T.Vaginalis P65 Antibody

What is Trichomonas vaginalis P65 and why is it significant in research?

The P65 protein is an essential adhesion protein of Trichomonas vaginalis, an anaerobic flagellated protozoan parasite that causes trichomoniasis, the most prevalent sexually transmitted disease worldwide. P65 plays a vital role in the pathogen's ability to adhere to and penetrate the vaginal tract surface epithelium . This protein is significant in research because it represents a key virulence factor that facilitates T. vaginalis colonization and infection. Understanding P65's structure and function provides insights into the fundamental mechanisms of T. vaginalis pathogenesis, potentially leading to improved diagnostic methods and therapeutic interventions. Research focusing on P65 has implications for addressing the approximately 280 million T. vaginalis infections that occur annually worldwide .

What types of T.Vaginalis P65 antibodies are available for research applications?

The primary type of T.Vaginalis P65 antibody documented in the research literature is a polyclonal antibody derived from rabbit IgG. This antibody is produced by immunizing rabbits with recombinant T. vaginalis P65 protein and subsequently purifying the antibodies through affinity chromatography techniques . The available antibody targets the P65 adhesion protein, which spans approximately 331 amino acids . While the search results specifically mention polyclonal antibodies, researchers should note that different preparations may vary in their specificity and applications. The antibody is typically formulated in a buffer containing 50mM glycine at pH 8.0 to maintain stability and functionality .

How should T.Vaginalis P65 antibody be stored and handled to maintain optimal activity?

To maintain optimal activity of T.Vaginalis P65 antibody, researchers should adhere to the following storage and handling guidelines:

• For short-term storage (2-4 weeks), store the antibody at 4°C in its original vial.

• For long-term storage, maintain the antibody at -20°C.

• To prevent protein degradation during extended storage, add a carrier protein such as 0.1% Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA).

• Avoid multiple freeze-thaw cycles as these can significantly diminish antibody activity and specificity.

• When working with the antibody, handle it as a clear, colorless sterile-filtered solution.

• Before use, allow the antibody to equilibrate to room temperature gradually and mix gently to ensure uniformity .

These precautions are essential for preserving the structural integrity and functional capacity of the antibody, ensuring consistent and reliable experimental results over time.

How can T.Vaginalis P65 antibody be used to study host-pathogen interactions in infection models?

T.Vaginalis P65 antibody serves as a valuable tool for investigating host-pathogen interactions through multiple experimental approaches:

Immunolocalization Studies:
Researchers can use the antibody for immunofluorescence microscopy to visualize P65 distribution during T. vaginalis attachment to host cells. The protocol typically involves:

• Fixing infected host cells with 4% paraformaldehyde for 15 minutes

• Permeabilizing with 0.25% Triton X-100 for 10 minutes

• Blocking with appropriate serum (typically 5% BSA)

• Incubating with T.Vaginalis P65 antibody at 1:100 dilution overnight at 4°C

• Detecting with fluorophore-conjugated secondary antibodies

• Counterstaining nuclei with DAPI and imaging with confocal microscopy

Co-immunoprecipitation Experiments:
The antibody can identify host receptors interacting with P65 by:

• Lysing infected host cells in appropriate buffer

• Precipitating with T.Vaginalis P65 antibody

• Analyzing precipitated complexes via Western blotting or mass spectrometry

Inhibition Assays:
Pre-incubating T. vaginalis with the antibody before infection can help quantify the contribution of P65 to adhesion and invasion processes, providing insights into the molecular mechanisms of pathogenesis .

What are the recommended protocols for using T.Vaginalis P65 antibody in Western blot analyses?

For optimal Western blot results when using T.Vaginalis P65 antibody, researchers should follow this methodological approach:

Sample Preparation:

• Harvest T. vaginalis trophozoites in late-logarithmic growth phase

• Wash cells twice with PBS

• Lyse cells using an appropriate lysis buffer containing protease inhibitors

• Centrifuge at 12,000 g to remove debris

• Quantify protein concentration using Bradford or BCA assay

Gel Electrophoresis and Transfer:

• Load 20-30 μg of protein per lane on a 10-12% SDS-PAGE gel

• Perform electrophoresis at 100-120V

• Transfer proteins to PVDF or nitrocellulose membrane at 100V for 1 hour or 30V overnight at 4°C

Antibody Incubation and Detection:

• Block membrane with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with T.Vaginalis P65 antibody at 1:1000-1:5000 dilution overnight at 4°C

• Wash 3-5 times with TBST

• Incubate with HRP-conjugated secondary antibody (typically anti-rabbit IgG) at 1:5000-1:10000 dilution for 1 hour at room temperature

• Wash 3-5 times with TBST

• Develop using ECL reagent and detect signals using appropriate imaging system

Expected Results:
The T.Vaginalis P65 protein should be detected at approximately 65 kDa, though post-translational modifications may cause slight variations in apparent molecular weight .

How can researchers use the T.Vaginalis P65 antibody to investigate inflammatory responses in cell culture models?

T.Vaginalis P65 antibody can be effectively used to investigate inflammatory responses through the following methodological approaches:

Cytokine Production Analysis:

• Culture macrophages or vaginal epithelial cells in appropriate media

• Pre-treat cells with or without MAPK inhibitors (e.g., SB203580 for p38, PD98059 for ERK)

• Co-incubate cells with T. vaginalis at various multiplicities of infection

• Collect supernatants at different time points (typically 18 hours post-infection)

• Measure proinflammatory cytokines (IL-6, TNF-α, IFN-γ) using ELISA

• In parallel, harvest cells for protein extraction

• Use T.Vaginalis P65 antibody in Western blotting to correlate P65 levels with cytokine production

Signaling Pathway Activation:

• Expose macrophages to T. vaginalis for specific time periods (0.5, 1, 2, and 4 hours)

• Extract proteins and analyze phosphorylation of p38, ERK, and NF-κB p65 via Western blotting

• Use T.Vaginalis P65 antibody to confirm the presence of the parasite protein

• Correlate parasite protein levels with the degree of signaling pathway activation

Immunofluorescence Analysis:

• Culture cells on coverslips and stimulate with T. vaginalis

• Fix and permeabilize cells

• Stain with phospho-specific antibodies for NF-κB p65 and T.Vaginalis P65 antibody

• Counterstain nuclei with DAPI

• Analyze nuclear translocation of NF-κB p65 using confocal microscopy

This approach allows researchers to establish direct connections between T. vaginalis infection, P65 expression, and the subsequent inflammatory cascade in host cells.

How does T.Vaginalis P65 interact with host TLR2 to trigger inflammatory responses and what methods can be used to study this interaction?

T.Vaginalis P65 appears to interact with host Toll-like receptor 2 (TLR2) to trigger inflammatory responses through complex signaling cascades. This interaction can be studied using several sophisticated approaches:

Co-immunoprecipitation and Protein Interaction Analysis:

• Express recombinant TLR2 and T.Vaginalis P65 in appropriate expression systems

• Perform pull-down assays using T.Vaginalis P65 antibody

• Analyze interacting partners by mass spectrometry or Western blotting

• Confirm direct interactions using surface plasmon resonance or biolayer interferometry

Cell Signaling Analysis:
Research demonstrates that T. vaginalis activates TLR2, which subsequently leads to phosphorylation of signaling proteins including p38, ERK, and NF-κB p65. This activation pattern can be analyzed by:

• Comparing wild-type and TLR2-/- macrophages after T. vaginalis exposure

• Measuring phosphorylation levels of p38, ERK, and p65 NF-κB

• Correlating these phosphorylation events with proinflammatory cytokine production

Experimental Data from TLR2 Pathway Activation:

This temporal pattern suggests that p38 activation precedes ERK activation, with both contributing to sustained NF-κB p65 signaling and subsequent proinflammatory cytokine production (IL-6, TNF-α, IFN-γ) .

What are the methodological approaches for investigating the role of T.Vaginalis P65 in the context of microbial symbiont interactions?

T. vaginalis frequently harbors microbial symbionts, particularly Mycoplasma hominis and Mycoplasma girerdii, which can modulate host immune responses. Investigating P65's role in these polymicrobial interactions requires specialized methodological approaches:

Differential Expression Analysis:

• Culture T. vaginalis isolates with and without Mycoplasma symbionts

• Extract total protein from both cultures

• Perform Western blot analysis using T.Vaginalis P65 antibody

• Quantify differences in P65 expression levels using densitometry

• Correlate expression with adhesion capacity and virulence

Co-localization Studies:

• Process T. vaginalis samples using immunofluorescence microscopy

• Use T.Vaginalis P65 antibody alongside Mycoplasma-specific antibodies

• Analyze potential co-localization patterns at the parasite surface

• Evaluate if symbionts alter P65 distribution or expression

Functional Modification Analysis:

• Compare cytokine profiles induced by T. vaginalis with and without symbionts

• Use specific inhibitors to block P65 function

• Measure alterations in the host inflammatory response, particularly IL-1 and IL-6 levels, which are significantly higher in symbiont-positive T. vaginalis infections

• Use TLR2-/- macrophage models to assess whether symbiont-induced modifications depend on TLR2 signaling

Comparative Host Response Data:

These approaches allow researchers to determine how microbial symbionts might influence T. vaginalis pathogenesis through modulation of P65 expression or function .

What are the challenges in interpreting Western blot data using T.Vaginalis P65 antibody and how can researchers overcome them?

Researchers face several specific challenges when interpreting Western blot data using T.Vaginalis P65 antibody, which can be addressed through methodological refinements:

Challenge 1: Cross-reactivity with host proteins

• Solution: Perform competitive inhibition studies by pre-incubating the antibody with recombinant P65 protein prior to Western blotting. True P65 signals will disappear while cross-reactive bands will remain.

• Alternative approach: Include both T. vaginalis-positive and negative samples to identify parasite-specific bands.

Challenge 2: Post-translational modifications affecting antibody recognition

• Solution: Use denaturing and non-denaturing conditions in parallel to determine if protein conformation affects antibody binding.

• Methodological refinement: Treat samples with phosphatase or glycosidase enzymes to remove modifications that might interfere with antibody recognition.

Challenge 3: Variable expression levels across different T. vaginalis strains

• Solution: Include multiple reference strains and clinical isolates to establish a range of normal expression.

• Data interpretation guide: Normalize P65 signals to a conserved T. vaginalis protein (e.g., α-tubulin) to account for differences in parasite load.

Challenge 4: Interference from microbial symbionts

• Solution: Culture T. vaginalis with antibiotics to eliminate bacterial symbionts and compare P65 expression before and after treatment.

• Control experiment: Specifically detect Mycoplasma presence using PCR and correlate with variations in P65 detection .

Troubleshooting Table for Western Blot Analysis:

By implementing these methodological refinements, researchers can generate more reliable and interpretable Western blot data when using T.Vaginalis P65 antibody .

How can T.Vaginalis P65 antibody be used to investigate the differential immune responses in TLR2-dependent and TLR2-independent pathways?

T.Vaginalis P65 antibody offers significant utility for dissecting the complex immune responses involving both TLR2-dependent and TLR2-independent pathways through several advanced methodological approaches:

Comparative Signaling Analysis in Wild-Type vs. TLR2-/- Models:

• Isolate peritoneal macrophages from both wild-type and TLR2-/- mice

• Stimulate with T. vaginalis at various MOIs (multiplicity of infection)

• Perform Western blot analysis at specific time points (0.5, 1, 2, and 4 hours)

• Probe for phosphorylation of multiple signaling molecules:

  • p38 MAPK

  • ERK1/2

  • NF-κB p65

  • Other potential TLR2-independent mediators

• Use T.Vaginalis P65 antibody to confirm equivalent parasite loads across experiments

• Quantify differences in activation kinetics between pathways

Pharmacological Inhibition Studies:

• Pretreat wild-type macrophages with specific inhibitors:

  • SB203580 (p38 inhibitor) at 30 μM

  • PD98059 (ERK inhibitor) at 40 μM

  • Inhibitors of TLR2-independent pathways

• Stimulate with T. vaginalis

• Measure cytokine production (IL-6, TNF-α, IFN-γ) via ELISA

• Correlate inhibitor effects with P65 expression and distribution

What methods can researchers use to study the potential therapeutic applications of targeting T.Vaginalis P65 for vaccine development?

Developing vaccines against T. vaginalis represents an important research direction, and the P65 protein offers a promising target. Researchers can employ several methodological approaches utilizing T.Vaginalis P65 antibody to advance vaccine development:

Epitope Mapping and Identification:

• Generate overlapping peptides spanning the P65 protein (aa 1-331)

• Screen peptides for antibody binding using ELISA or peptide arrays

• Identify immunodominant epitopes that elicit strong antibody responses

• Validate epitopes using competitive binding assays with the T.Vaginalis P65 antibody

Neutralization Assays:

• Pre-incubate T. vaginalis with various concentrations of T.Vaginalis P65 antibody

• Introduce treated parasites to vaginal epithelial cell monolayers

• Measure adhesion inhibition through microscopic analysis or radiolabeling techniques

• Quantify the minimum antibody concentration required for significant inhibition

Immunogenicity Assessment:

• Immunize animal models with recombinant P65 protein or peptide conjugates

• Collect sera at defined intervals post-immunization

• Compare induced antibodies with commercial T.Vaginalis P65 antibody for:

  • Epitope specificity (using competitive ELISAs)

  • Functional activity (using adhesion inhibition assays)

  • Isotype distribution (using isotype-specific secondary antibodies)

Vaccine Formulation Optimization:

• Test multiple adjuvant combinations with recombinant P65

• Measure antibody titers using standardized ELISAs

• Assess functional antibody activity through inhibition assays

• Evaluate memory response through challenge studies

• Use T.Vaginalis P65 antibody as a positive control throughout testing

Research with recombinant T.Vaginalis P65 protein (aa 1-331) has already demonstrated its utility for vaccine development studies . By employing the T.Vaginalis P65 antibody as a reference standard, researchers can systematically evaluate candidate vaccines for their ability to elicit protective antibody responses against this key adhesion protein.

How can researchers design experiments to study the interactions between T.Vaginalis P65 and other virulence factors in the context of host immune evasion?

T. vaginalis employs multiple virulence factors that work in concert to establish infection and evade host immunity. Designing experiments to understand how P65 interacts with other virulence determinants requires sophisticated methodological approaches:

Co-expression and Co-localization Analysis:

• Perform dual immunofluorescence staining of T. vaginalis using:

  • T.Vaginalis P65 antibody

  • Antibodies against other virulence factors (e.g., cysteine proteases, adhesins)

• Analyze distribution patterns during different stages of host-parasite interaction

• Quantify co-localization coefficients using confocal microscopy and image analysis software

• Determine whether virulence factors cluster together at host-parasite interface

Temporal Expression Profiling:

• Expose T. vaginalis to host cells or host-derived factors

• Collect parasites at defined time points (0, 1, 2, 4, 8, 24 hours)

• Perform Western blot analysis for P65 and other virulence factors

• Create temporal expression maps to identify coordinated expression patterns

Functional Interdependence Studies:

• Generate gene knockout or knockdown parasites for specific virulence factors

• Assess P65 expression and distribution using T.Vaginalis P65 antibody

• Measure adhesion capacity and host cell cytotoxicity

• Determine whether loss of one virulence factor affects P65 functionality

Immune Evasion Mechanism Analysis:

• Expose macrophages to wild-type and modified T. vaginalis strains

• Monitor phagocytosis rates and parasite survival

• Assess NF-κB p65 nuclear translocation and inflammatory cytokine production

• Use T.Vaginalis P65 antibody to correlate parasite protein levels with immune modulation

Research data has revealed key interactions:
When T. vaginalis harbors symbiotic microorganisms like Mycoplasma hominis, the inflammatory response is significantly enhanced, with IL-1 and IL-6 levels several-fold higher compared to symbiont-free parasites . Similarly, T. vaginalis virus (TVV) has been shown to modulate host responses, potentially through interactions with parasite virulence factors including P65 . These observations suggest complex interactions between P65 and other factors that collectively influence pathogenesis and immune evasion.

What are the recommended experimental controls when using T.Vaginalis P65 antibody in various immunological assays?

When designing experiments utilizing T.Vaginalis P65 antibody, incorporating proper controls is essential for generating reliable and interpretable data. The following methodological approach outlines critical controls for various immunological assays:

For Western Blot Analysis:

• Positive Control: Include recombinant T.Vaginalis P65 protein (aa 1-331) as a reference standard

• Negative Control: Use protein extracts from non-T. vaginalis protozoa (e.g., Giardia lamblia)

• Isotype Control: Include normal rabbit IgG at the same concentration as the P65 antibody

• Peptide Competition Control: Pre-incubate P65 antibody with excess recombinant P65 protein before Western blotting

• Loading Control: Probe for a constitutively expressed T. vaginalis protein (e.g., α-tubulin)

For Immunofluorescence:

• Primary Antibody Omission: Process samples without primary antibody to detect non-specific secondary antibody binding

• Isotype Control: Use normal rabbit IgG to assess background staining

• Uninfected Cell Control: Include host cells without T. vaginalis to identify cross-reactivity

• Fixed Parasite Control: Analyze fixed T. vaginalis without host cells to establish baseline staining patterns

For ELISA and Cytokine Assays:

• Positive Stimulation Control: Include Pam3CSK4 (10 μg/ml) as a known TLR2 agonist

• Negative Control: Use media-only treatment without parasites

• Inhibitor Controls: Include appropriate vehicle controls for inhibitor experiments

• Cell Viability Control: Ensure >95% viability using trypan blue exclusion after inhibitor treatments

These methodological controls ensure that any observed effects are specifically attributable to T.Vaginalis P65 and not experimental artifacts or non-specific interactions.

How can researchers optimize immunoprecipitation protocols using T.Vaginalis P65 antibody to identify novel interaction partners?

Optimizing immunoprecipitation (IP) protocols with T.Vaginalis P65 antibody requires careful methodological considerations to maximize specificity while maintaining protein-protein interactions:

Pre-IP Sample Preparation:

• Harvest late-logarithmic phase T. vaginalis trophozoites (the optimal stage for protein expression)

• Wash cells 3 times with ice-cold PBS to remove media components

• Select appropriate lysis buffer based on interaction strength:

  • For strong interactions: RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS)

  • For weak interactions: Gentler NP-40 buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40)

• Include protease and phosphatase inhibitor cocktails to preserve protein integrity

• Perform cell lysis on ice for 30 minutes with periodic gentle mixing

• Centrifuge at 14,000 g for 15 minutes at 4°C to remove debris

Antibody Binding Optimization:

• Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

• Determine optimal antibody concentration through titration (typically 2-5 μg per mg of total protein)

• Incubate cleared lysate with T.Vaginalis P65 antibody overnight at 4°C with gentle rotation

• Add pre-washed Protein A/G beads and incubate for 2-4 hours at 4°C

Washing and Elution Strategy:

• Perform increasingly stringent washes to reduce non-specific binding:

  • First wash: Lysis buffer

  • Second wash: Lysis buffer with higher salt (250 mM NaCl)

  • Third wash: Lysis buffer with reduced detergent

• Elute bound proteins using one of these methods:

  • Gentle: Non-denaturing elution with excess P65 peptide

  • Standard: SDS sample buffer heating at 95°C for 5 minutes

Controls and Validation:

• Include isotype control (normal rabbit IgG) IP processed identically

• Perform reverse IP with antibodies against suspected interaction partners

• Validate interactions through orthogonal methods:

  • Co-localization by immunofluorescence

  • Proximity ligation assay (PLA)

  • Biolayer interferometry with purified components

This optimized methodology has been successfully applied to investigate interactions between pathogen proteins and host immune components, particularly in the context of TLR2 pathway activation .

What future research directions could benefit from T.Vaginalis P65 antibody applications in understanding trichomoniasis pathogenesis?

T.Vaginalis P65 antibody opens numerous avenues for future research that could significantly advance our understanding of trichomoniasis pathogenesis:

Investigation of P65's Role in Host-Microbiome Interactions:

• Apply T.Vaginalis P65 antibody to study how the vaginal microbiota influences P65 expression and function

• Develop co-culture systems with different bacteria to assess modulation of P65-mediated adhesion

• Investigate whether P65 expression changes in response to lactobacilli vs. bacteria associated with bacterial vaginosis

• Determine if P65 interacts directly with bacterial surface components

Development of Point-of-Care Diagnostics:

• Evaluate T.Vaginalis P65 antibody pairs for sandwich ELISA development

• Assess the sensitivity and specificity of P65 detection in clinical specimens

• Develop lateral flow immunoassays using anti-P65 antibodies for rapid diagnosis

• Compare P65-based detection methods with current diagnostic approaches

Delineation of Structure-Function Relationships:

• Map functional domains within P65 using deletion mutants and the antibody

• Identify specific regions responsible for host cell binding

• Determine whether post-translational modifications affect antibody recognition

• Engineer modified P65 variants to study effects on virulence

Exploration of P65's Role in Mixed Infections:

• Investigate whether HIV infection alters P65 expression or function

• Study the impact of other STIs on P65-mediated T. vaginalis adhesion

• Determine if P65 contributes to the increased HIV acquisition risk associated with trichomoniasis

• Develop in vitro models of polymicrobial infection involving T. vaginalis

Therapeutic Development:

• Screen for small molecule inhibitors that disrupt P65-host cell interactions

• Develop neutralizing antibodies based on epitope mapping with the T.Vaginalis P65 antibody

• Test peptide vaccines targeting key P65 epitopes

• Evaluate combination approaches targeting multiple virulence factors simultaneously