TLR5 Antibody, Biotin conjugated

What is TLR5 and why is it significant in immunological research?

TLR5 (Toll-like receptor 5) is a pattern recognition receptor that plays a crucial role in the innate immune response to microbial agents. It specifically mediates detection of bacterial flagellins and acts through the MyD88 and TRAF6 signaling pathway, ultimately leading to NF-kappa-B activation, cytokine secretion, and inflammatory response . TLR5 is primarily expressed on the cell membrane and is found in various cell types including intestinal epithelial cells, dendritic cells, and certain immune cell populations .

The significance of TLR5 in immunological research stems from its role as a key mediator of host-microbe interactions, particularly in recognizing flagellated bacteria. Recent research has expanded its importance to include potential applications in:

• Cancer immunotherapy approaches utilizing TLR5 agonists like CBLB502/entolimod

• Radioprotection through TLR5-mediated activation of pro-survival pathways

• Vaccine adjuvant development leveraging TLR5's ability to enhance immune responses

• Understanding intestinal barrier function and microbiota sensing mechanisms

TLR5 signaling represents a critical pathway in the complex interplay between host immunity and both pathogenic and commensal bacteria, making it an important target for therapeutic development and basic research .

What are the key specifications of biotin-conjugated TLR5 antibodies?

Biotin-conjugated TLR5 antibodies come with specific characteristics that researchers should consider when selecting the appropriate reagent for their experiments. The following table summarizes typical specifications:

When selecting a biotin-conjugated TLR5 antibody, researchers should carefully consider the match between the antibody specifications and their experimental requirements, particularly regarding species reactivity, validated applications, and the specific epitope recognized by the antibody .

How does biotin conjugation enhance the utility of TLR5 antibodies in research applications?

Biotin conjugation significantly expands the versatility and sensitivity of TLR5 antibodies through several mechanisms:

First, the biotin-streptavidin interaction is one of the strongest non-covalent biological interactions known (Kd ≈ 10^-15 M), providing exceptional stability in complex experimental systems. This allows for robust detection even when target proteins are expressed at low levels .

Second, the amplification capabilities of the biotin-streptavidin system enhance signal detection. Each biotin-conjugated antibody can bind multiple streptavidin molecules, each carrying multiple reporter molecules (enzymes, fluorophores, etc.), creating an amplification cascade. This is particularly valuable when studying TLR5 expression in tissues where expression may be limited or in flow cytometry applications requiring increased sensitivity .

Third, biotin conjugation provides exceptional flexibility in experimental design. The same biotin-conjugated TLR5 antibody can be paired with different streptavidin conjugates (HRP, fluorophores, gold particles) allowing researchers to adapt to various detection systems without changing the primary antibody. This is demonstrated in protocols utilizing biotin-labeled antibodies for diverse applications ranging from Western blotting to pull-down assays with magnetic streptavidin beads .

Finally, biotin-conjugated antibodies facilitate multiplexing in imaging and flow cytometry. When combined with directly labeled antibodies against other targets, researchers can develop comprehensive panels to study TLR5 in the context of multiple markers simultaneously .

What are the validated applications for biotin-conjugated TLR5 antibodies?

Biotin-conjugated TLR5 antibodies have been validated across multiple research applications, each with specific optimization parameters:

Western Blotting (WB): Biotin-conjugated TLR5 antibodies have demonstrated efficacy in detecting TLR5 protein (95-97 kDa) in cell lysates. Optimal dilution ranges from 1:300-5000, with validation in cell lines like HT29 . When using these antibodies for WB, streptavidin-HRP is typically employed as the detection reagent, providing sensitive visualization of bands corresponding to TLR5.

Enzyme-Linked Immunosorbent Assay (ELISA): These antibodies have been validated for ELISA applications at dilutions of 1:500-1000 . Both sandwich and direct ELISA formats can be adapted for TLR5 detection, with the biotin-conjugated antibody serving as either a capture or detection antibody depending on the assay design.

Immunohistochemistry (IHC-P): For paraffin-embedded tissue sections, biotin-conjugated TLR5 antibodies are effective at dilutions between 1:200-400 . Successful staining typically reveals membrane localization consistent with TLR5's known subcellular distribution.

Immunocytochemistry (ICC) and Immunofluorescence (IF): These applications have been validated using various cell lines including THP-1, with typical working concentrations of 10 μg/ml . The biotin tag allows for customizable detection with various fluorophore-conjugated streptavidin molecules.

Pull-down Assays: Biotin-conjugated antibodies have been successfully used in conjunction with streptavidin magnetic beads to purify TLR5 protein complexes from cell culture systems . This approach enables isolation of TLR5 and its associated proteins for further characterization.

Flow Cytometry: While not explicitly mentioned in all product datasheets, biotin-conjugated antibodies are well-suited for flow cytometry applications, where they can be detected with fluorophore-conjugated streptavidin.

Each application requires specific optimization parameters including antibody concentration, incubation time, and detection system to achieve optimal signal-to-noise ratios .

What is the optimal protocol for using biotin-conjugated TLR5 antibodies in Western blot analysis?

The following protocol has been optimized for Western blot detection of TLR5 using biotin-conjugated antibodies based on successful experimental approaches:

Sample Preparation:

• Prepare cell lysates from relevant cell lines (e.g., HT29, THP-1) or tissue samples in RIPA buffer containing protease inhibitors

• Determine protein concentration using standard methods (BCA or Bradford assay)

• Mix 20-50 μg of protein with Laemmli buffer containing reducing agent

• Heat samples at 95°C for 5 minutes

Gel Electrophoresis and Transfer:

• Separate proteins using 8-10% SDS-PAGE (TLR5 is ~95-97 kDa)

• Transfer to PVDF or nitrocellulose membrane using standard conditions

• Verify transfer efficiency using reversible staining (Ponceau S)

Blocking and Antibody Incubation:

• Block membrane with 5% BSA in TBS-T for 1 hour at room temperature (avoid milk-based blocking buffers as they may contain endogenous biotin)

• Dilute biotin-conjugated TLR5 antibody in blocking buffer (1:300-2000 depending on product specifications)

• Incubate overnight at 4°C with gentle agitation

• Wash 3-5 times with TBS-T, 5 minutes each

Detection:

• Incubate with streptavidin-HRP (1:5000-1:20000) in TBS-T for 1 hour at room temperature

• Wash 3-5 times with TBS-T, 5 minutes each

• Apply chemiluminescent substrate and detect signal

Expected Results:

• Human TLR5: Single band at approximately 97 kDa

• Mouse TLR5: Single band at approximately 95 kDa

• Potential glycosylated forms may appear as higher molecular weight bands

Western blot validation of TLR5 antibodies has been demonstrated in cell lines like HT29, where clear bands of the expected molecular weight are visible, confirming specificity and suitable working dilutions for the biotin-conjugated antibody .

How should researchers optimize immunofluorescence protocols using biotin-conjugated TLR5 antibodies?

Optimizing immunofluorescence protocols with biotin-conjugated TLR5 antibodies requires careful attention to several key parameters:

Sample Preparation:

• Culture cells on coverslips or prepare tissue sections (4-6 μm)

• Fix cells with 4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes (for intracellular domains)

• For tissue sections, perform appropriate antigen retrieval as TLR5 epitopes may be masked by fixation

Blocking:

• Block with 5% normal serum (matched to secondary antibody host) or BSA in PBS for 30-60 minutes at room temperature

• Critical step: Include avidin/biotin blocking to minimize background from endogenous biotin, particularly in tissues with high biotin content

Antibody Incubation:

• Apply biotin-conjugated TLR5 antibody at 10 μg/ml (the validated concentration for immunofluorescence)

• For co-localization studies, simultaneously apply antibodies against other targets of interest

• Incubate overnight at 4°C or 1-2 hours at room temperature in a humidified chamber

Detection:

• Wash 3-5 times with PBS, 5 minutes each

• Apply fluorophore-conjugated streptavidin (e.g., Alexa Fluor 488, 555, or 647)

• Incubate for 1 hour at room temperature protected from light

• Wash 3-5 times with PBS

• Counterstain nuclei with DAPI (1 μg/ml) for 5-10 minutes

Mounting and Imaging:

• Mount with anti-fade mounting medium

• For optimal results, image using confocal microscopy to precisely localize TLR5 expression

• TLR5 should show predominantly membrane localization with some intracellular staining

Successful immunofluorescence with biotin-conjugated TLR5 antibodies has been demonstrated in cell lines like THP-1, where specific membrane staining is visible . When optimizing, titrate both the primary antibody and streptavidin-fluorophore concentrations to achieve optimal signal-to-noise ratio for your specific application.

How can biotin-conjugated TLR5 antibodies be used to study TLR5-flagellin interactions?

Studying TLR5-flagellin interactions represents a fundamental aspect of innate immunity research, and biotin-conjugated TLR5 antibodies offer several sophisticated approaches to investigate these interactions:

Competitive Binding Assays:

• Develop a solid-phase binding assay with immobilized recombinant TLR5

• Pre-incubate with varying concentrations of biotin-conjugated TLR5 antibody

• Add labeled flagellin and measure binding

• Generate inhibition curves to determine if the antibody competes with flagellin for the same binding site

This approach can determine whether your TLR5 antibody recognizes epitopes involved in flagellin binding, which is particularly relevant for studying TLR5 agonists like CBLB502/entolimod .

Cell-Based Functional Assays:

• Utilize reporter cell lines expressing TLR5 and an NF-κB reporter construct

• Pre-treat cells with biotin-conjugated TLR5 antibody at varying concentrations

• Stimulate with flagellin or synthetic TLR5 agonists like GP532

• Measure reporter activity to assess antibody blocking potential

This approach allows quantitative assessment of how the antibody affects TLR5 signaling function, particularly useful when evaluating the mechanisms of TLR5 agonists in immune activation .

Co-immunoprecipitation Studies:

• Treat cells with flagellin or TLR5 agonists

• Lyse cells and immunoprecipitate using biotin-conjugated TLR5 antibody coupled to streptavidin beads

• Analyze precipitated complexes for associated proteins including MyD88, TRAF6, and other signaling components

• Compare complex formation under different stimulation conditions

This technique, validated using magnetic streptavidin beads for purifying TLR5 protein complexes, enables researchers to track signaling complex assembly following receptor activation .

Live Cell Imaging:

• Label cells with biotin-conjugated TLR5 antibody and fluorescent streptavidin

• Add fluorescently labeled flagellin

• Perform time-lapse confocal microscopy to track co-localization and internalization dynamics

This approach provides direct visualization of TLR5-flagellin interactions in living cells, offering insights into the spatial and temporal dynamics of receptor engagement.

These methodologies can be particularly valuable when investigating novel TLR5 agonists such as the deimmunized and pharmacologically optimized variant GP532, which demonstrates enhanced properties compared to earlier generation agonists like entolimod .

What experimental approaches can be used to investigate TLR5's role in cancer immunotherapy using biotin-conjugated antibodies?

TLR5's emerging role in cancer immunotherapy, particularly through agonists like CBLB502/entolimod, can be investigated using several experimental approaches with biotin-conjugated TLR5 antibodies:

Expression Profiling in Cancer Models:

• Use biotin-conjugated TLR5 antibodies to screen tumor cell lines, patient-derived xenografts, and clinical samples via immunohistochemistry or flow cytometry

• Correlate TLR5 expression patterns with tumor characteristics and clinical outcomes

• Identify tumor types most likely to respond to TLR5-targeted therapies

This approach establishes the foundation for targeted therapy development by identifying eligible patient populations .

Engineered CAR-T/NK Cell Systems:

• Use biotin-conjugated antibodies to validate TLR5 expression in engineered CAR-NK systems designed to secrete TLR5 agonists

• Confirm inducible expression of TLR5 agonists like CBLB502 following CAR engagement

• Track the distribution of secreted TLR5 agonists in the tumor microenvironment

This methodology has been validated in systems where CAR133 constructs were engineered with inducible TLR5 agonist (i502/CBLB502) expression cassettes under control of NFAT-IL2 minimal promoters, creating TRUCK cells that selectively deposit CBLB502 at tumor sites .

Mechanistic Studies of TLR5 Agonist Function:

• Perform co-staining with biotin-conjugated TLR5 antibodies and markers of immune activation

• Track changes in TLR5 localization and expression following agonist treatment

• Use pull-down approaches to identify TLR5-associated proteins in the tumor microenvironment

This approach helps elucidate how TLR5 agonists like GP532 (an optimized variant of entolimod) exert their anti-tumor effects by mobilizing endogenous immune responses .

Cancer Stem Cell Targeting:

• Use biotin-conjugated TLR5 antibodies in combination with cancer stem cell markers like CD133

• Isolate and characterize TLR5-expressing cancer stem cell populations

• Evaluate the effect of TLR5 agonists on cancer stem cell survival and differentiation

This strategy addresses the challenge of eliminating cancer stem cells, which are considered primary sources of cancer progression and recurrence, particularly in colorectal cancer models .

These experimental approaches leverage the versatility of biotin-conjugated TLR5 antibodies to investigate multiple aspects of TLR5's role in cancer immunotherapy, from expression profiling to functional characterization of novel therapeutic strategies.

How can researchers evaluate potential cross-reactivity or specificity issues with biotin-conjugated TLR5 antibodies?

Evaluating specificity and potential cross-reactivity is crucial for generating reliable data with biotin-conjugated TLR5 antibodies. Researchers should implement the following comprehensive validation approaches:

Genetic Knockdown/Knockout Controls:

• Perform siRNA/shRNA knockdown of TLR5 in relevant cell lines

• Compare antibody staining between knockdown and control cells using flow cytometry, Western blot, or immunofluorescence

• Quantify reduction in signal corresponding to knockdown efficiency

• For definitive validation, include TLR5 knockout cells or tissues when available

This approach provides functional validation of antibody specificity by demonstrating reduced signal when the target protein is depleted.

Peptide Competition Assays:

• Pre-incubate biotin-conjugated TLR5 antibody with excess immunizing peptide

• Apply the antibody-peptide mixture in parallel with unblocked antibody

• Compare signal between blocked and unblocked conditions

• Specific antibodies will show significantly reduced staining when pre-incubated with their cognate peptide

This methodology directly tests whether the antibody binds to its intended epitope by competitive blocking.

Cross-Reactivity Assessment:

• Test antibody against recombinant proteins or cells expressing other TLR family members

• Compare staining patterns across multiple species if the antibody claims cross-reactivity

• Validate species reactivity claims (e.g., human, mouse, rat) through comparative analysis

When evaluating biotin-conjugated TLR5 antibodies, researchers should be particularly aware of potential cross-reactivity with:

• Other TLR family members that share structural homology

• Proteins containing leucine-rich repeat domains similar to TLR5

• Endogenous biotin-containing proteins that might generate background signal

Multi-technique Validation:

• Confirm consistent results across multiple detection methods (WB, IF, ELISA)

• Compare band patterns in Western blot with expected molecular weight (~95-97 kDa)

• Verify subcellular localization is consistent with TLR5's known distribution (primarily cell membrane)

Control for Biotin-Specific Issues:

• Include avidin/biotin blocking steps to eliminate signal from endogenous biotin

• Run parallel experiments with non-biotinylated antibodies against the same target

• Include appropriate negative controls (isotype controls with biotin conjugation)

These validation approaches ensure that experimental observations reflect genuine TLR5 detection rather than artifacts or cross-reactivity, essential for generating reproducible and reliable research outcomes.

What are common challenges when using biotin-conjugated TLR5 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with biotin-conjugated TLR5 antibodies. The following table presents these challenges along with evidence-based solutions:

Specific Troubleshooting for TLR5 Detection:

For optimal detection of TLR5 using biotin-conjugated antibodies, consider these specific parameters based on experimental evidence:

• Membrane Preparation: TLR5 is primarily localized to the cell membrane , so sample preparation methods that preserve membrane integrity are critical for consistent results.

• Epitope Accessibility: The immunogen ranges used for antibody generation (e.g., 701-810/873 for mouse TLR5 or 682-850AA for human TLR5 ) should be considered when troubleshooting, as these regions must be accessible for antibody binding.

• Species-Specific Optimization: While many TLR5 antibodies show cross-reactivity between human and mouse, optimization may be required when switching between species, particularly for predicted reactivity in rat models .

• Storage Conditions: Maintain antibody storage at -20°C and avoid repeated freeze-thaw cycles to preserve biotin conjugation integrity and antibody function .

Implementing these evidence-based solutions will help researchers overcome common challenges associated with biotin-conjugated TLR5 antibodies, leading to more consistent and reliable experimental outcomes.

How can researchers optimize detection conditions for low TLR5 expression levels?

Detecting low levels of TLR5 expression presents a significant challenge in many experimental systems. The following optimization strategies leverage the advantages of biotin-conjugated antibodies to enhance sensitivity:

Signal Amplification Approaches:

• Multi-layer Detection Systems:

  • Implement a three-step detection method: biotin-conjugated primary antibody → streptavidin-biotin-peroxidase complex → amplified substrate

  • This approach provides exponential signal enhancement compared to standard two-step methods

  • Particularly effective for tissues with limited TLR5 expression

• Tyramide Signal Amplification (TSA):

  • Use biotin-conjugated TLR5 antibody followed by streptavidin-HRP

  • Add biotinylated tyramide which becomes activated by peroxidase and deposits additional biotin molecules around the antigen

  • Detect with a second round of streptavidin-reporter

  • This technique can increase sensitivity by 10-100 fold

Sample and Protocol Optimization:

• Antigen Retrieval Enhancement:

  • Extend antigen retrieval time for fixed tissues (15-20 minutes)

  • Test both citrate (pH 6.0) and EDTA (pH 9.0) based buffers to determine optimal epitope exposure

  • Consider protein crosslinker reversal techniques for heavily fixed samples

• Concentration and Incubation Optimization:

  • Increase antibody concentration while monitoring background (up to 10 μg/ml for IF applications)

  • Extend primary antibody incubation to 48 hours at 4°C for tissue sections

  • Incorporate gentle agitation during incubations to improve antibody penetration

• Reduce Background Interference:

  • Implement stringent blocking of endogenous biotin using commercial avidin/biotin blocking kits

  • Use hydrogen peroxide treatment to quench endogenous peroxidases prior to detection

  • Incorporate longer and more frequent washing steps to improve signal-to-noise ratio

Detection System Selection:

• Highly Sensitive Substrates:

  • For colorimetric detection, use DAB with nickel enhancement

  • For chemiluminescence, select femto-grade substrates with extended signal duration

  • For fluorescence, choose quantum dot-conjugated streptavidin for photostable, bright signals

• Imaging Optimization:

  • Use confocal microscopy with increased photomultiplier sensitivity

  • Implement deconvolution algorithms to enhance signal detection

  • Consider super-resolution techniques for precise localization of low abundance receptors

These strategies have proven effective for detecting TLR5 in challenging samples, including those with minimal expression levels. By combining several approaches, researchers can achieve reliable detection even in systems where TLR5 is expressed at levels below standard detection thresholds .

How are biotin-conjugated TLR5 antibodies being used in CAR-T/NK cell therapy development?

Biotin-conjugated TLR5 antibodies are playing increasingly important roles in the development and characterization of next-generation CAR-T and CAR-NK cell therapies, particularly those incorporating TLR5 agonists as immunomodulatory components:

Verification of Inducible TLR5 Agonist Expression:
CAR-T/NK cells engineered with inducible TLR5 agonist expression systems (referred to as "TRUCK" cells) require precise characterization to confirm functionality. Biotin-conjugated TLR5 antibodies are used to:

• Verify expression of the TLR5 agonist (e.g., CBLB502/entolimod) following CAR engagement

• Quantify agonist secretion using ELISA-based approaches with biotin-conjugated antibodies

• Confirm biological activity of the secreted agonist through binding studies

This approach has been validated in systems where CD133-targeting CAR-NK cells were engineered to express CBLB502 under control of the NFAT-IL2 minimal promoter, creating cells that selectively deposit the TLR5 agonist into tumor tissue following antigen recognition .

Characterization of TLR5 Expression in Immune Cells:
Understanding TLR5 expression patterns on various immune cell populations is critical for predicting responses to TLR5 agonist therapy:

• Flow cytometric analysis using biotin-conjugated TLR5 antibodies helps characterize expression on T cells, NK cells, and dendritic cells

• Co-staining with activation markers reveals correlations between TLR5 expression and functional status

• Comparison of TLR5 expression before and after cell engineering processes ensures maintenance of receptor density

Monitoring TLR5 Agonist Distribution:
Biotin-conjugated TLR5 antibodies facilitate studies tracking the distribution of secreted TLR5 agonists in the tumor microenvironment:

• Tissue sections from tumor models treated with TRUCK cells can be analyzed using immunohistochemistry

• Co-localization with immune cell markers helps identify cells responding to the locally delivered TLR5 agonist

• Quantification of TLR5 agonist concentration at tumor sites versus systemic circulation validates the targeting approach

This methodology supports the core advantage of the TRUCK approach: "selectively depositing CBLB502 into the tumor tissue while avoiding systemic immune cell activation," a critical factor in minimizing toxicity while maximizing therapeutic efficacy .

These applications demonstrate how biotin-conjugated TLR5 antibodies contribute to advancing CAR-T/NK cell therapies beyond conventional approaches, particularly for challenging malignancies like colorectal cancer where targeting cancer stem cells through TLR5-mediated immune activation represents a promising strategy .

What future applications are being developed for TLR5 antibodies in conjunction with TLR5 agonist therapeutics?

The development of next-generation TLR5 agonists like GP532, an optimized variant of entolimod with reduced immunogenicity, is opening new research avenues where biotin-conjugated TLR5 antibodies serve as essential tools:

Companion Diagnostic Development:
Biotin-conjugated TLR5 antibodies are being evaluated for companion diagnostic applications to identify patients likely to respond to TLR5 agonist therapy:

• Immunohistochemical protocols using biotin-conjugated antibodies can assess TLR5 expression in tumor biopsies

• Flow cytometry panels incorporating these antibodies can evaluate immune cell responsiveness

• Standardized assays could potentially predict response to TLR5 agonists like GP532, addressing the challenge of patient stratification

Pharmacodynamic Biomarker Monitoring:
Emerging applications include using biotin-conjugated TLR5 antibodies to track receptor modulation during treatment:

• Serial biopsies analyzed with quantitative immunohistochemistry can monitor changes in TLR5 expression

• Flow cytometry of peripheral blood mononuclear cells can detect systemic effects on immune cell TLR5 expression

• These approaches help establish correlations between receptor dynamics and clinical outcomes

Neutralizing Antibody Assessment:
A critical challenge with first-generation TLR5 agonists like entolimod was the development of neutralizing antibodies, limiting multi-dose applications. GP532 was designed to overcome this limitation:

• Biotin-conjugated TLR5 antibodies are being used to develop competition assays that detect neutralizing antibodies

• These assays help evaluate the effectiveness of deimmunization strategies in next-generation agonists

• Monitoring patient samples for neutralizing antibody development helps optimize dosing schedules

As noted in the literature, the TLR5 agonist entolimod "induced a rapid neutralizing immune response, presumably due to immune memory from prior exposure to flagellated enterobacteria," making this application particularly relevant for clinical translation .

Structure-Function Relationship Studies:
Biotin-conjugated TLR5 antibodies targeting specific epitopes are facilitating structure-function studies:

• Epitope mapping reveals receptor domains critical for agonist binding

• Competition studies identify antibodies that enhance or inhibit agonist activity

• These insights guide the rational design of next-generation TLR5 agonists with optimized pharmacological properties

These emerging applications represent the convergence of antibody-based research tools with therapeutic development, illustrating how biotin-conjugated TLR5 antibodies contribute to advancing novel immunotherapeutic approaches from bench to bedside .