OR2H1 Antibody

What is OR2H1 and why is it significant in cancer research?

OR2H1 is an olfactory receptor from the G-protein coupled receptor (GPCR) family with transmembrane domains that interact with odorant molecules in olfactory neurons. Its significance in cancer research stems from its unique expression pattern – widely expressed in various solid epithelial tumors while showing limited expression in normal tissues (primarily restricted to testis). This selective expression profile makes OR2H1 a potentially valuable target for cancer immunotherapy, particularly for CAR T cell approaches targeting solid tumors .

What types of cancer express OR2H1?

OR2H1 expression has been documented across multiple solid tumor types with varying frequencies:

• Intrahepatic cholangiocarcinoma: ~69% of cases

• Prostate cancer: >38% of cases

• Serous endometrial carcinoma: ~27% of cases

• Ovarian carcinomas: 20% of cases (8/40 in immunohistochemistry studies)

• Lung carcinomas: 13% of cases (8/60)

• Cholangiocarcinomas: 59% of cases (43/73)

• Colon cancer: ~4% of cases

Immunohistochemistry, RT-QPCR, and RNAscope analyses have confirmed OR2H1 expression in pancreatic, ovarian, lung, and breast cancers of various histologies .

What methods are available for detecting OR2H1 expression in tissue samples?

Multiple complementary methods have been validated for detecting OR2H1 expression:

For RT-QPCR, researchers have successfully used primers (Forward: 5'-TCACTCAGTACAGCTCCCATGC-3'; Reverse: 5'-TTCAGTTCTTGCAATTAAGTCAGACTCT-3') with GAPDH as a reference gene for normalization .

How can I validate the specificity of an OR2H1 antibody for my research?

Validating OR2H1 antibody specificity requires a multi-faceted approach:

• Gene knockout controls: Utilize CRISPR/Cas9-mediated OR2H1 knockout cells as a negative control to confirm antibody specificity. This approach has been successfully employed to validate OR2H1 detection in published studies .

• Multiple detection methods: Compare protein detection via immunohistochemistry with mRNA expression data (RT-QPCR or RNAscope). Concordance between protein and mRNA detection strengthens confidence in antibody specificity.

• Tissue panel screening: Screen a diverse panel of normal tissues known to be OR2H1-negative (based on databases like GTEX Portal and The Human Protein Atlas) alongside positive controls (testis tissue or known OR2H1-expressing tumor cells).

• Blocking experiments: Conduct peptide competition assays using the synthetic peptide immunogen to confirm binding specificity of the antibody.

• Antibody validation in multiple applications: Verify consistent results across Western blot, IHC, and flow cytometry applications, confirming expected molecular weight and cellular localization patterns .

What factors affect OR2H1 antibody performance in immunohistochemistry of FFPE tissue?

Several critical factors impact OR2H1 antibody performance in FFPE tissue staining:

• Antigen retrieval method: Studies have shown that heat-induced epitope retrieval is essential for OR2H1 detection in FFPE tissues. The precise pH and buffer composition should be optimized based on the specific antibody.

• Antibody concentration: Titration experiments are necessary to determine optimal antibody concentration, typically ranging from 1:500 to 1:2000 for primary antibodies .

• Incubation conditions: Temperature and duration significantly impact staining quality. Overnight incubation at 4°C often yields better signal-to-noise ratio than shorter incubations at room temperature.

• Detection system: The choice between chromogenic (DAB) versus fluorescent detection should be based on the expression level of OR2H1 and the need for multiplexing with other markers.

• Tissue fixation variables: Fixation time, type of fixative, and tissue processing methods all influence epitope preservation. When possible, standardize these parameters across all samples being compared .

• Controls: Include testis tissue as a positive control and ileum as a negative control, as established in published research protocols .

How can OR2H1 expression be quantified at the protein level?

Quantification of OR2H1 protein can be approached through several methodologies:

• Fluorescence-based flow cytometry: Conjugate OR2H1 antibody to fluorophores (such as PE) and quantify relative expression levels using calibration beads. This allows estimation of receptor density per cell.

• Western blot densitometry: Semi-quantitative analysis using housekeeping proteins (like GAPDH) as loading controls can provide relative expression levels across samples.

• IHC scoring systems: Implement H-score or Allred scoring systems that account for both staining intensity and percentage of positive cells. Digital pathology platforms can provide objective quantification of staining intensity.

• Quantitative mass spectrometry: For absolute quantification, targeted proteomics approaches using labeled peptide standards can determine exact copy numbers of OR2H1 protein.

For optimal results, researchers should combine multiple quantification methods to validate findings across different platforms .

How should I design experiments to investigate OR2H1's functional role in cancer cells?

Designing experiments to elucidate OR2H1's functional role requires a comprehensive approach:

• Gene modulation strategies:

  • CRISPR/Cas9 knockout: Generate complete OR2H1 knockout cell lines

  • shRNA/siRNA: Create transient and stable knockdown models

  • Overexpression models: Introduce OR2H1 in cell lines with low/no expression

• Functional assays:

  • Cell proliferation: Measure growth rates using real-time cell analysis systems

  • Metabolism: Assess glucose uptake using fluorescent glucose analogs (e.g., 2-NBDG)

  • Migration/invasion: Determine metastatic potential using Boyden chamber assays

  • Colony formation: Evaluate clonogenic potential in 2D and 3D cultures

• Signaling pathway analysis:

  • Investigate G-protein coupled signaling pathways typically associated with olfactory receptors

  • Assess metabolic signaling changes, as OR2H1 has been implicated in glucose metabolism

  • Examine potential cross-talk with oncogenic pathways

• In vivo models:

  • Xenograft studies comparing OR2H1 wildtype versus knockout cells

  • Patient-derived xenografts to maintain tumor heterogeneity

  • Metastasis models to evaluate impact on tumor spread

• Multiomics approach:

  • Transcriptomics to identify downstream gene expression changes

  • Metabolomics to characterize alterations in cancer cell metabolism

  • Proteomics to map protein interaction networks

What controls are essential when evaluating OR2H1 antibody specificity in immunohistochemistry?

When evaluating OR2H1 antibody specificity for immunohistochemistry, these controls are essential:

• Positive tissue controls:

  • Testis tissue (known to express OR2H1)

  • Validated OR2H1-positive tumor samples (cholangiocarcinoma, ovarian cancer)

• Negative tissue controls:

  • Ileum tissue (confirmed OR2H1-negative)

  • Panel of normal tissues (58 normal tissue types have been confirmed OR2H1-negative)

• Cellular controls:

  • Cell lines with known OR2H1 expression status

  • Isogenic cell lines with CRISPR/Cas9-mediated OR2H1 knockout

• Technical controls:

  • Secondary antibody-only control (omit primary antibody)

  • Isotype control antibody (same species and isotype as OR2H1 antibody)

  • Peptide competition (pre-incubation with immunizing peptide)

• Method validation controls:

  • Parallel RNAscope analysis for OR2H1 mRNA detection

  • Western blot verification of antibody specificity

These controls collectively ensure that any observed staining is specific to OR2H1 rather than technical artifacts or non-specific binding .

How can I determine the optimal cutoff for OR2H1 positivity in clinical tumor samples?

Determining the optimal cutoff for OR2H1 positivity requires a systematic approach:

• Baseline expression determination:

  • Analyze a large panel of normal tissues (>50) to establish background levels

  • Quantify expression in positive control tissues (testis) to establish reference levels

• Statistical approaches to cutoff determination:

  • ROC curve analysis comparing tumor vs. normal tissue staining

  • Survival analysis using various cutpoints to identify clinically meaningful thresholds

  • Tertile or quartile classification based on expression distribution within a cohort

• Validation across detection methods:

  • Compare IHC positivity with RT-QPCR quantification

  • Validate with RNAscope to confirm RNA-protein correlation

  • Correlate with functional outcomes in experimental models

• Clinical outcome correlation:

  • Retrospective analysis linking expression levels to patient survival

  • Association with response to standard therapies

  • Stratification by tumor type and stage

• Standardization considerations:

  • Implement digital pathology quantification for objective scoring

  • Use H-score or Allred scoring systems that account for both intensity and percentage

  • Consider tissue-specific thresholds based on background expression

Based on published research, cutoffs for positivity have varied by tumor type, with cholangiocarcinomas showing a 59% positivity rate compared to 13% in lung carcinomas using the same evaluation criteria .

What is the optimal protocol for OR2H1 western blot analysis?

For optimal western blot detection of OR2H1, follow this detailed protocol:

• Sample preparation:

  • Lyse cells or tissues in RIPA buffer supplemented with protease inhibitors

  • For membrane proteins like OR2H1, include brief sonication steps (3 x 10s pulses)

  • Determine protein concentration using BCA or Bradford assay

• Gel electrophoresis:

  • Load 20-40μg total protein per lane

  • Use 10-12% polyacrylamide gels to properly resolve OR2H1 (~35 kDa molecular weight)

  • Include positive controls (testis tissue lysate) and negative controls

• Transfer conditions:

  • Semi-dry or wet transfer to PVDF membrane (preferred over nitrocellulose for OR2H1)

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking and antibody incubation:

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

  • Incubate with primary OR2H1 antibody at 1:500-1:2000 dilution overnight at 4°C

  • Wash 3x5 minutes with TBST

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Detection and analysis:

  • Develop using enhanced chemiluminescence (ECL) reagents

  • Expect OR2H1 band at approximately 35 kDa

  • Strip and reprobe for loading control (GAPDH or β-actin)

  • For quantification, normalize OR2H1 signal to loading control

• Validation steps:

  • Confirm specificity using OR2H1 knockout lysates

  • Consider using recombinant OR2H1 protein as positive control

How can I optimize immunohistochemistry protocols for OR2H1 detection in various tumor types?

Optimizing immunohistochemistry for OR2H1 across tumor types requires systematic protocol refinement:

• Fixation optimization:

  • Standard 10% neutral buffered formalin fixation for 24-48 hours

  • Consistent processing conditions to ensure comparable results

• Antigen retrieval optimization:

  • Test multiple retrieval methods: citrate buffer (pH 6.0), EDTA buffer (pH 9.0), and enzymatic retrieval

  • Optimize retrieval time (10-30 minutes) and temperature

• Blocking optimization:

  • Test 3-5% BSA, 5-10% normal serum, or commercial blocking reagents

  • Include avidin/biotin blocking if using biotin-based detection systems

• Antibody titration:

  • Perform serial dilutions (1:100 to 1:2000) of OR2H1 antibody

  • Determine optimal concentration that maximizes specific signal while minimizing background

  • Consider extended incubation times (overnight at 4°C) for weak signals

• Detection system selection:

  • For low expression: Use amplification systems (e.g., tyramide signal amplification)

  • For routine detection: Use polymer-based detection systems

  • For multiplex staining: Consider fluorescent secondary antibodies

• Tumor-specific adaptations:

  • Lung tissue: Additional peroxidase blocking (3% H₂O₂, 15 minutes)

  • Highly pigmented tumors: Consider azure B treatment to reduce melanin interference

  • Necrotic areas: Implement careful region-of-interest selection

• Counterstain optimization:

  • Adjust hematoxylin counterstaining time (30 seconds to 2 minutes) based on tumor type

  • For challenging tissues, consider nuclear fast red as an alternative counterstain

Based on published protocols, successful detection of OR2H1 has been achieved using DAB chromogen development following antigen retrieval in a variety of epithelial tumors .

What are the best strategies for quantifying OR2H1 mRNA expression via RT-QPCR?

For optimal quantification of OR2H1 mRNA expression via RT-QPCR:

• RNA isolation optimization:

  • For FFPE samples: Use specialized kits designed for degraded RNA

  • For fresh/frozen tissues: Standard TRIzol or column-based methods

  • Include DNase treatment to eliminate genomic DNA contamination

• cDNA synthesis considerations:

  • Use SuperScriptTM IV First-Strand Synthesis System or comparable high-efficiency reverse transcriptases

  • Include no-RT controls to detect genomic DNA contamination

  • Consider gene-specific primers for reverse transcription if expression is low

• Primer design and validation:

  • Validated OR2H1-specific primers:

    • Forward: 5'-TCACTCAGTACAGCTCCCATGC-3'

    • Reverse: 5'-TTCAGTTCTTGCAATTAAGTCAGACTCT-3'

  • Design primers spanning exon-exon junctions to avoid genomic amplification

  • Verify primer specificity through melt curve analysis and sequencing of amplicons

• Reference gene selection:

  • GAPDH has been validated as a suitable reference gene for OR2H1 studies

    • Forward: 5'-CCTGCACCACCAACTGCTTA-3'

    • Reverse: 5'-AGTGATGGCATGGACTGTGGT-3'

  • Consider multiple reference genes (ACTB, B2M, HPRT1) for more robust normalization

  • Validate reference gene stability across your specific sample types

• Quantification strategy:

  • Absolute quantification: Generate standard curves using plasmids containing OR2H1

  • Relative quantification: Use 2^(-ΔΔCt) method with appropriate calibrator samples

  • Include inter-run calibrators when analyzing large sample sets across multiple plates

• Data validation:

  • Verify findings using OR2H1 protein detection methods

  • Compare with RNAscope results for tissue-level validation

  • Include positive controls (testis tissue) and negative controls (ileum tissue)

Why might I observe discrepancies between OR2H1 mRNA and protein detection in my samples?

Discrepancies between OR2H1 mRNA and protein detection can arise from multiple factors:

• Post-transcriptional regulation:

  • MicroRNA-mediated suppression of OR2H1 translation

  • RNA binding proteins affecting mRNA stability or translation efficiency

  • Alternative splicing generating protein isoforms not detected by certain antibodies

• Methodological limitations:

  • Different sensitivity thresholds between RT-QPCR (typically more sensitive) and IHC/WB

  • Antibody epitope accessibility issues in fixed tissues

  • RNA quality differences affecting RT-QPCR reliability

• Biological heterogeneity:

  • Intratumoral heterogeneity leading to sampling discrepancies

  • Temporal variations in OR2H1 expression during tumor progression

  • Microenvironmental factors influencing protein but not mRNA levels

• Technical considerations:

  • FFPE fixation causing RNA degradation but preserving protein epitopes

  • Antibody cross-reactivity with related olfactory receptors

  • Reference gene instability affecting RT-QPCR normalization

• Resolution strategies:

  • Perform single-cell analyses to account for heterogeneity

  • Use multiple detection methods on the same sample

  • Implement RNAscope as a bridge technology that provides spatial context for mRNA

  • Consider protein half-life vs. mRNA stability differences

Research shows OR2H1 detection rates are comparable between mRNA-based and protein-based methods in most tumor types, but discrepancies can occur in highly heterogeneous samples .

What approaches can resolve weak or inconsistent OR2H1 antibody signals in western blots?

To address weak or inconsistent OR2H1 signals in western blots:

• Sample preparation optimization:

  • Use membrane protein extraction kits specific for GPCRs

  • Avoid repeated freeze-thaw cycles of protein lysates

  • Include protease inhibitor cocktails optimized for membrane proteins

  • Consider non-denaturing conditions if epitope conformation is critical

• Loading adjustments:

  • Increase total protein loading (up to 50-80μg for low abundance targets)

  • Concentrate samples using TCA precipitation or similar methods

  • Load positive controls (testis lysate) at various dilutions

• Detection enhancement strategies:

  • Switch to high-sensitivity ECL substrates

  • Extend primary antibody incubation (up to 48 hours at 4°C)

  • Use signal amplification systems (biotin-streptavidin)

  • Consider fluorescent secondary antibodies with longer exposure times

• Antibody optimization:

  • Try alternative OR2H1 antibodies targeting different epitopes

  • Reduce antibody dilution (1:200-1:500)

  • Add 0.05% Tween-20 to antibody dilution buffer to reduce non-specific binding

  • Test different blocking buffers (BSA vs. milk vs. commercial blockers)

• Transfer optimization:

  • For membrane proteins like OR2H1, use PVDF membranes

  • Add 0.1% SDS to transfer buffer to improve elution of hydrophobic proteins

  • Reduce methanol concentration in transfer buffer to 10%

  • Consider longer transfer times at lower voltage

• Sample handling guidelines:

  • Maintain strict cold chain throughout preparation

  • Prepare fresh samples whenever possible

  • Include reducing agents (DTT or β-mercaptoethanol) in sample buffer

How can I address non-specific binding issues when using OR2H1 antibodies in immunostaining?

To minimize non-specific binding in OR2H1 immunostaining:

• Blocking optimization:

  • Test multiple blocking agents: 5-10% normal serum matching secondary antibody species

  • Include 0.1-0.3% Triton X-100 for permeabilization

  • Consider dual blocking with both serum and BSA

  • Add 0.1% gelatin to blocking buffer for highly autofluorescent tissues

• Antibody concentration adjustment:

  • Titrate primary antibody across broader range (1:100-1:5000)

  • Increase washing duration and number of washes (5x5 minutes)

  • Dilute antibodies in blocking buffer rather than PBS/TBS alone

  • Pre-absorb antibody with tissue powder from negative tissues

• Protocol modifications:

  • Implement endogenous peroxidase quenching (3% H₂O₂, 10 minutes)

  • Add avidin/biotin blocking for biotin-based detection systems

  • Include mouse-on-mouse blocking for mouse antibodies on mouse tissues

  • Reduce secondary antibody concentration

• Advanced techniques:

  • Use monovalent Fab fragments to block endogenous immunoglobulins

  • Implement protein A/G pre-treatment to block endogenous immunoglobulins

  • Consider direct conjugated primary antibodies to eliminate secondary antibody issues

  • Use isotype-matched control antibodies to identify non-specific binding patterns

• Validation strategies:

  • Compare staining patterns with RNAscope results

  • Test antibody on known negative tissues (confirmed via RT-QPCR)

  • Perform peptide competition assays

  • Include CRISPR/Cas9 OR2H1 knockout samples as definitive negative controls

How can OR2H1 antibodies be utilized in developing CAR T cell therapies for solid tumors?

OR2H1 antibodies are instrumental in CAR T cell therapy development through multiple applications:

• Target validation and screening:

  • Evaluate OR2H1 expression across tumor types and normal tissues

  • Quantify receptor density using antibody-based flow cytometry

  • Screen patient biopsies to identify suitable candidates for OR2H1-targeted therapy

• CAR design and optimization:

  • Derive single-chain variable fragments (scFvs) from OR2H1 antibodies

  • Screen antibody libraries to identify optimal OR2H1-binding domains

  • Test various antibody-derived binding domains for CAR construction

• Preclinical efficacy assessment:

  • Monitor target engagement via competitive binding assays

  • Assess on-target/off-tumor binding in tissue cross-reactivity studies

  • Evaluate CAR T cell infiltration in tumor models using OR2H1 co-staining

• Patient selection biomarkers:

  • Develop IHC-based companion diagnostics for patient stratification

  • Establish quantitative thresholds for OR2H1 positivity

  • Create standardized scoring systems for clinical implementation

• Therapy monitoring:

  • Track changes in OR2H1 expression during treatment

  • Identify escape mechanisms through epitope mapping

  • Monitor emergence of OR2H1-negative tumor subpopulations

Research has demonstrated that scFvs derived from OR2H1 antibodies can successfully redirect T cells against OR2H1-expressing tumors, showing cytotoxic activity both in vitro and in vivo against ovarian, lung, and other epithelial tumors .

What is the significance of OR2H1's role in glucose metabolism for cancer research?

OR2H1's involvement in glucose metabolism has significant implications for cancer research:

• Metabolic targeting strategies:

  • OR2H1 knockout via CRISPR/Cas9 has demonstrated reduced glucose uptake in cancer cells

  • The 2-NBDG assay reveals functional consequences of OR2H1 expression on glucose metabolism

  • This metabolic role suggests OR2H1 may contribute to the Warburg effect in tumors

• Dual-targeting approaches:

  • Combined targeting of OR2H1 and glucose metabolism pathways may yield synergistic effects

  • Glycolysis inhibitors could potentially sensitize tumors to OR2H1-targeted therapies

  • Metabolic imaging could serve as a biomarker for OR2H1 functional activity

• Mechanistic research directions:

  • Investigation of signaling pathways connecting OR2H1 to glucose transporters

  • Analysis of how OR2H1 affects metabolic enzyme expression or activity

  • Exploration of potential role in hypoxia response and metabolic adaptation

• Clinical correlations:

  • Assessment of relationship between OR2H1 expression and FDG-PET avidity

  • Evaluation of OR2H1 status in treatment-resistant tumors with altered metabolism

  • Analysis of metabolic signatures as predictive biomarkers for OR2H1-targeted therapies

• Therapeutic implications:

  • Development of bispecific antibodies targeting both OR2H1 and metabolic enzymes

  • Design of OR2H1 antagonists that may disrupt metabolic signaling

  • Exploration of combination strategies with metabolic inhibitors

CRISPR/Cas9-mediated ablation of OR2H1, followed by glucose uptake analysis using fluorescent glucose analogs, has confirmed OR2H1's tumor-promoting role in glucose metabolism, suggesting novel therapeutic opportunities through metabolic disruption .

How can multiplexed detection of OR2H1 with other biomarkers enhance cancer research?

Multiplexed detection of OR2H1 with other biomarkers can significantly advance cancer research:

• Tumor heterogeneity characterization:

  • Co-stain OR2H1 with other tumor markers to identify distinct subpopulations

  • Quantify spatial relationships between OR2H1+ cells and stromal/immune components

  • Correlate OR2H1 expression with regions of hypoxia, proliferation, or apoptosis

• Pathway analysis applications:

  • Multiplex OR2H1 with glucose transporters (GLUT1/3) to validate metabolic correlations

  • Co-stain with phospho-proteins in relevant signaling pathways

  • Evaluate relationship with hypoxia markers (HIF-1α) and metabolic enzymes

• Immune contexture evaluation:

  • Assess OR2H1 expression relative to tumor-infiltrating lymphocytes

  • Correlate with immune checkpoint molecules (PD-L1, CTLA-4)

  • Evaluate relationship with myeloid cell infiltration patterns

• Technical approaches:

  • Multiplex immunofluorescence with tyramide signal amplification

  • Cyclic immunofluorescence for high-parameter tissue analysis

  • Mass cytometry imaging (IMC) for highly multiplexed protein detection

  • Digital spatial profiling for quantitative spatial analysis

• Clinical applications:

  • Develop prognostic algorithms incorporating OR2H1 with established biomarkers

  • Create patient stratification approaches for combination therapies

  • Design precision medicine strategies based on multiplexed biomarker profiles

Researchers have successfully implemented multiplexed detection approaches combining OR2H1 with other cancer biomarkers to enhance understanding of its biological context and therapeutic implications in various epithelial tumors .