tolC Antibody

What is TolC and why is it significant for antibody development?

TolC is an outer membrane protein that forms a critical component of the AcrAB-TolC efflux pump system in Gram-negative bacteria such as Escherichia coli. This tripartite system plays a fundamental role in antimicrobial resistance by extruding diverse compounds, effectively reducing the intracellular concentration of antibiotics and other toxic substances . The significance of TolC as an antibody target stems from its external exposure on the bacterial surface, making it accessible to antibodies in both research and potential therapeutic applications. Recent research has confirmed that TolC is immunogenic, capable of activating macrophages, T cells, and B cells, leading to the production of protective antibodies against E. coli . This immunogenicity makes TolC an attractive target for researchers developing diagnostic tools, studying bacterial resistance mechanisms, and exploring novel therapeutic approaches to combat antimicrobial resistance.

What structural features of TolC are important to consider when selecting epitopes for antibody development?

TolC forms a distinctive channel structure with a periplasmic domain consisting of coiled-coil alpha-barrel regions that are critical for its function. When selecting epitopes for antibody development, researchers should consider the following structural elements:

• The periplasmic tip of TolC, which interacts with AcrA in the assembled pump complex

• The alpha-barrel domain, which contains residues involved in substrate specificity

• External regions with high predicted antigenicity and flexibility

How do I interpret contradictory results when using TolC antibodies across different bacterial strains?

Contradictory results when using TolC antibodies across different bacterial strains may stem from several factors that require methodological consideration. First, sequence variations in the TolC protein between bacterial species and even strains can affect epitope recognition - even minor amino acid differences in key binding regions can dramatically alter antibody affinity . Second, differences in TolC expression levels between strains under various growth conditions can lead to variable signal intensity in assays. Third, the accessibility of epitopes may differ depending on the conformation of TolC, which can be influenced by the assembly state of the efflux pump complex.

To address these contradictions systematically:

• Perform sequence alignment analysis of TolC across your bacterial strains of interest to identify potential epitope variations

• Validate your antibody against positive and negative control strains, including TolC knockout mutants

• Use orthogonal detection methods, such as mass spectrometry, to confirm TolC expression levels

• Consider using multiple antibodies targeting different epitopes of TolC to improve detection reliability

• Standardize growth conditions and sample preparation protocols to minimize variation

When reporting contradictory results, thoroughly document experimental conditions and bacterial strain characteristics to help the research community understand the context-specific nature of your findings .

What are the most effective protocols for validating a new TolC antibody?

Validating a TolC antibody requires a comprehensive approach to ensure specificity, sensitivity, and reproducibility. Based on established practices in antibody validation, the following protocol is recommended:

Genetic validation:

• Test the antibody against wild-type bacteria and TolC knockout mutants to confirm specificity

• Use strains with known TolC mutations to assess epitope recognition

Multiple technique validation:

• Western blot: Confirm the antibody detects a protein of the correct molecular weight (~52 kDa for TolC)

• Immunofluorescence: Verify localization at the bacterial outer membrane

• Flow cytometry: Assess binding to intact bacteria

• ELISA: Determine binding affinity and sensitivity

Cross-reactivity testing:

• Test against related bacterial species with homologous TolC proteins

• Assess potential cross-reactivity with other outer membrane proteins

Functional validation:

• Determine if the antibody affects TolC-dependent functions such as antibiotic resistance

• Assess impact on bacterial growth in the presence of known efflux substrates

As noted in search result , validation should be tailored to your specific experimental needs: "The level of validation you undertake may depend on how well validated the antibody already is, and how critical the antibody is to your experiment. Is the antibody just one of a panel used to identify a process? Perhaps you don't need extensive validation. Does your seminal finding rest on its specificity? It will be vital to ensure the antibody is well validated."

How can I optimize immunofluorescence protocols for visualizing TolC in intact bacterial cells?

Optimizing immunofluorescence protocols for TolC visualization requires careful attention to bacterial sample preparation, fixation methods, and imaging parameters:

Sample preparation:

• Culture bacteria in appropriate media to mid-log phase for optimal TolC expression

• Wash cells gently in phosphate-buffered saline (PBS) to remove media components that might interfere with antibody binding

• Consider using minimal media to reduce autofluorescence

Fixation and permeabilization:

• Use paraformaldehyde (2-4%) for fixation to preserve cellular structures

• Test different permeabilization approaches: lysozyme treatment (0.1 mg/ml, 5-15 minutes) works well for exposing periplasmic epitopes without disrupting membrane integrity

• For TolC's extracellular epitopes, permeabilization may be minimized to reduce background

Antibody incubation:

• Block with 1-3% BSA in PBS to reduce non-specific binding

• Optimize primary antibody dilution (typically 1:100 to 1:1000) and incubation time (1-4 hours at room temperature or overnight at 4°C)

• Use fluorophore-conjugated secondary antibodies with excitation/emission spectra distinct from bacterial autofluorescence

• Include multiple washing steps (at least 3 × 5 minutes) after each antibody incubation

Controls:

• Include a TolC knockout strain as a negative control

• Use secondary antibody-only controls to assess background

• Include a cytoplasmic or inner membrane marker to help interpret TolC localization

Imaging considerations:

• Use confocal microscopy for better resolution of membrane-localized signals

• Adjust laser power and gain settings to minimize photobleaching while maintaining signal clarity

• Capture Z-stacks to fully visualize the three-dimensional distribution of TolC around the bacterial cell

This methodology can be further refined based on specific bacterial strains and experimental objectives.

What approaches can I use to detect epitope-specific binding of TolC antibodies?

Several sophisticated approaches can be employed to detect and characterize epitope-specific binding of TolC antibodies:

Peptide array analysis:

• Synthesize overlapping peptides (15-20 amino acids) spanning the TolC sequence

• Test antibody binding to identify linear epitopes

• Analyze binding patterns to reveal epitope preferences

Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

• Compare deuterium uptake patterns of TolC in the presence and absence of antibody

• Regions with reduced deuterium exchange upon antibody binding indicate epitope locations

• This approach is particularly valuable for conformational epitopes

Site-directed mutagenesis:

• Introduce point mutations in predicted epitope regions

• Test antibody binding to mutant TolC proteins

• Reduced binding to specific mutants confirms epitope involvement

Cryo-electron microscopy:

• Visualize antibody-TolC complexes at near-atomic resolution

• Similar to the approach used to determine the structure of AcrAB-TolC pump (3.6 Å resolution)

• Can reveal precise binding interfaces and conformational changes induced by antibody binding

Computational prediction and validation:

• Use epitope prediction tools such as BepiPred-2.0 and Karplus & Schulz Flexibility Prediction

• Validate predictions with experimental data

• As demonstrated in the TolC study, flexibility analysis can help identify potential antigenic regions

These approaches can be combined to build a comprehensive understanding of TolC antibody binding characteristics, which is essential for both research applications and therapeutic development.

How can TolC antibodies be used to study the assembly and dynamics of the AcrAB-TolC efflux pump?

TolC antibodies offer powerful tools for investigating the assembly dynamics and structural transitions of the AcrAB-TolC efflux pump system. Recent cryo-EM studies have revealed that TolC adopts a fully opened state via tip-to-tip interactions with AcrA in the assembled pump complex . Researchers can exploit antibodies to probe these interactions through several sophisticated approaches:

Conformation-specific antibody development:

• Generate antibodies that specifically recognize different conformational states of TolC (closed, intermediate, or open)

• Use these antibodies to track conformational changes during pump assembly and drug transport

• The near-atomic resolution cryo-EM structures of AcrAB-TolC in both resting and drug transport states provide structural templates for designing such antibodies

Real-time monitoring of pump assembly:

• Use fluorescently labeled Fab fragments against TolC to monitor pump assembly kinetics in live cells

• Perform fluorescence resonance energy transfer (FRET) experiments with differentially labeled antibodies against TolC and AcrA/AcrB to study component interactions

• Correlate assembly dynamics with efflux activity under various conditions

Inhibition studies:

• Use antibodies targeting specific domains of TolC to block interactions with AcrA

• Compare effects with known small-molecule inhibitors like MBX3132, which locks the pump in a T-saturated state

• Map functional domains through differential inhibition patterns

Single-molecule tracking:

• Apply super-resolution microscopy with antibody-based labeling to track individual TolC molecules in the bacterial membrane

• Analyze diffusion patterns to understand recruitment into functional pump complexes

• Correlate with antibiotic exposure to reveal real-time adaptive responses

These approaches provide mechanistic insights beyond static structural information, revealing how the pump assembles, functions, and responds to inhibitors or substrates in physiologically relevant conditions.

What is the evidence for anti-TolC antibodies enhancing antibiotic efficacy, and how might this be exploited?

Evidence for anti-TolC antibodies enhancing antibiotic efficacy comes from both mechanistic studies of efflux pump function and immunological research. This represents a promising strategy for combating antimicrobial resistance:

Mechanistic evidence:

• Mutations affecting the electrostatic properties of the TolC channel, particularly D371V, significantly impact bacterial growth even without antibiotics and cause hyper-susceptibility to efflux-substrates

• This suggests that antibodies binding similar regions might disrupt TolC function more effectively than simple deletion of the gene

• Research indicates that "inhibition of TolC functionality is less well-tolerated than deletion of tolC, and such inhibition may have an antibacterial effect"

Immunological evidence:

• Immunization of mice with TolC produced protective antibodies that increased bacterial uptake by macrophages in vitro and improved survival rates by 60% following E. coli infection

• Human plasma contains natural anti-TolC IgG and IgA antibodies, with infected patients showing increased anti-TolC IgM levels

• TolC immunization stimulates production of multiple antibody isotypes (IgM, IgG1, IgG2) that can bind live bacteria

Potential exploitation strategies:

• Antibody-antibiotic combination therapy:

  • Co-administer anti-TolC antibodies with conventional antibiotics

  • Target antibodies to regions involved in substrate selectivity to enhance accumulation of specific antibiotics

• Bispecific antibody development:

  • Create antibodies that simultaneously bind TolC and recruit immune effectors

  • Design constructs that recognize both TolC and AcrA to disrupt pump assembly

• Vaccine approach:

  • Develop TolC-based vaccines to stimulate protective antibody production

  • Target conserved epitopes to provide protection against multiple Gram-negative pathogens

• Antibody-guided drug delivery:

  • Use anti-TolC antibodies to deliver antibiotic payloads directly to bacterial surfaces

  • Enhance local antibiotic concentration while reducing systemic exposure

This evidence suggests that targeting TolC with antibodies represents a multi-faceted strategy that both disrupts efflux pump function directly and enhances immune-mediated bacterial clearance.

How do mutations in TolC affect antibody binding and what does this reveal about structure-function relationships?

Mutations in TolC can significantly affect antibody binding, providing valuable insights into structure-function relationships of this critical efflux component. Research has revealed several important patterns:

Effects of periplasmic tip mutations:

• Mutations at the periplasmic tip of TolC alter interactions with AcrA in the assembled pump

• Antibodies targeting wild-type epitopes in this region may show reduced binding to mutant variants

• Changes in binding affinity can be used to map critical interaction residues between TolC and other pump components

Electrostatic alterations:

• The D371V mutation significantly impacts channel function by altering electrostatic properties

• Antibodies recognizing this region may show differential binding to mutant and wild-type TolC

• Correlation between antibody binding patterns and functional impacts reveals electrostatic requirements for channel operation

Higher-barrel region mutations:

• Some functional TolC mutations are located higher up the alpha-barrel, away from the proposed PAP-docking sites

• These findings challenge the "tip-to-tip" model of PAP-TolC interaction

• Antibodies binding to these unexpected functional regions can help refine structural models of pump assembly

Substrate specificity effects:

• TolC mutations can cause antibiotic-specific phenotypes, suggesting TolC plays a role in substrate selectivity

• Differential antibody binding to TolC variants correlates with altered substrate profiles

• This reveals that "substrate specificity may not be determined solely by the transporter protein or the PAP, but may reside at least partially with the TolC-channel"

By systematically mapping mutations, antibody binding patterns, and functional outcomes, researchers can develop more accurate models of TolC's role in efflux pump assembly and substrate recognition. This approach has revealed TolC's "possible new role in vetting of efflux substrates, alongside its established role in tripartite complex assembly" .

What immune responses does TolC protein elicit and how does this inform antibody development?

TolC protein elicits diverse immune responses that provide valuable guidance for antibody development strategies. Recent immunological research has characterized these responses in detail:

Humoral immune response:

• Human plasma contains natural anti-TolC IgG and IgA antibodies, indicating ongoing exposure to this bacterial protein

• Patients with Gram-negative infections show elevated anti-TolC IgM levels compared to control subjects

• In mouse models, TolC immunization stimulates production of multiple antibody isotypes, with higher levels of IgG1 and IgG2 among the IgG subclasses

Cellular immune response:

• TolC protein stimulates macrophages to produce nitric oxide and inflammatory cytokines (IL-6, TNF-α) in vitro

• Lymph node cells from TolC-immunized mice show increased T cell proliferation upon re-stimulation

• These cells also produce IFNγ, indicating a robust cell-mediated immune response

Functional effects of immune response:

• Anti-TolC IgG from immunized mice can bind to live E. coli bacteria

• This binding enhances bacterial uptake by macrophages in vitro

• TolC-immunized mice show a 60% increase in survival rate following E. coli infection

Implications for antibody development:

• Epitope selection:

  • Focus on epitopes recognized by protective antibodies from immunized animals

  • Target regions that enable binding to live bacteria rather than just isolated protein

  • Consider conserved epitopes to provide cross-protection against multiple species

• Antibody isotype consideration:

  • Design strategies to elicit complementary IgG subclasses (IgG1, IgG2) for optimal protection

  • Consider applications requiring specific isotypes (e.g., IgG3 for complement activation)

• Functional screening:

  • Screen candidate antibodies for their ability to enhance phagocytosis

  • Test antibodies for their impact on bacterial survival in relevant infection models

  • Evaluate combinations with antimicrobials for synergistic effects

This immunological profile confirms that "TolC is immunogenic, activating macrophages, T and B cells, leading to the production of protective antibodies against E. coli" , making it a promising target for both therapeutic antibodies and vaccine development.

How should I design experiments to evaluate the protective efficacy of anti-TolC antibodies?

Designing experiments to evaluate the protective efficacy of anti-TolC antibodies requires a multi-tiered approach that addresses both in vitro and in vivo aspects of protection:

In vitro assessments:

• Bacterial binding assays:

  • Flow cytometry to quantify antibody binding to live bacteria

  • Immunofluorescence microscopy to visualize binding patterns

  • Comparison across multiple bacterial strains to assess cross-reactivity

• Functional inhibition assays:

  • Measure antibiotic accumulation in bacteria in the presence of anti-TolC antibodies

  • Determine minimal inhibitory concentrations (MICs) of antibiotics with and without antibodies

  • Assess efflux pump substrate retention using fluorescent dyes (e.g., ethidium bromide)

• Immune effector assays:

  • Opsonophagocytosis assays with macrophages or neutrophils

  • Complement-dependent cytotoxicity (CDC) assessment

  • Antibody-dependent cellular cytotoxicity (ADCC) with relevant immune cells

In vivo experimental design:

• Immunization protocols:

  • Follow established models such as the one described in the research: primary immunization followed by booster at day 14

  • Consider routes of administration (intraperitoneal, intranasal, oral) based on target infection site

  • Compare active immunization with passive antibody transfer

• Challenge models:

  • Use standardized bacterial challenge doses (e.g., 2 × LD50 as used in the referenced study)

  • Evaluate different routes of infection relevant to natural infection processes

  • Monitor survival rates, bacterial burden, and inflammatory markers

• Combination therapy assessment:

  • Test antibodies in combination with standard antibiotic treatments

  • Evaluate potential for dose reduction of antibiotics

  • Assess impact on emergence of resistance during treatment

Control groups and variables:

• Essential controls:

  • Isotype-matched irrelevant antibodies

  • Anti-TolC antibodies against TolC-knockout bacteria

  • Comparison with antibiotics alone and untreated groups

• Variables to consider:

  • Antibody dose and timing relative to infection

  • Bacterial inoculum size and growth phase

  • Host factors (immunocompetent vs. immunocompromised models)

This experimental framework allows for comprehensive evaluation of protective efficacy while establishing mechanistic understanding of how anti-TolC antibodies confer protection.

What are the technical considerations for developing monoclonal versus polyclonal antibodies against TolC?

Developing monoclonal versus polyclonal antibodies against TolC involves distinct technical considerations that impact their research and therapeutic applications:

Monoclonal Antibody Development:

• Epitope selection:

  • Target highly specific epitopes in functional domains of TolC

  • Consider accessibility in the assembled AcrAB-TolC complex

  • Use structural data from cryo-EM studies (3.6 Å resolution) to identify critical binding sites

• Hybridoma screening strategy:

  • Design functional screens to identify clones that inhibit efflux pump activity

  • Test for binding to both recombinant TolC and intact bacteria

  • Screen for cross-reactivity with TolC from multiple species if broader coverage is desired

• Validation challenges:

  • Confirm epitope specificity through competition assays

  • Verify recognition of native TolC in its membrane environment

  • Test against TolC mutants to confirm epitope identity

• Production considerations:

  • Optimize hybridoma culture conditions for consistent antibody production

  • Consider recombinant production for humanized versions if therapeutic applications are planned

  • Ensure lot-to-lot consistency through rigorous quality control

Polyclonal Antibody Development:

• Immunization strategies:

  • Use either whole heat-inactivated bacteria or purified TolC protein

  • Consider multiple routes (intranasal, intraperitoneal, oral) as demonstrated in the research

  • Design boosting schedule based on antibody titer monitoring

• Adjuvant selection:

  • Use appropriate adjuvants for bacterial proteins (Alum was used in the referenced study)

  • Balance immunogenicity enhancement with minimal adverse effects

  • Consider adjuvants that promote desired antibody isotypes (IgG1, IgG2)

• Purification approaches:

  • Affinity purification using recombinant TolC to isolate specific antibodies

  • Consider epitope-specific purification for enriching antibodies against functional domains

  • Remove cross-reactive antibodies using bacterial lysates from TolC knockout strains

• Batch variability management:

  • Implement pooling strategies to reduce animal-to-animal variation

  • Develop reference standards for batch comparison

  • Establish functional benchmarks for acceptable lot release

Comparative advantages for research applications:

When selecting between these approaches, researchers should consider their specific experimental needs, balancing the high specificity and reproducibility of monoclonals against the broader epitope coverage and potentially more robust functional effects of polyclonals.

What are the most common pitfalls when using TolC antibodies and how can they be addressed?

Researchers working with TolC antibodies frequently encounter several challenges that can compromise experimental results. Understanding these pitfalls and implementing appropriate solutions is crucial for successful applications:

False negative results in intact bacteria

• Cause: Limited epitope accessibility due to LPS or capsule interference

• Solution:

  • Gentle pre-treatment with EDTA to increase outer membrane permeability

  • Use of specialized fixation protocols that preserve epitope structure while improving accessibility

  • Selection of antibodies targeting more exposed regions of TolC

Cross-reactivity with other outer membrane proteins

• Cause: Structural similarity between TolC and other beta-barrel proteins

• Solution:

  • Rigorous validation using TolC knockout strains as negative controls

  • Pre-absorption of antibodies with lysates from TolC knockout bacteria

  • Epitope mapping to select antibodies targeting unique TolC regions

Strain-specific variations in detection

• Cause: Sequence variations in TolC across bacterial species and strains

• Solution:

  • Sequence alignment analysis before selecting antibodies

  • Use of multiple antibodies targeting conserved epitopes

  • Customization of protocols for specific bacterial strains

Inconsistent results between detection methods

• Cause: Conformational differences in TolC preparation across techniques

• Solution:

  • Use different antibodies optimized for each application (Western blot vs. flow cytometry)

  • Standardize sample preparation protocols

  • Include positive controls for each detection method

Non-specific binding in complex samples

• Cause: Matrix effects from clinical or environmental samples

• Solution:

  • Optimize blocking conditions (1-3% BSA or 5% milk is often effective)

  • Include multiple washing steps with detergent-containing buffers

  • Use more specific secondary detection systems

Poor correlation between antibody binding and functional inhibition

• Cause: Binding to non-functional epitopes

• Solution:

  • Screen antibodies for functional effects on efflux activity

  • Target regions known to be critical for TolC function (e.g., D371 region)

  • Use combinations of antibodies targeting different functional domains

As with all antibody work, the guiding principle should be thorough validation: "Taking a bit of time choosing an antibody can stop you wasting anything from a few days to several months at the bench. This is especially true with new researchers - you don't want to end up blaming yourself for a failed experiment, when in reality it is the antibodies fault!"

How do I troubleshoot inconsistent results when using TolC antibodies in different assays?

Troubleshooting inconsistent results across different assays requires a systematic approach to identify and address assay-specific variables affecting TolC antibody performance:

Cross-assay comparison and analysis:

• Document all variables:

  • Create a detailed comparison chart of protocols across assays

  • Note antibody concentrations, incubation times, and buffer compositions

  • Record sample preparation methods for each assay

• Perform parallel validation:

  • Run positive and negative controls across all assays simultaneously

  • Use the same antibody lot and protein preparation

  • Standardize as many conditions as possible

Assay-specific troubleshooting:

• Western blot inconsistencies:

  • Problem: Detection in Western blot but not in flow cytometry

  • Possible cause: Antibody recognizes denatured epitope not accessible in native conformation

  • Solution: Use native PAGE or dot blot as intermediate tests to confirm epitope conformation dependence

• Immunofluorescence issues:

  • Problem: Signal in flow cytometry but not in microscopy

  • Possible cause: Epitope masking by fixation method

  • Solution: Test multiple fixation approaches (paraformaldehyde, methanol, acetone) to preserve epitope structure

• ELISA discrepancies:

  • Problem: Strong ELISA signal but weak cell-based detection

  • Possible cause: Plate-bound TolC presents epitopes differently than membrane-embedded protein

  • Solution: Develop membrane-based ELISA using bacterial membrane preparations

Antibody characterization:

• Epitope accessibility analysis:

  • Map the location of recognized epitopes on TolC structure

  • Correlate with accessibility in different sample preparations

  • Consider using epitope-specific competing peptides to confirm binding specificity

• Antibody modifications:

  • Fragment antibodies to improve penetration in certain assays

  • Test direct labeling versus secondary detection systems

  • Optimize antibody concentration for each specific assay

Sample preparation standardization:

• Bacterial growth conditions:

  • Standardize growth phase for harvesting (mid-log phase recommended)

  • Use consistent media and temperature conditions

  • Document any induction protocols for TolC expression

• Protein extraction methods:

  • Compare gentle versus harsh extraction methods

  • Evaluate native versus denaturing conditions

  • Consider detergent effects on epitope presentation

By systematically addressing these variables and documenting their effects, researchers can develop optimized protocols for each assay while understanding the underlying causes of cross-assay variations.

How can I distinguish between specific and non-specific binding when using TolC antibodies?

Distinguishing between specific and non-specific binding is critical for generating reliable data with TolC antibodies. A comprehensive approach combines appropriate controls, optimization techniques, and validation methods:

Essential control experiments:

• Genetic controls:

  • TolC knockout strain comparison is the gold standard

  • Strains with known TolC mutations affecting specific epitopes

  • Heterologous expression systems with controlled TolC levels

• Antibody controls:

  • Isotype-matched irrelevant antibodies at identical concentrations

  • Pre-immune serum for polyclonal antibodies

  • Secondary antibody-only controls to assess background

• Competition assays:

  • Pre-incubation with purified TolC protein should reduce specific signal

  • Titration of competing antigen provides quantitative measure of specificity

  • Peptide competition with epitope-specific fragments

Optimization strategies:

• Blocking optimization:

  • Test multiple blocking agents (BSA, milk, normal serum)

  • Optimize blocking time and temperature

  • Consider pre-adsorption of antibodies with bacterial lysates lacking TolC

• Washing stringency:

  • Increase number and duration of washing steps

  • Add low concentrations of detergent (0.05-0.1% Tween-20)

  • Use higher salt concentrations to reduce ionic interactions

• Antibody dilution optimization:

  • Perform detailed titration curves for each application

  • Determine signal-to-noise ratio at each concentration

  • Find minimum concentration giving acceptable specific signal

Quantitative validation methods:

• Signal-to-noise ratio analysis:

  • Calculate the ratio between signal in TolC-positive and TolC-negative samples

  • Ratios >10 typically indicate high specificity

  • Plot ratio across antibody dilutions to identify optimal concentration

• Two-color flow cytometry:

  • Co-stain with antibodies against known outer membrane proteins

  • Compare staining patterns to confirm membrane localization

  • Use scatter properties to exclude dead cells (which often show non-specific binding)

• Immunoprecipitation followed by mass spectrometry:

  • Perform pull-down experiments and analyze by MS

  • Confirm TolC as the predominant precipitated protein

  • Identify any cross-reactive proteins for further optimization

When evaluating commercial antibodies, consider that "Estimates vary, but it is suggested that up to half of commercial antibodies may not be fit for purpose..." . Therefore, investing time in thorough validation is essential for establishing reliable TolC detection methods.