 4, 6, 8, 12, 16, 24 hpi)

  • Use synchronized infection (high MOI of ~5)

  • Apply genome-wide transcriptomics (RNA-seq or CAGE-seq)

• Nascent RNA labeling:

  • Use metabolic labeling with 4-thiouridine (4sU)

  • Capture newly synthesized RNA at each time point

  • Distinguish primary transcription from RNA accumulation

• Single-cell approaches:

  • Apply scRNA-seq to capture cell-to-cell variability

  • Identify distinct infection phases within population

  • Correlate with viral protein expression by flow cytometry

• Quantitative analysis frameworks:

  • Calculate the percentage of reads per gene detected at late vs. early timepoints

  • Cluster genes based on expression patterns (e.g., H-H, L-H, L-M, M-M, LM-LM clusters)

  • Correlate with genome replication by qPCR of viral DNA

For a comprehensive understanding, integrate transcriptomic data with measurements of viral genome replication, as the ratio of viral transcripts increases approximately 2-fold in GRG and 8-fold in BA71V from early to late infection, which correlates with a 15-fold increase in GRG genome copy numbers and a 30-fold increase in BA71V genomes .

How can antibodies against BA71V proteins be applied in advanced research applications?

Antibodies against BA71V proteins can be applied in multiple advanced research contexts:

Each application requires specific validation steps to ensure antibodies perform optimally in the selected experimental context.

How might single-cell approaches advance our understanding of BA71V infection dynamics?

Single-cell technologies offer powerful new insights into ASFV infection:

• Single-cell RNA sequencing (scRNA-seq):

  • Reveals heterogeneity in cellular responses to infection

  • Identifies distinct cell states during infection progression

  • Allows precise temporal ordering of infection events

• Spatial transcriptomics:

  • Maps infection dynamics within tissue context

  • Identifies local microenvironmental factors influencing infection

  • Correlates with immune cell infiltration patterns

• CyTOF and spectral flow cytometry:

  • Enables high-dimensional profiling of protein expression

  • Allows simultaneous detection of multiple viral and host proteins

  • Reveals rare cell populations with distinct infection phenotypes

• Integration with BA71V-126 antibody applications:

  • Use antibodies to sort infected cells at different stages

  • Combine protein and RNA detection in the same cells

  • Track infection progression at single-cell resolution

These approaches can address fundamental questions about why some cells support productive infection while others are resistant, even with standardized MOI.

What are the implications of ASFV promoter architecture for synthetic biology applications?

Understanding ASFV promoter architecture enables several synthetic biology applications:

• Engineered expression systems:

  • Design synthetic promoters with predictable temporal activity

  • Create attenuated virus strains with altered gene expression

  • Develop reporter viruses for tracking infection

• Vaccine development strategies:

  • Modulate antigen expression timing and levels

  • Engineer strains with enhanced immunogenicity

  • Create temperature-sensitive variants through promoter modification

• ASFV-based vector development:

  • Utilize ASFV promoters in heterologous expression systems

  • Develop large-capacity vectors for gene delivery

  • Create chimeric promoters with novel properties

The detailed understanding of early promoter motifs (EPM) with their conserved sequence signatures positioned 9-10 nucleotides upstream of the transcription start site provides a foundation for rational design of synthetic regulatory elements .