Ba71V-93 Antibody Pair

What is the BA71V-93 Antibody Pair and how does it relate to ASFV research?

The BA71V-93 Antibody Pair is a detection tool developed for research on African Swine Fever Virus, specifically targeting epitopes from the BA71V strain. BA71V is a well-characterized non-pathogenic ASFV strain that has been adapted to grow in Vero cells, making it valuable for laboratory research. Unlike virulent field strains, BA71V does not cause disease in pigs and lacks the ability to induce specific immune responses in vivo, as demonstrated in controlled experiments . This antibody pair likely consists of capture and detection antibodies that recognize specific viral proteins, enabling the quantification of viral particles or viral proteins in research samples.

What are the primary applications of the BA71V-93 Antibody Pair in ASFV research?

This antibody pair can be utilized in several critical research applications including:

• Monitoring viral replication kinetics in cell culture systems

• Quantifying viral load in experimental samples

• Validating attenuation in candidate vaccine strains

• Differentiating vaccinated from infected animals (DIVA strategies)

• Tracking virus distribution in tissue samples

The antibody pair would be particularly valuable when studying attenuated ASFV strains like BA71ΔCD2, which have shown promise as vaccine candidates while maintaining genetic stability during in vitro propagation .

How does the CD2v deletion affect antibody detection in ASFV research?

The deletion of the CD2v protein (encoded by the EP402R gene) creates important considerations for antibody-based detection systems. CD2v functions as a hemagglutinin in ASFV and mediates hemadsorption, a characteristic used in traditional ASFV diagnostics. When working with CD2v-deleted strains like BA71ΔCD2, researchers must note that:

• These viruses cannot be detected via hemadsorption assays

• Specific antibody pairs targeting non-CD2v epitopes are necessary

• Immunological testing can function as a DIVA strategy since "BA71ΔCD2 does not induce antibodies against CD2v, thus making it easy to differentiate vaccinated from infected pigs"

This property creates both challenges and opportunities when designing detection systems for ASFV research.

How can researchers optimize experimental design when using the BA71V-93 Antibody Pair for cross-protection studies?

When designing cross-protection studies that utilize the BA71V-93 Antibody Pair, researchers should implement the following methodological considerations:

• Control selection: Include appropriate controls such as samples from pigs inoculated with virulent BA71, non-pathogenic BA71V, and deletion mutants like BA71ΔCD2 to establish baseline detection parameters.

• Sampling timeline: Design a strategic sampling schedule based on known ASFV kinetics. For instance, viremia from virulent BA71 becomes detectable by qPCR from day 4 post-infection and increases progressively until death, while attenuated strains like BA71ΔCD2 may not produce detectable viremia .

• Parallel assay validation: Always run parallel detection methods (qPCR, ELISA, ELISPOT) alongside antibody pair detection to validate results and resolve potential discrepancies.

• Challenge strain considerations: When testing cross-protection, include samples from heterologous challenges (like E75 or Georgia 2007/1) to verify antibody pair sensitivity across genetically diverse ASFV strains .

Research has shown that protection against ASFV challenges correlates with specific immune responses, though not perfectly at the individual animal level. For example, some pigs showing high numbers of specific T cells have still succumbed to challenge, while others with non-detectable antibodies have survived .

What approaches can resolve contradictory data between antibody detection results and clinical outcomes in ASFV research?

Researchers frequently encounter scenarios where antibody-based detection results do not perfectly align with clinical outcomes or protection status. To resolve these discrepancies:

• Implement multi-parameter immune analysis: Combine antibody detection with functional T-cell assays. Research shows that "protection afforded by BA71ΔCD2 correlated with the presence of specific humoral and cellular responses before BA71 challenge" , yet exceptions exist at the individual animal level.

• Analyze CD8+ T-cell cross-reactivity: Conduct CFSE proliferation assays using PBMCs obtained post-vaccination and stimulated with different ASFV strains. This approach revealed that "every single pig inoculated with BA71ΔCD2 showed specific CD8+ T cells in their blood, capable of proliferating in vitro in response to either BA71 or E75" .

• Dose-response relationship analysis: Systematically examine protection outcomes across different vaccination doses. For example:

This data demonstrates a clear dose-dependent protection pattern that can help contextualize antibody detection results .

How can the BA71V-93 Antibody Pair be utilized to investigate immunodominant ASFV epitopes?

To leverage this antibody pair for epitope mapping research:

• Competitive binding assays: Use the antibody pair alongside sera from ASFV-surviving pigs to identify potential immunodominant regions through competition studies.

• Cross-competition with known immunodominant antigens: Several ASFV antigens have been "described as constantly recognized by sera from pigs surviving ASFV infection" . Compare antibody pair binding in the presence of these proteins.

• T-cell epitope correlation: Investigate whether epitopes recognized by the antibody pair correlate with regions recognized by cross-reactive CD8+ T cells. This is particularly relevant since "immunodominant cytotoxic T-lymphocyte (CTL) determinants have so far remained elusive" .

• CD2v immunomodulation assessment: The research indicates that "CD2v [can] inhibit mitogen-dependent lymphocyte proliferation" , potentially contributing to immunodominance patterns. The antibody pair could help track CD2v presence/absence and correlate it with broader T-cell response patterns.

What are the optimal sample preparation protocols when using the BA71V-93 Antibody Pair with different porcine tissue types?

When preparing different porcine tissue samples for analysis with the BA71V-93 Antibody Pair, researchers should adopt tissue-specific protocols:

• Blood/Serum samples:

  • Collect blood during peak viremia periods (typically 4-7 days post-infection for virulent strains)

  • Process within 4 hours of collection to prevent degradation

  • Separate serum via centrifugation (10 minutes at 2,000g)

  • If using for viremia quantification, refer to established protocols that can detect "high viremia detectable in serum by quantitative PCR (qPCR) from day 4 p.i."

• Macrophage preparations:

  • For porcine alveolar macrophages (PAMs), follow lung lavage protocols

  • Process immediately or cryopreserve in 10% DMSO

  • Note that both "BA71 and BA71ΔCD2 could grow equally well in the COS-1 cell line and in porcine alveolar macrophages (PAMs) without any effect on their genetic stability, pathogenicity, or immunogenicity"

• Tissue homogenates:

  • Collect target tissues (lymph nodes, spleen, tonsil) immediately post-mortem

  • Homogenize in appropriate buffer with protease inhibitors

  • Filter through 0.45μm filter before applying antibody detection protocols

Selection of appropriate positive and negative controls is critical for each tissue type to establish detection thresholds.

How can researchers troubleshoot inconsistent results when implementing the BA71V-93 Antibody Pair in different assay formats?

When facing inconsistent results across different assay formats, follow this structured troubleshooting approach:

• Verify antibody integrity:

  • Test antibody functionality using a standardized positive control

  • Perform titration series to identify optimal working concentrations

  • Confirm storage conditions have been maintained properly

• Evaluate detection sensitivity limits:

  • Determine the lower limit of detection using serial dilutions

  • Compare with PCR-based detection which has shown sensitivity for detecting "viremia from day 7 to 24 p.i., about 4 logs lower than in the control pigs"

  • Establish whether inconsistencies occur at specific viral load thresholds

• Assess matrix interference:

  • Different sample types may contain inhibitory components

  • Implement sample dilution series to identify potential inhibition

  • Consider sample pre-treatment methods to reduce background

• Cross-format standardization:

  • Develop a common set of reference standards usable across all assay formats

  • Calculate correction factors for inter-format conversion

  • Implement Bland-Altman analysis to characterize systematic differences between methods

Research has shown that immune response detection methods like ELISA and IFN-γ ELISPOT can show variable correlation with protection , suggesting multiple detection approaches may be necessary for comprehensive analysis.

How does the BA71V-93 Antibody Pair compare with other detection methods for analyzing cross-protective immune responses?

When evaluating cross-protective immune responses, researchers should understand the comparative advantages of different detection methods:

Research demonstrates that comprehensive immune monitoring requires multiple approaches, as "protection afforded by BA71ΔCD2 correlated with the presence of specific humoral and cellular responses" , yet individual metrics alone were insufficient to predict protection.

What are the critical considerations when using the BA71V-93 Antibody Pair to differentiate between vaccine candidates of varying attenuation?

When using this antibody pair to evaluate and differentiate ASFV vaccine candidates, researchers should:

• Establish attenuation baseline profiles:

  • Compare detection profiles across known virulence variants:

    • Virulent strains (BA71, E75): Cause "clinical signs associated with acute ASF, including fever and high viremia"

    • Attenuated candidates (BA71ΔCD2): Show no clinical signs or detectable viremia

    • Non-replicating candidates (BA71ΔTK, BA71ΔTKv220i): Complete attenuation with "no recombinant virus detectable at any time postinfection"

• Correlate antibody detection with protection metrics:

  • Track antibody detection parameters alongside:

    • Clinical protection: "Pigs inoculated with the intermediate or high doses of BA71ΔCD2 did not show significant ASF clinical signs or viremia"

    • Cross-protection capability: "Immunization with either 3.3 × 10⁴ or 10⁶ PFU of BA71ΔCD2 fully protected all 12 pigs against heterologous E75 lethal challenge"

    • Cellular immunity induction: "The ability of BA71ΔCD2 to induce cross-reactive CD8+ T cells contrasts with the E75-restricted repertoire induced by the classically attenuated E75CV1 virus"

• Assess genetic stability correlation:

  • Monitor detection consistency across cell passage numbers

  • Note that "the genetic stability observed for BA71ΔCD2 after 20 consecutive passages in COS-1 cells" demonstrates long-term consistency

• Evaluate DIVA capability:

  • Determine whether the antibody pair can distinguish between vaccinated and infected animals

  • Integrate with other DIVA strategies since "BA71ΔCD2 does not induce antibodies against CD2v, thus making it easy to differentiate vaccinated from infected pigs"

How can the BA71V-93 Antibody Pair contribute to identifying new correlates of protection against ASFV?

To leverage this antibody pair for developing new correlates of protection:

• Integrated biomarker discovery:

  • Combine antibody detection data with transcriptomic and proteomic profiling

  • Correlate detection results with survival outcomes after challenge

  • Identify early antibody recognition patterns that predict protection

• Cross-protective epitope targeting:

  • Use the antibody pair to screen for conserved epitopes across ASFV genotypes

  • Investigate whether these regions correlate with "the ability of BA71ΔCD2 to induce CD8+ T cells that specifically recognized both viruses in vitro"

• Mechanistic studies of CD2v role:

  • Employ the antibody pair to investigate CD2v-dependent and independent protection mechanisms

  • Explore whether "the already-described ability of CD2v to inhibit mitogen-dependent lymphocyte proliferation might contribute to the immunodominance observed"

• Dose-optimization studies:

  • Utilize the antibody pair to establish minimum protective antigen thresholds

  • Reference existing dose-response data showing that protection is "dose dependent" with significant differences between 10³ PFU (partial protection) and higher doses (complete protection)

Developing robust correlates of protection will address the current challenge where "a lack of total correlation was observed between protection and the level of ASFV-specific immunity present at the time of challenge" .

What refinements in experimental protocols can enhance the utility of the BA71V-93 Antibody Pair in DIVA strategies?

To optimize DIVA applications of this antibody pair:

• Multiplex DIVA platforms:

  • Combine this antibody pair with assays detecting CD2v antibodies

  • Develop integrated testing algorithms that account for "the natural presence of nonhemadsorbing ASFV strains in certain regions of the world"

• Field-applicable protocol modifications:

  • Adapt laboratory protocols for field conditions

  • Develop sample stabilization methods for remote collection

  • Validate against diverse field strains, particularly given that "there is still room for improvement, mainly from the biosafety point of view and regarding its ability to induce an immune response distinguishable from those detectable in infected animals"

• Reference standard development:

  • Establish international reference standards for antibody detection

  • Create calibrated positive controls at defined thresholds

  • Validate against sera panels from different vaccination and challenge scenarios

Refinements in these areas would address the ongoing need for "more DIVA markers in the future, besides the presence of antibodies against the positive marker" .