OR10H1 Antibody

What is OR10H1 and why is it significant in cancer research?

OR10H1 (Olfactory Receptor Family 10 Subfamily H Member 1) is a G-protein coupled receptor predominantly expressed in the human urinary bladder with notably higher expression in bladder cancer tissues at both mRNA and protein levels . The receptor belongs to the larger family of olfactory receptors, which are traditionally associated with olfactory epithelium but are now recognized to be ectopically expressed in various tissues and implicated in several diseases including cancer . OR10H1 demonstrates tissue specificity, being prominently expressed in bladder tissue but barely expressed in other tissues, making it particularly relevant for bladder cancer research . Significantly higher amounts of OR10H1 transcripts have been detected in the urine of bladder cancer patients compared to control subjects, suggesting its potential utility as a non-invasive biomarker . This bladder-specific expression pattern, coupled with its altered expression in cancer tissues, positions OR10H1 as both a potential biomarker and therapeutic target for bladder cancer .

What types of OR10H1 antibodies are available for research applications?

Commercial OR10H1 antibodies are available in various formats suitable for different research applications, with rabbit polyclonal antibodies being commonly used in published studies . According to the available search results, antibodies targeting the C-terminal region of the OR10H1 protein are available and have been validated for research use . These antibodies are typically provided in liquid form, purified from rabbit antiserum by affinity chromatography using epitope-specific immunogen . The commercially available antibodies have been tested for applications including Western blotting (WB) and ELISA, with recommended dilutions typically ranging from 1/500 to 1/3000 for WB and approximately 1/20000 for ELISA applications . These antibodies are formulated in PBS buffer (pH 7.4, 150 mM NaCl) with 0.02% sodium azide and 50% glycerol for stability and are typically stored at -20°C to maintain their efficacy . Researchers should note that optimal dilutions may need to be determined empirically for each specific experimental condition and cell line.

How has the specificity of OR10H1 antibodies been validated in research?

The specificity of OR10H1 antibodies has been rigorously validated through multiple complementary approaches in research settings . One key validation method involved immunocytochemical staining in OR10H1-transfected Hana3A cells, which demonstrated specific detection of the receptor in cells expressing the OR10H1 construct but not in control cells . Furthermore, the antibody specificity was confirmed through immunohistochemical staining of bladder cancer tissues, which revealed strong expression in carcinoma cells but not in stromal cells, with weaker expression observed in normal urothelium, consistent with the expected expression pattern . Additionally, validation experiments showed agreement between protein detection using the antibody and mRNA expression levels determined by RT-PCR and RNA-Seq analysis across various bladder cancer cell lines and tissue samples . The concordance between different detection methods (immunohistochemistry, RT-PCR, and RNA-Seq data) provides strong evidence for the specificity of the OR10H1 antibodies used in these studies . This multi-platform validation approach ensures researchers can confidently use these antibodies for investigating OR10H1 expression and function.

How can OR10H1 antibodies be optimized for detecting low expression levels in normal bladder tissue?

Detecting low-level OR10H1 expression in normal bladder tissue requires careful optimization of antibody protocols to enhance sensitivity while maintaining specificity . Researchers should consider implementing signal amplification techniques such as tyramide signal amplification or polymer-based detection systems, which can significantly increase detection sensitivity for immunohistochemical applications . Based on published studies, antigen retrieval methods should be carefully optimized, as OR10H1 is a membrane protein that may require specialized retrieval conditions; heat-induced epitope retrieval in a citrate or EDTA buffer has shown effectiveness in previous bladder tissue studies . For Western blot applications with low expression samples, increasing protein loading (50-100 μg), extending primary antibody incubation time (overnight at 4°C), and using high-sensitivity chemiluminescent substrates can improve detection . When working with urine samples, where OR10H1 transcript levels may be very low in healthy controls, researchers should optimize RNA extraction protocols specifically for urine sediments and consider using digital PCR or nested PCR approaches rather than standard qRT-PCR for improved sensitivity . Additionally, enrichment of urothelial cells from the urine samples prior to analysis may increase the detection rate in samples with very low expression levels.

What are the critical considerations when using OR10H1 antibodies for comparative studies between cancer and normal tissues?

When designing comparative studies between cancerous and normal tissues using OR10H1 antibodies, researchers must address several critical considerations to ensure valid comparisons . First, standardization of tissue processing, fixation times, and storage conditions is essential, as variations can significantly affect antibody binding efficiency and create artificial differences in staining intensity that are not reflective of true biological variation . Second, researchers should implement appropriate normalization strategies, including the use of housekeeping proteins as loading controls for Western blots and reference genes like TBP for qRT-PCR, which has been successfully used in published OR10H1 studies . Third, blinded quantification of staining intensity using digital image analysis software rather than subjective scoring is strongly recommended to reduce observer bias, particularly when differences between normal and cancer tissues may be subtle . Fourth, the heterogeneity of OR10H1 expression within different areas of the same tumor and between patients necessitates adequate sampling and inclusion of sufficient biological replicates; studies have shown variable expression levels across different bladder cancer cell lines and patient samples . Finally, researchers should validate findings using multiple detection methods (e.g., combining immunohistochemistry with qRT-PCR or Western blotting) and include appropriate positive controls (such as BFTC905 cells) and negative controls (antibody omission and isotype controls) to ensure the reliability of comparative analyses .

How can researchers differentiate between OR10H1 and other closely related olfactory receptors when using antibodies?

Differentiating between OR10H1 and other structurally similar olfactory receptors requires careful antibody selection and validation due to the high sequence homology within the olfactory receptor family . Researchers should prioritize antibodies targeting the C-terminal region of OR10H1, as this area typically exhibits greater sequence divergence between related receptors; commercial antibodies specifically designed against C-terminal epitopes of OR10H1 are available and have been validated in previous studies . Cross-reactivity testing is essential and should include pre-absorption controls with the immunizing peptide as well as testing on cells or tissues known to express related olfactory receptors but not OR10H1 . For definitive validation, researchers can employ genetic approaches such as siRNA knockdown or CRISPR-Cas9 knockout of OR10H1 in positive cell lines (such as BFTC905), followed by antibody staining to confirm specificity . In Western blot applications, high-resolution SDS-PAGE gels should be used to separate potentially similar-sized olfactory receptors, along with careful analysis of band patterns and molecular weights . For tissue analyses, correlating antibody staining patterns with mRNA expression data from RT-PCR using primers that span exon junctions (as described in the studies using 269 bp PCR products spanning both exons of OR10H1) provides an additional layer of validation for antibody specificity . This multi-modal approach ensures that the detected signals are truly specific to OR10H1 and not related olfactory receptors.

What are the optimal conditions for using OR10H1 antibodies in immunohistochemistry of bladder tissues?

Optimizing immunohistochemistry (IHC) protocols for OR10H1 detection in bladder tissues requires careful attention to several key parameters based on published research methodologies . For fixation, 10% neutral buffered formalin for 24-48 hours has been successfully used in bladder tissue studies, with overfixation potentially masking epitopes and underfixation causing tissue degradation . Antigen retrieval is critical; heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95-98°C for 20 minutes has proven effective for OR10H1 detection in bladder cancer tissues, though each laboratory should optimize this step for their specific tissue processing methods . For primary antibody incubation, dilutions between 1:200 and 1:500 have been reported in the literature, with overnight incubation at 4°C providing optimal staining intensity while minimizing background . A polymer-based detection system rather than biotin-streptavidin is recommended to avoid endogenous biotin interference in bladder tissues, and hematoxylin counterstaining should be kept light to avoid obscuring membranous OR10H1 staining . Positive controls should include BFTC905 bladder cancer cells, which have been validated to express OR10H1 at detectable levels by multiple methods . Negative controls should include both antibody omission and ideally tissues known to be negative for OR10H1 expression based on transcriptomic data . For quantification, researchers should employ digital image analysis with membrane-specific algorithms rather than general intensity measurements, as OR10H1 is primarily localized to the cell membrane .

What protocol modifications are needed for detecting OR10H1 in urine samples from bladder cancer patients?

Detecting OR10H1 in urine samples requires specific methodological adaptations to overcome challenges associated with this complex biological fluid . For RNA-based detection, researchers should collect fresh morning urine samples (50-100 mL) and process them within 30 minutes or add RNA preservation buffers immediately after collection to prevent RNA degradation . Urine samples should be centrifuged at 1,500-2,000g for 10 minutes to pellet cellular components, followed by RNA extraction using specialized kits optimized for urine sediments, as standard protocols may yield insufficient RNA . Published studies have successfully used RT-PCR with primers spanning both exons of OR10H1 (generating a 269 bp product) to avoid genomic DNA contamination artifacts, which is particularly important in urine samples with potentially low target abundance . For qRT-PCR analysis, the delta Cq method with TBP as a reference gene has been validated, though digital PCR may offer improved sensitivity for very low abundance targets . For protein-based detection in urine, researchers should consider concentrating proteins by ultrafiltration or precipitation before Western blot analysis, and using high-sensitivity detection systems with prolonged exposure times . Antibody-based ELISA approaches may also be viable, with recommended antibody dilutions of approximately 1:20000, though significant optimization may be required for urine samples specifically . Researchers should also establish proper normalization methods for urine analyses, such as creatinine normalization or using the ratio of OR10H1 to housekeeping genes, to account for variations in urine concentration between patients .

How should researchers optimize Western blot protocols for OR10H1 detection in different cell lines?

Western blot optimization for OR10H1 detection across different cell lines requires systematic adjustment of multiple parameters to accommodate varying expression levels and sample types . Sample preparation should include efficient membrane protein extraction using specialized buffers containing 1-2% nonionic detergents (such as Triton X-100 or NP-40) to solubilize OR10H1, which is a membrane-associated G-protein coupled receptor . Protein loading should be optimized based on expression level: 20-30 μg may be sufficient for high-expressing lines like BFTC905, while poorly differentiated cell lines with lower expression may require 50-100 μg of total protein . SDS-PAGE separation should use 10-12% gels with extended run times to ensure good resolution of the OR10H1 protein band, and transfer conditions should be optimized for membrane proteins (typically overnight transfer at lower voltage) . For primary antibody incubation, dilutions between 1:500 and 1:3000 have been reported as effective, with the optimal dilution needing to be determined empirically for each cell line; overnight incubation at 4°C generally provides better results than shorter incubations . Blocking solutions containing 5% non-fat dry milk in TBST are typically sufficient, though 5% BSA may provide lower background in some cases . For detection, enhanced chemiluminescence systems are recommended, with exposure times adjusted according to expression levels: shorter (1-2 minute) exposures for high-expressing lines and longer (5-15 minute) exposures for low-expressing lines . Stripping and reprobing membranes for housekeeping proteins should be performed carefully to avoid loss of OR10H1 signal, and whenever possible, researchers should use separate gels for target and loading control detection .

How should researchers interpret conflicting OR10H1 expression data between protein and mRNA levels?

When encountering discrepancies between OR10H1 protein detection (via antibody-based methods) and mRNA expression data, researchers should apply a systematic analytical approach to resolve these conflicts . First, evaluate the temporal relationship between mRNA and protein expression, as time lags between transcription and translation can create apparent discrepancies; this is particularly relevant in differentiation studies where OR10H1 expression changes significantly during urothelial cell differentiation . Second, consider post-transcriptional regulation mechanisms such as miRNA-mediated suppression or mRNA stability issues, which may cause high mRNA levels but low protein expression; targeted experiments examining these mechanisms can help explain discrepancies . Third, assess post-translational modifications or protein stability issues that might affect antibody epitope recognition or protein half-life; varying protein extraction methods or using multiple antibodies targeting different epitopes can help resolve such issues . Fourth, examine the sensitivity limitations of each detection method; RNA-Seq and qRT-PCR can often detect lower expression levels than antibody-based methods, potentially creating false disparities when OR10H1 is expressed below the antibody detection threshold . Finally, consider technical variabilities such as differences in normalization methods between RNA (typically using TBP as reference gene) and protein (using housekeeping proteins) quantification approaches . To resolve these conflicts conclusively, researchers should implement orthogonal validation approaches, such as in situ hybridization to visualize mRNA in the same tissues used for immunohistochemistry, or recombinant expression systems to calibrate detection methods .

How can researchers determine if OR10H1 antibody staining patterns correlate with functional activity of the receptor?

Correlating OR10H1 antibody staining patterns with functional receptor activity requires integration of multiple experimental approaches and careful data interpretation . Researchers should first establish baseline correlations between antibody staining intensity/patterns and mRNA expression levels across multiple cell lines with varying OR10H1 expression (such as BFTC905, RT112, and 5637 cell lines) to validate the antibody's ability to reflect expression differences . To link expression to function, calcium imaging experiments using FURA-2-AM can be performed following the protocols described in published studies, where cells are exposed to the OR10H1 agonist Sandranol (100-500 μM) while monitoring calcium influx; these results can then be correlated with antibody staining intensity in the same cell populations . cAMP measurement assays provide another functional readout, as OR10H1 activation by Sandranol triggers a concentration-dependent increase in cAMP levels; researchers can correlate the magnitude of this response with antibody staining intensity across different cell lines or patient-derived samples . Functional consequences such as changes in cell proliferation, migration, and viability following Sandranol stimulation can also be measured and correlated with receptor expression levels determined by antibody staining . For in situ analyses of tissue samples, researchers can employ dual-labeling approaches combining OR10H1 antibody staining with markers of downstream signaling activation (such as phospho-CREB for cAMP pathway activation) to assess correlations between receptor presence and activity within the same tissue section . Finally, genetic manipulation approaches (siRNA knockdown or overexpression) followed by antibody staining and functional assays can establish causal relationships between staining intensity and functional responses .

How can OR10H1 antibodies be used to evaluate the receptor as a potential biomarker in bladder cancer?

Employing OR10H1 antibodies for biomarker evaluation requires a systematic approach spanning multiple clinical and analytical dimensions . Researchers should first establish a standardized immunohistochemistry protocol with defined scoring criteria for OR10H1 staining in tissue samples, using the validated antibodies against the C-terminal region of the protein as described in published studies . A comprehensive biomarker validation study should include tissue microarrays containing bladder tumors of different grades and stages, matched normal tissues, and metastatic lesions to assess expression patterns across disease progression . Correlation analyses between OR10H1 expression and clinicopathological parameters (tumor grade, stage, recurrence, progression, and survival outcomes) are essential; published data already suggest significantly higher expression in bladder cancer tissues compared to normal tissues (average FPKM values of 3.8 versus 0.3) . For liquid biopsy applications, researchers should develop protocols for detecting OR10H1 protein in urine samples using antibody-based methods such as ELISA or Western blotting, complementing the existing RT-PCR approaches that have successfully detected higher OR10H1 transcript levels in cancer patients' urine . Multimarker panels combining OR10H1 with other bladder cancer biomarkers should be evaluated for improved sensitivity and specificity compared to single markers . Additionally, longitudinal studies tracking OR10H1 expression before and after treatment, during follow-up, and in cases of recurrence are necessary to establish its utility for monitoring disease status . Cross-platform validation comparing antibody-based detection with mRNA-based methods and functional assays will strengthen the biomarker validation process and help establish clinically relevant cutoff values for positivity .

What approaches can researchers use to study the interaction between OR10H1 antibodies and receptor function in cancer cell models?

Investigating interactions between OR10H1 antibodies and receptor function requires specialized experimental designs that can distinguish between different modes of antibody action . Researchers can employ neutralization assays where cells are pre-treated with OR10H1 antibodies before stimulation with the known agonist Sandranol, followed by measurement of calcium influx using FURA-2-AM fluorimetric imaging or cAMP accumulation assays to determine if the antibody blocks ligand binding or receptor activation . Receptor internalization studies using fluorescently-labeled OR10H1 antibodies combined with confocal microscopy time-lapse imaging can reveal whether antibody binding triggers receptor endocytosis, potentially altering cell surface expression levels and responsiveness to ligands . For mechanistic studies, researchers can investigate whether OR10H1 antibodies themselves can act as agonists or antagonists by monitoring downstream signaling events such as cAMP production, calcium mobilization, or ATP/serotonin secretion following antibody treatment alone, compared to known agonist Sandranol . Long-term functional effects can be assessed by treating bladder cancer cell lines (such as BFTC905) with OR10H1 antibodies over extended periods (24-72 hours) and measuring changes in cell proliferation, migration, viability, and morphology, similar to the effects documented with Sandranol stimulation . Advanced approaches may include using domain-specific antibodies targeting different regions of OR10H1 to map functionally important epitopes, or developing bispecific antibodies that could simultaneously target OR10H1 and components of the immune system to enhance potential therapeutic applications . These diverse approaches can help researchers understand whether OR10H1 antibodies merely serve as detection tools or could potentially modulate receptor function in ways relevant to cancer biology and therapy .

How can OR10H1 antibodies be optimized for investigating the receptor's role in novel therapeutic approaches for bladder cancer?

Optimizing OR10H1 antibodies for therapeutic research applications involves several specialized adaptations and validation steps beyond standard detection protocols . Researchers should first develop and characterize function-modulating antibodies that can either block OR10H1 activation (antagonistic) or stimulate the receptor (agonistic) by screening antibody candidates for their effects on Sandranol-induced calcium and cAMP responses in bladder cancer cell lines like BFTC905 . For potential antibody-drug conjugate (ADC) development, internalization kinetics of OR10H1 antibodies should be quantified using fluorescently-labeled antibodies and live-cell imaging to determine their suitability as delivery vehicles for cytotoxic payloads . Cancer-specificity validation is critical; researchers should comprehensively profile OR10H1 antibody binding across a panel of normal tissues to confirm the preferential binding to bladder cancer cells observed in published studies, where OR10H1 showed significantly higher expression in cancer versus normal tissues . For in vivo applications, antibodies may need to be humanized or developed as fragments (Fab, scFv) with optimized pharmacokinetic properties, and should be tested for stability in urine to assess potential for intravesical administration, a common route for bladder cancer therapies . Combination studies investigating synergistic effects between OR10H1-targeting antibodies and established bladder cancer treatments (BCG, chemotherapy, immunotherapy) should be conducted in appropriate cell and animal models, with readouts including cell viability, apoptosis induction, cell cycle changes, and immune cell activation . Researchers should also explore antibody-based imaging applications, developing fluorescently-labeled or radiolabeled OR10H1 antibodies for tumor visualization in preclinical models, leveraging the receptor's overexpression in bladder cancer tissues . These optimizations will help translate OR10H1 antibodies from research tools to potential therapeutic and diagnostic agents for bladder cancer.