FBA1 Antibody

What is FBA1 and where is it found in different organisms?

FBA1 (fructose-bisphosphate aldolase 1) is a glycolytic enzyme that catalyzes reversible aldol reactions during gluconeogenesis and glycolysis. This enzyme exists across multiple kingdoms with varying functions:

• In plants such as Arabidopsis thaliana, it functions as a chloroplastic enzyme involved in carbon fixation and metabolic pathways .

• In fungi, particularly Candida species, Fba1 serves as both a metabolic enzyme and a potential diagnostic marker for invasive infections .

• In bacteria, particularly Group A Streptococcus (GAS), the FbaA homolog functions not only as a metabolic enzyme but also as a virulence factor that interacts with host immune regulatory proteins .

The conservation of this enzyme across diverse organisms makes it both a valuable research target and creates challenges for developing species-specific antibodies.

What are the most effective validation methods for FBA1 antibodies?

Effective validation of FBA1 antibodies requires multiple orthogonal approaches to confirm specificity and functionality:

• Western blotting: Should demonstrate bands at the expected molecular weight (~74-77 kDa for some isoforms, though post-translational modifications may result in apparent weights of ~100 kDa) .

• Knockout validation: Essential for confirming antibody specificity, as demonstrated by YCharOS initiative which applies comprehensive knockout characterization to hundreds of antibodies .

• Immunoprecipitation (IP): Helps confirm native protein recognition and can identify interaction partners .

• Immunofluorescence (IF): Validates subcellular localization patterns consistent with known biology .

• Cross-reactivity testing: Particularly important when working with conserved proteins like FBA1 across different species .

How should FBA1 antibodies be stored to maintain optimal activity?

For maximum stability and performance, FBA1 antibodies require careful handling:

• Store lyophilized antibodies in a manual defrost freezer to prevent degradation from temperature fluctuations .

• Avoid repeated freeze-thaw cycles which can cause protein denaturation and reduced activity .

• Upon receipt, immediately transfer products shipped at 4°C to the recommended storage temperature .

• For reconstituted antibodies, aliquoting into single-use volumes helps minimize the number of freeze-thaw cycles.

• Follow manufacturer recommendations for specific antibody formulations, as storage conditions may vary between preparations.

Proper storage significantly extends antibody shelf-life and maintains consistent experimental performance.

What applications are FBA1 antibodies most commonly used for in research?

FBA1 antibodies serve diverse research applications across multiple disciplines:

• Diagnostic applications: Detection of invasive candidiasis through ELISA-based assays with reported sensitivity of 87.1% and specificity of 92.8% for anti-Fba1 antibodies .

• Host-pathogen interaction studies: Investigating how bacterial FbaA interacts with human complement regulatory proteins (factor H and FHL-1) to evade immune responses .

• Metabolic pathway investigation: Studying glycolysis and gluconeogenesis regulation in various organisms .

• Subcellular localization studies: Determining the cellular distribution of FBA1 in different tissues and under various conditions .

• Protein-protein interaction analyses: Identifying binding partners through co-immunoprecipitation experiments .

These applications highlight the versatility of FBA1 antibodies as research tools across multiple biological contexts.

How do specificity and cross-reactivity issues affect experimental design when using FBA1 antibodies?

Cross-reactivity represents a significant challenge when working with FBA1 antibodies due to the high conservation of this enzyme across species:

The PHY0406S antibody against Arabidopsis thaliana FBA1 cross-reacts with homologs in multiple plant species including Brassica napus, Solanum lycopersicum, Gossypium raimondii, and others . Additionally, the synthetic peptide used for immunization shares 100% homology with FBA2 (AT4G38970), creating potential for isoform cross-reactivity .

For experimental design, researchers should:

• Include appropriate controls including knockout/knockdown samples when possible.

• Validate antibody specificity in their specific experimental system prior to main studies.

• Consider using epitope-tagged versions of the protein when studying specific isoforms.

• Employ multiple antibodies recognizing different epitopes to increase confidence in results.

• Use complementary detection methods (e.g., mass spectrometry) to confirm antibody-based findings.

The development of computational approaches for predicting antibody specificity, as described by researchers using biophysics-informed models, may help address these challenges in the future .

What are the implications of FbaA binding to complement regulatory proteins in pathogenic organisms?

The interaction between bacterial FbaA and human complement regulatory proteins represents a sophisticated immune evasion mechanism with significant research implications:

FbaA in Group A Streptococcus binds to human factor H (FH) and factor H-like protein 1 (FHL-1), which helps the bacteria evade complement-mediated killing and promotes invasion of host cells . This binding involves a 16-amino-acid region corresponding to residues 97-112 of FbaA .

This pathogenic mechanism suggests several research and therapeutic avenues:

• Development of monoclonal antibodies that block the FbaA-FH interaction could prevent bacterial immune evasion and reduce virulence .

• The FbaA MAb2 antibody has been shown to inhibit FH binding to GAS, reducing bacterial invasion of A549 epithelial cells in experimental models .

• These findings indicate FbaA could serve as a vaccine target, as supported by studies showing that FbaA immunization provides protection against GAS challenge in mice .

• The mechanism may be relevant to other pathogens that exploit similar immune evasion strategies.

Understanding this interaction has both basic research value in host-pathogen biology and translational potential for infectious disease interventions.

How can epitope mapping enhance the utility of FBA1 antibodies in research applications?

Detailed epitope mapping of FBA1 antibodies provides critical information for optimizing research applications:

Research on FbaA MAb2 identified its binding epitope as amino acid residues 95-118, with key interaction residues I101, T103, P105, D106, and L110 determined through phage display epitope library screening . This epitope overlaps with the FH and FHL-1 binding site (residues 97-112) .

Epitope mapping offers several research advantages:

• Functional blocking antibodies: When epitopes overlap with functional domains (as with FbaA MAb2), antibodies can block specific protein-protein interactions.

• Compatibility in detection panels: Knowing epitope locations helps design antibody panels where multiple antibodies can bind simultaneously.

• Cross-reactivity prediction: Epitope sequence conservation across species helps predict potential cross-reactivity.

• Increased reproducibility: Detailed epitope knowledge improves method standardization across laboratories.

Modern approaches combine traditional mapping techniques with computational methods, including biophysics-informed models that associate "each potential ligand a distinct binding mode, which enables the prediction and generation of specific variants beyond those observed in the experiments" .

What diagnostic value do anti-FBA1 antibodies offer for fungal infection detection?

Anti-FBA1 antibodies show promising diagnostic utility for invasive candidiasis detection:

A comprehensive evaluation of antibodies against recombinant Candida albicans fructose-bisphosphate aldolase (Fba1) as serological markers revealed impressive performance metrics:

Table 1: Diagnostic performance of anti-Fba1 and anti-Eno antibodies for invasive candidiasis

Notable advantages of this approach include:

• Early detection potential: Positive antibody tests were obtained before blood culture results for 51.1% of patients using anti-Fba1 .

• Higher sensitivity than conventional blood cultures.

• Combined testing with anti-Eno antibodies improved sensitivity to 90.1% and negative predictive value to 97.1% .

• Specificity for Candida genus without cross-reactivity to other pathogens causing bacteremia or invasive aspergillosis .

These findings suggest that anti-Fba1 antibody detection could significantly improve early diagnosis of invasive candidiasis, particularly when combined with other biomarkers.

How are advances in AI and computational approaches transforming antibody design for targets like FBA1?

Recent computational advances are revolutionizing antibody design approaches:

The Baker Lab has developed RFdiffusion, an AI system fine-tuned to design human-like antibodies with customized binding properties . This approach focuses on designing antibody loops—the flexible regions responsible for antigen binding—producing novel antibody blueprints that can bind specified targets .

Key advantages for FBA1 antibody research include:

• Design of antibodies with precise specificity profiles: This could address cross-reactivity challenges common with conserved targets like FBA1.

• Customized binding properties: The ability to generate antibodies "with either specific high affinity for a particular target ligand, or with cross-specificity for multiple target ligands" .

• Reduced experimental iterations: Computational pre-screening reduces the number of antibodies that need experimental validation.

• Disentanglement of binding modes: As demonstrated in phage display experiments, computational approaches can identify "different binding modes, each associated with a particular ligand against which the antibodies are either selected or not" .

These computational approaches complement traditional antibody development methods, potentially accelerating the creation of research and diagnostic antibodies with superior performance characteristics.

Case Study: Inhibition of Pathogen-Host Interactions Using FbaA-Specific Monoclonal Antibodies

A detailed investigation of FbaA-specific monoclonal antibodies demonstrated their ability to disrupt bacterial immune evasion strategies:

Researchers identified FbaA MAb2, which binds to amino acid residues 95-118 of FbaA in Group A Streptococcus. Through functional studies including competitive inhibition ELISA and immunofluorescence microscopy, they demonstrated that this antibody could block the interaction between FbaA and human factor H .

The invasion assay revealed significant functional consequences:

Table 2: Impact of FbaA MAb2 on GAS invasion of A549 epithelial cells

These findings demonstrate that:

• FH binding enhances bacterial invasion of epithelial cells

• FbaA MAb2 significantly inhibits this process by blocking the FbaA-FH interaction

• Monoclonal antibodies targeting specific epitopes can functionally interfere with bacterial virulence mechanisms

This approach represents a potential therapeutic strategy for addressing bacterial infections that exploit host immune regulatory proteins.

Research Finding: Diagnostic Performance of Anti-Fba1 in Different Patient Populations

The diagnostic utility of anti-Fba1 antibodies varies across different patient populations:

Table 3: Positivity rates of anti-Eno and anti-Fba1 antibodies in different patient groups

This data highlights several important research considerations:

• Anti-Fba1 demonstrates higher sensitivity than anti-Eno across most patient groups

• Gender differences may exist in antibody response, with females showing higher anti-Fba1 positivity rates

• Low cross-reactivity with other fungal or bacterial infections supports specificity for Candida

• Combined testing approaches can maximize diagnostic accuracy

These findings inform both diagnostic algorithm development and research design when using anti-Fba1 antibodies for patient classification.

How might standardized antibody validation initiatives improve FBA1 antibody reliability?

The YCharOS initiative represents a model for improving antibody validation through open science collaboration:

This effort has already characterized 812 antibodies against 78 proteins using knockout validation approaches across multiple applications including Western blot, immunoprecipitation, and immunofluorescence . Similar approaches applied to FBA1 antibodies would address reproducibility challenges in the field.

Implementing comprehensive validation would involve:

• Systematic testing in knockout/knockdown models across different species

• Standardized reporting of validation data in publications

• Industry-academic partnerships to improve antibody production standards

• Public repositories of validation data accessible to researchers

The impact of such initiatives is already evident, with "the number of antibodies that have either been withdrawn or had their recommended usage altered by the vendor" increasing as validation data becomes available .

How can biophysics-informed computational models enhance antibody specificity for FBA1 research?

Emerging computational approaches offer promising solutions to FBA1 antibody specificity challenges:

Biophysics-informed models that associate distinct binding modes with specific ligands can "enable the prediction and generation of specific variants beyond those observed in the experiments" . These models have successfully disentangled binding modes "even when they are associated with chemically very similar ligands" .

For FBA1 research, these approaches could:

• Design antibodies that distinguish between closely related FBA1 isoforms

• Create reagents specific to particular species despite high sequence conservation

• Generate antibodies targeting specific functional domains

• Mitigate experimental artifacts and selection biases

The combination of these computational methods with high-throughput experimental validation represents a powerful approach for next-generation FBA1 antibody development.