THBD Antibody

What is THBD antibody and what protein does it recognize?

THBD antibody recognizes Thrombomodulin, a transmembrane glycoprotein with natural anticoagulant properties. It forms a complex with thrombin on endothelial cells, facilitating the conversion of protein C to activated protein C (APC), which prevents excessive clotting while ensuring proper wound healing processes . Commercially available antibodies typically recognize a protein of 75kDa, although predicted band size in some protocols is specified as 60 kDa . Thrombomodulin is also known by several alternative names including CD141, THRM, THBD, TM, and Fetomodulin .

In which cell types and tissues is THBD normally expressed?

THBD expression is restricted to specific cell types and tissues. Research has established that it is primarily expressed in endothelial and mesothelial cells . Additional expression has been documented in synovial lining cells and syncytio-trophoblasts of placenta . For experimental validation, positive Western blot detection has been confirmed in several cell types including A431 cells, human placenta tissue, THP-1 cells, and HUVEC cells . In immunohistochemistry applications, positive detection has been reported in human lung cancer tissue, human placenta tissue, and human tonsillitis tissue . These expression patterns have implications for experimental design when studying THBD functionality.

What are the recommended applications for THBD antibodies in research?

Based on validated research protocols, THBD antibodies have demonstrated utility across multiple experimental applications. They are suitable for Western blotting (recommended dilution 1:2000-1:10000), immunohistochemistry using paraffin-embedded tissues (recommended dilution 1:500-1:2000), protein array analysis, and immunoprecipitation . For Western blot applications, researchers should note that specific antibody clones have been validated against recombinant Thrombomodulin protein and THP1 cell lysate . When performing immunohistochemistry, optimal results are achieved with antigen retrieval using TE buffer pH 9.0, though citrate buffer pH 6.0 may serve as an alternative . Researchers should titrate the antibody in their specific testing systems to obtain optimal results, as performance may be sample-dependent .

What are the key considerations for optimizing immunohistochemistry with THBD antibodies?

When optimizing immunohistochemistry protocols using THBD antibodies, researchers should address several critical factors. First, tissue fixation and processing: formalin-fixed, paraffin-embedded tissues have been successfully used with validated antibody concentrations of 2 μg/ml . Second, antigen retrieval: optimal results typically require TE buffer pH 9.0, although citrate buffer pH 6.0 has been used as an alternative . Third, antibody concentration: titration is essential, with recommended dilution ranges of 1:500-1:2000 . Fourth, detection systems: must be optimized based on tissue type and expected expression levels. Finally, include appropriate positive controls (human placenta, tonsillitis tissue, lung cancer tissue) and negative controls (antibody diluent only, isotype control) . Researchers should also be aware that THBD expression patterns vary by tissue type, with expression predominantly in endothelial cells, mesothelial cells, and specific epithelial tissues .

How can researchers investigate THBD's role as a receptor for Human Cytomegalovirus (HCMV)?

Investigating THBD's role as an HCMV receptor requires specialized experimental approaches. Researchers should design viral entry inhibition assays using soluble THBD proteins and THBD-specific antibodies to assess their ability to block viral infection in epithelial and endothelial cells . A dose-response design is critical, as studies have shown dose-dependent inhibition of HCMV infection . To evaluate THBD's independent receptor function, researchers should employ cell systems with knockout or overexpression models - for example, studies have demonstrated that THBD overexpression in NRP2 knockout cells markedly increases viral entry . Confirmation of Pentamer-specific infection can be achieved using Pentamer-specific monoclonal antibodies (e.g., 8I21 mAb) at concentrations of 0.1 μg/ml . Finally, viral spread assays should be conducted to determine if THBD-expressing cells can propagate the infection to surrounding cells . These methodological approaches allow for comprehensive assessment of THBD's role in HCMV pathogenesis.

What methodological approaches can differentiate between THBD and NRP2 as HCMV receptors?

To differentiate between THBD and NRP2 as HCMV receptors, researchers should implement a multi-faceted experimental approach. First, perform competition assays between recombinant THBD and NRP2 proteins, as research has shown that a combination of these proteins inhibits HCMV entry to higher levels than NRP2 alone . Second, utilize genetic manipulation in cell culture models - specifically, compare HCMV infection rates in wild-type cells, NRP2 knockout cells, and NRP2 knockout cells with THBD overexpression . Third, employ receptor-specific blocking antibodies independently and in combination to assess their effects on viral entry. Fourth, conduct co-immunoprecipitation experiments to determine if THBD and NRP2 physically interact during viral entry. Finally, perform quantitative analyses of viral entry in cells expressing varying levels of each receptor. This comprehensive approach will elucidate the independent and potentially cooperative roles of THBD and NRP2 in HCMV infection, which has been suggested by research showing that THBD and NRP2 may function as independent receptors or co-receptors .

What are the key considerations for developing multiplexed assays using THBD antibodies?

Developing effective multiplexed assays with THBD antibodies requires careful optimization of several parameters. First, protein biotinylation ratios significantly impact assay performance - research indicates optimal biotinylation ratios of 50:1 for target proteins yield minimal background while maintaining high signal across the testing range . Second, antibody concentration must be carefully titrated; studies suggest starting concentrations of 0.5μg/mL for monoclonal antibodies when detecting THBD and related proteins . Third, sample dilution factors are critical - for serum samples, a 1:5000 dilution has proven effective in maintaining sensitivity while limiting background interference . Fourth, researchers must evaluate and control for cross-reactivity between detection antibodies; for instance, documented cross-reactivity between anti-IgG3 and IgG1 requires specific validation steps . Finally, platform selection impacts sensitivity - Meso Scale Discovery (MSD) platforms have been successfully employed for multiplexed detection of THBD alongside other proteins of interest . These methodological considerations enable researchers to develop robust multiplexed assays for investigating THBD in complex biological samples.

How can researchers address contradictory findings related to THBD molecular weight in their experiments?

Researchers encountering discrepancies in THBD molecular weight (reported as 60kDa in some sources and 75kDa in others ) should implement systematic troubleshooting approaches. First, conduct simultaneous analysis of different tissue and cell types, as THBD may undergo differential post-translational modifications across cell types. Second, employ multiple antibody clones targeting different epitopes, as antibodies recognizing different domains may detect variants with distinct molecular weights. Third, use both reducing and non-reducing conditions in Western blot analysis, as disulfide bonds may affect protein migration. Fourth, perform deglycosylation experiments with enzymes such as PNGase F, as THBD is a glycoprotein and glycosylation patterns may vary by tissue source. Fifth, validate findings by mass spectrometry to definitively identify the protein and any post-translational modifications. This methodological approach allows researchers to systematically resolve molecular weight discrepancies and ensure accurate interpretation of their experimental results.

What experimental design is optimal for investigating THBD's dual role in coagulation and viral pathogenesis?

Investigating THBD's dual functionality requires a comprehensive experimental design that addresses both coagulation and viral entry mechanisms. For coagulation studies, researchers should implement protein C activation assays using purified components (thrombin, protein C, and recombinant THBD) with chromogenic substrates to quantify activated protein C generation . In parallel, viral entry assays should be conducted using reporter viruses (e.g., GFP-expressing HCMV) in cell systems with manipulated THBD expression . To examine potential interplay between these functions, researchers should design competition experiments where thrombin and HCMV simultaneously compete for THBD binding. Domain-specific antibodies or recombinant THBD fragments can help identify which protein domains are essential for each function. Additionally, site-directed mutagenesis of specific THBD residues can determine if the same binding sites mediate both functions or if they are structurally distinct. Finally, translational relevance can be assessed by correlating THBD expression levels with coagulation parameters and viral susceptibility in patient-derived samples. This integrated experimental approach allows for comprehensive investigation of THBD's multifunctional nature.

What controls should be included when using THBD antibodies in research?

A robust experimental design with THBD antibodies requires comprehensive controls to ensure valid and reproducible results. For positive controls, researchers should include known THBD-expressing samples appropriate to their application: HUVEC or THP-1 cells, human placenta tissue for Western blot applications; and human placenta, tonsillitis, or lung cancer tissue for immunohistochemistry . For negative controls, implement multiple strategies: isotype-matched control antibodies at identical concentrations to the THBD antibody; secondary antibody-only controls to evaluate non-specific binding; and THBD-negative tissues or cell lines. When performing immunohistochemistry, include peptide competition controls where the antibody is pre-incubated with the immunizing peptide to confirm specificity . For Western blot applications, molecular weight markers must be used to confirm the expected band size (60-75kDa) . When developing novel assays, validate antibody specificity through protein array testing against a diverse protein panel, as has been done with testing against 19,000 different full-length human proteins . This systematic approach to controls minimizes false positives and ensures experimental rigor.

How should researchers quantify THBD expression in different experimental systems?

For accurate THBD quantification across experimental systems, researchers should employ multiple complementary methodologies. For protein-level quantification in Western blot applications, use densitometry with appropriate normalization to housekeeping proteins (β-actin, GAPDH), and develop standard curves using recombinant THBD at known concentrations . For immunohistochemistry quantification, implement digital pathology approaches with software-based intensity scoring (H-score method or 0-3+ scoring system) and account for both staining intensity and percentage of positive cells . For high-throughput screening, targeted mass spectrometry using immuno-MRM (Multiple Reaction Monitoring) provides absolute quantification with high specificity . When comparing expression across different tissue or cell types, researchers should standardize sample preparation, fixation protocols, and antigen retrieval methods . For multi-site studies, include common reference standards across all testing sites. Statistical analysis should include tests of normality and appropriate parametric or non-parametric tests based on data distribution. This comprehensive approach ensures reliable quantification of THBD across diverse experimental contexts.

How can THBD antibodies be utilized in cancer research and diagnostics?

THBD antibodies offer significant utility in cancer research and diagnostics through multiple methodological applications. For tumor classification, immunohistochemical staining with optimized protocols (1:500-1:2000 dilution) can distinguish vascular tumors, as THBD is present in almost all benign vascular tumors and the majority of malignant vascular tumors (Kaposi's sarcoma, angiosarcoma, and epithelioid hemangioendothelioma) . In mesothelioma diagnostics, THBD serves as a marker for mesothelial cells and malignant mesotheliomas, providing diagnostic value when used in antibody panels . For prognostic studies, researchers should correlate THBD expression levels with patient outcomes using tissue microarrays and standardized scoring systems. In mechanistic studies investigating THBD's role in tumor biology, combine THBD antibodies with functional assays such as cell migration, invasion, and angiogenesis models. For liquid biopsy applications, develop ELISA or multiplex assays to detect soluble THBD in patient serum as a potential biomarker. This diverse methodological toolkit enables researchers to comprehensively investigate THBD's significance in oncology, from basic mechanisms to clinical applications.

What are the methodological approaches for studying THBD in immune response modulation?

Investigating THBD's role in immune modulation requires specialized experimental approaches focusing on its interactions with immune cells and pathways. Researchers should implement co-culture systems where THBD-expressing cells (like endothelial cells) are cultured with immune cells (monocytes, T cells) to assess modulation of cytokine production, measured by ELISA or multiplex cytokine assays. Flow cytometry with fluorochrome-conjugated THBD antibodies can evaluate THBD expression on different immune cell populations, particularly after inflammatory stimulation. For mechanistic studies, researchers should design protein interaction assays to identify THBD binding partners on immune cells, complemented by signaling pathway analysis (phospho-flow cytometry or Western blotting for key signaling molecules). In vivo models with THBD manipulation (knockout, knockdown, or overexpression) allow assessment of immune responses to pathogens or inflammatory stimuli. Recent research has expanded THBD's known functions beyond coagulation to include viral receptor activity , suggesting it may have broader immune regulatory roles. This comprehensive experimental approach enables detailed characterization of THBD's immunomodulatory functions.

What are common troubleshooting approaches for inconsistent THBD antibody performance?

When encountering inconsistent results with THBD antibodies, researchers should implement a systematic troubleshooting protocol. For Western blot applications showing weak or absent signals, first verify protein transfer efficiency using reversible staining methods, then optimize primary antibody concentration (1:2000-1:10000) and incubation conditions (time, temperature) . For high background in immunohistochemistry, implement additional blocking steps, optimize antibody dilution (1:500-1:2000), and evaluate alternative antigen retrieval methods (comparing TE buffer pH 9.0 with citrate buffer pH 6.0) . For cross-reactivity issues, conduct peptide competition assays and consider alternative antibody clones. When faced with inconsistent results between applications, note that some antibody clones may perform differently across techniques - for example, an antibody might work well in Western blot but poorly in immunohistochemistry. Sample preparation issues can be addressed by comparing fresh versus archived samples and optimizing fixation protocols. For clinical samples, consider patient-specific variables like medication use that might affect THBD expression. This methodical approach allows researchers to identify and resolve specific factors affecting antibody performance.

How can researchers optimize THBD detection in complex biological samples?

Optimizing THBD detection in complex biological samples requires specialized methodological approaches. For serum or plasma samples, implement sample pre-treatment steps including heat inactivation of complement and pre-clearing with protein A/G beads to remove interfering immunoglobulins. Optimize antibody concentration through careful titration, with research suggesting starting dilutions of 1:5000 for serum samples . For tissues with high extracellular matrix content, incorporate additional permeabilization steps and extended incubation times. When dealing with samples containing multiple cell types, consider laser capture microdissection to isolate specific cell populations prior to analysis. For low abundance detection, implement signal amplification systems such as tyramide signal amplification or quantum dots. When analyzing clinical samples with variable preservation, standardize fixation and processing protocols or adjust antibody concentrations accordingly. Multiplexed detection can be optimized by carefully selecting antibodies raised in different host species and fluorophores with minimal spectral overlap. Finally, when working with tissues known for high autofluorescence (such as brain or liver), incorporate quenching steps using Sudan Black B or commercial autofluorescence quenchers. These targeted optimization strategies enhance detection sensitivity and specificity in challenging biological contexts.