CD247 Recombinant Monoclonal Antibody

What is CD247 and why is it important in immunological research?

CD247 (also known as CD3ζ or TCRζ) is a 163 amino acid protein with a predicted molecular weight of 18kDa that belongs to the CD3Z/FCER1G family. It has a short extracellular domain of only nine amino acids that is conserved between mouse and human, and contains 6-7 intracellular tyrosines that serve as potential phosphorylation sites . CD247 is primarily expressed in T cells and NKT cells, with reports of expression in CD16+ human NK cells as well .

The protein is crucial for mediating signal transduction after TCR engagement, interacting with key signaling molecules like ZAP70 and the TCR alpha/beta chains . Given its fundamental role in T-cell activation, CD247 has become a significant target in immunological research, particularly in studies involving T-cell function, autoimmune diseases, and cancer immunotherapy.

What types of CD247 antibodies are available for research applications?

Several types of CD247 antibodies are available for research:

• Recombinant monoclonal antibodies - Such as the ZooMAb® Rabbit Monoclonal Antibody, which offers enhanced specificity, affinity, reproducibility, and stability compared to conventional monoclonals

• Polyclonal antibodies - Such as affinity-isolated antibodies available for various applications

• Fluorophore-conjugated antibodies - Including PE-conjugated and other fluorescent-labeled antibodies for flow cytometry and imaging applications

Each antibody type has specific advantages depending on the experimental design and research questions being addressed.

What are the key differences between conventional and recombinant CD247 monoclonal antibodies?

Recombinant CD247 monoclonal antibodies offer significantly enhanced specificity, affinity, reproducibility, and stability over conventional monoclonals, making them particularly valuable for demanding research applications .

How should I design experiments to validate CD247 antibody specificity in my model system?

To validate CD247 antibody specificity in your model system:

• Western blot analysis: Run parallel samples of positive controls (such as Jurkat cells or human/mouse spleen tissue lysates) alongside your experimental samples . Look for a specific band at approximately 16 kDa, which corresponds to the observed molecular weight of CD247 .

• Immunoprecipitation validation: Perform IP using 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate from a known CD247-expressing cell line like Jurkat cells . This helps confirm the antibody's ability to bind native CD247.

• Orthogonal validation: Utilize orthogonal RNA sequencing data to correlate protein expression with transcript levels . This enhanced validation approach helps confirm the specificity of antibody binding.

• Knockout/knockdown controls: If possible, include CD247 knockout or knockdown samples as negative controls to confirm antibody specificity.

• Cross-reactivity testing: If working with non-human samples, test antibody reactivity across species of interest (human, mouse, rat) as reactivity can vary .

What are the optimal conditions for using CD247 antibodies in Western blot applications?

For optimal Western blot results with CD247 antibodies:

• Sample preparation: Use fresh lysates from Jurkat cells, human spleen tissue, or mouse spleen tissue as positive controls .

• Antibody dilution:

  • For polyclonal antibodies: Use dilutions between 1:500-1:3000

  • For monoclonal antibodies: A 1:10,000 dilution has been shown to effectively detect CD247 in Jurkat cell lysate

• Protein amount: Load 15-20 μg of total protein per lane for standard detection.

• Expected band size: Look for a specific band at approximately 16 kDa (observed molecular weight of CD247) .

• Buffer conditions: Use standard TBST buffer (Tris-buffered saline with 0.1% Tween-20) for washing and antibody dilution.

• Blocking solution: 5% non-fat dry milk or BSA in TBST is typically effective for reducing background.

• Incubation time: Primary antibody incubation overnight at 4°C generally yields optimal results.

• Detection system: Both chemiluminescence and fluorescence-based detection systems work well with CD247 antibodies.

What protocol should be followed for CD247 detection by immunohistochemistry?

For effective immunohistochemical detection of CD247:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections from appropriate samples such as human tonsil, which serves as an excellent positive control .

• Antigen retrieval:

  • Primary option: Use TE buffer at pH 9.0 for optimal epitope exposure

  • Alternative option: Citrate buffer at pH 6.0 may also be effective

• Antibody dilution:

  • For polyclonal antibodies: Use at 1:50-1:500 dilution

  • For monoclonal antibodies: A 1:1,000 dilution has been shown to effectively detect CD247 in human tonsil tissue sections

• Detection system: Use a polymer-based detection system compatible with rabbit primary antibodies.

• Counterstaining: Hematoxylin counterstaining provides good nuclear contrast.

• Controls: Always include positive control tissues (tonsil, spleen) and negative controls (primary antibody omission) to validate staining specificity.

• Expected result: CD247 should show membrane and cytoplasmic staining pattern in T-cells, particularly in T-cell zones of lymphoid tissues.

How can CD247 antibodies be utilized in flow cytometry for T-cell functional studies?

CD247 antibodies can be effectively employed in flow cytometry for T-cell functional studies through the following approaches:

• Surface vs. intracellular staining: While CD247 has a short extracellular domain, most applications require intracellular staining to access the larger cytoplasmic portion of the protein. For intracellular staining, use 0.40 μg per 10^6 cells in a 100 μl suspension .

• Multi-parameter analysis: Combine CD247 antibodies with other T-cell markers (e.g., CD3, CD4, CD8) and activation markers (e.g., CD69, CD25) to assess T-cell subpopulations and their activation status simultaneously.

• Phospho-flow analysis: Use phospho-specific CD247 antibodies to detect phosphorylation of the intracellular tyrosine residues following TCR engagement, providing a direct measurement of T-cell activation.

• Stimulation experiments: Design experiments comparing CD247 expression and phosphorylation before and after T-cell stimulation with anti-CD3/CD28 antibodies, PMA/ionomycin, or antigen-specific stimuli.

• Compensation and controls: When using fluorochrome-conjugated antibodies like PE anti-CD247, proper compensation is essential. Human peripheral blood lymphocytes can serve as positive controls .

• Data analysis: Analyze CD247 expression in relation to T-cell functionality markers to correlate signaling capacity with functional outcomes like cytokine production or proliferation.

This approach allows researchers to investigate TCR signaling dynamics in various contexts, including autoimmune diseases, cancer immunotherapy responses, and basic T-cell biology.

What are the considerations when using CD247 antibodies for studying T-cell receptor downregulation in disease states?

When studying T-cell receptor downregulation in disease states using CD247 antibodies, several important considerations should be addressed:

• Disease-specific contexts: CD247 downregulation has been observed in various conditions including systemic lupus erythematosus (SLE), chronic obstructive pulmonary disease (COPD), and cancer . The experimental design should account for disease-specific mechanisms of TCR downregulation.

• Epitope accessibility: In disease states, conformational changes or protein interactions may affect epitope accessibility. Select antibodies recognizing different epitopes to ensure detection regardless of conformational state.

• Correlation with functional assays: Combine CD247 expression analysis with functional T-cell assays (proliferation, cytokine production) to establish relationships between receptor downregulation and functional impairment.

• Sample handling: T-cell receptor components can be sensitive to processing conditions. Standardize sample collection, processing time, and storage conditions to minimize artifactual changes in CD247 expression.

• Quantification methods: Employ quantitative approaches such as mean fluorescence intensity (MFI) measurements in flow cytometry or quantitative Western blotting with appropriate normalization to accurately assess CD247 downregulation.

• Single-cell analysis: Consider single-cell approaches to distinguish between global downregulation across all T-cells versus selective downregulation in specific T-cell subsets.

• Genetic variability: Be aware that polymorphisms in CD247 may influence antibody binding and disease susceptibility . Genotyping samples may provide additional context for interpreting CD247 expression data.

These considerations help ensure that observed changes in CD247 expression represent true biological phenomena rather than technical artifacts, and enable meaningful comparisons across different disease contexts.

How can CD247 antibodies be incorporated into CAR-T cell research protocols?

CD247 antibodies serve critical functions in Chimeric Antigen Receptor T (CAR-T) cell research:

• CAR-T construct validation: Western blotting with CD247 antibodies can confirm the expression of CAR constructs containing the CD3ζ signaling domain. The expected molecular weight will be larger than native CD247 due to the fusion with other CAR components .

• Signaling pathway analysis: Following CAR activation, phospho-specific CD247 antibodies can be used to monitor the phosphorylation of ITAM motifs within the CD3ζ domain, providing direct evidence of successful CAR signaling initiation.

• Immunoprecipitation studies: Use CD247 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) to pull down CAR complexes and identify associated signaling proteins through subsequent mass spectrometry or Western blot analysis .

• Microscopy-based studies: Immunofluorescence with CD247 antibodies (1:50-1:500 dilution) can visualize CAR clustering and localization during target cell engagement, particularly when combined with fluorescently-labeled target antigens .

• Quality control: Flow cytometry with CD247 antibodies can assess CAR expression levels across manufactured CAR-T cell batches, ensuring consistent product quality.

• In vivo monitoring: For animal studies, tissue sections can be analyzed using immunohistochemistry with CD247 antibodies (1:50-1:500 dilution) to track CAR-T cell infiltration into tumor tissues .

• Correlative studies: Analyzing CD247/CD3ζ expression levels or phosphorylation status in relation to CAR-T efficacy can identify potential biomarkers of treatment response.

This multifaceted approach provides comprehensive insights into CAR-T cell biology, from manufacturing quality control to mechanistic understanding of therapeutic efficacy.

What are common challenges in CD247 detection and how can they be addressed?

When troubleshooting, remember that CD247 is a 16 kDa protein that may exhibit minor variations in molecular weight due to post-translational modifications. Additionally, CD247 expression can be downregulated in certain disease states, which may affect detection sensitivity in clinical samples.

How should researchers interpret discrepancies between CD247 protein expression and functional T-cell assays?

When faced with discrepancies between CD247 protein expression data and functional T-cell assays, researchers should consider several interpretive frameworks:

• Post-translational regulation: CD247 function is heavily regulated by phosphorylation of its six to seven intracellular tyrosine residues . Normal protein levels with impaired function may reflect altered phosphorylation rather than expression defects. Consider complementing expression analysis with phospho-specific antibodies.

• Protein localization effects: CD247 must be properly assembled into the TCR complex to function. Immunofluorescence microscopy (using dilutions of 1:50-1:500) can reveal whether CD247 is correctly localized, even when total protein levels appear normal.

• Compensatory mechanisms: Other signaling molecules may partially compensate for CD247 deficiencies. Analyze multiple components of the TCR signaling pathway simultaneously to identify compensatory upregulation.

• Threshold effects: T-cell function may require a minimum threshold of CD247 expression rather than showing linear correlation with expression levels. Careful titration experiments can help establish this threshold.

• Temporal dynamics: CD247 downregulation may be transient in response to activation. Time-course experiments comparing protein expression and functional readouts at multiple time points can reveal these dynamics.

• Subpopulation heterogeneity: Flow cytometry analysis of CD247 in conjunction with subset markers can determine if functional defects are restricted to specific T-cell subpopulations, which might be masked in bulk protein analysis.

• Technical considerations: Ensure that antibodies used for detection recognize functionally relevant epitopes. For instance, antibodies targeting the C-terminal half may provide more functionally relevant information .

By systematically exploring these possibilities, researchers can develop a more comprehensive understanding of the relationship between CD247 expression and T-cell functionality in their experimental system.

What statistical approaches are most appropriate for analyzing CD247 expression across different experimental conditions?

When analyzing CD247 expression across different experimental conditions, consider these statistical approaches:

• For comparing expression levels between groups:

  • For normally distributed data: Use paired or unpaired t-tests (two groups) or ANOVA with appropriate post-hoc tests (multiple groups)

  • For non-normally distributed data: Use non-parametric alternatives such as Mann-Whitney U (two groups) or Kruskal-Wallis tests (multiple groups)

  • Include power analysis to ensure adequate sample size for detecting biologically relevant differences

• For correlation analyses:

  • Use Pearson correlation for linear relationships between CD247 expression and other parameters (e.g., T-cell function markers) if data is normally distributed

  • Use Spearman rank correlation for non-parametric relationships

  • Consider multivariate regression when examining the relationship between CD247 expression and multiple variables

• For time-course experiments:

  • Apply repeated measures ANOVA or mixed-effects models to account for within-subject correlations

  • Consider area under the curve (AUC) analysis to summarize dynamic changes in CD247 expression

• For flow cytometry data:

  • Use coefficient of variation (CV) to assess staining consistency

  • Compare mean fluorescence intensity (MFI) rather than percent positive cells for more sensitive detection of expression differences

  • Consider biexponential transformation for visualizing wide dynamic ranges of expression

• For image analysis data:

  • Use integrated density measurements to quantify immunofluorescence or immunohistochemistry staining

  • Apply appropriate background correction methods

  • Consider spatial statistics for analyzing distribution patterns of CD247 in tissue sections

• For threshold-based analyses:

  • Use receiver operating characteristic (ROC) curves to establish optimal CD247 expression thresholds for predicting functional outcomes

  • Calculate sensitivity and specificity at various thresholds

• For all analyses:

  • Control for multiple comparisons using methods such as Bonferroni correction or false discovery rate (FDR)

  • Report effect sizes alongside p-values to indicate biological significance

  • Include appropriate visualization (box plots, scatter plots) to represent data distribution

These approaches ensure robust analysis of CD247 expression data, facilitating meaningful interpretation of experimental results.

How is CD247 expression analysis contributing to our understanding of autoimmune diseases?

CD247 expression analysis has provided significant insights into autoimmune disease mechanisms:

• Systemic Lupus Erythematosus (SLE): Research has revealed that SLE is associated with deficiency in CD247, a component of the TCR-CD3 complex. Comprehensive analysis showed that more than half of SLE patients tested exhibited CD247 deficiency . This downregulation contributes to T-cell dysfunction and may be involved in the autoimmune pathogenesis of SLE.

• Rheumatoid Arthritis (RA): Studies have identified associations between CD247 polymorphisms and RA susceptibility . CD247 expression analysis in RA patients has revealed altered TCR signaling that may contribute to inappropriate T-cell responses against self-antigens.

• Mechanistic insights across autoimmune conditions: CD247 downregulation appears to be a common feature across multiple autoimmune diseases, suggesting a shared mechanism of T-cell dysfunction. Myeloid-derived suppressor cells (MDSCs) have been implicated in mediating T-cell dysfunction through CD247 downregulation , providing a potential therapeutic target.

• Biomarker potential: Quantitative analysis of CD247 expression using flow cytometry or immunohistochemistry is being explored as a potential biomarker for disease activity and treatment response in various autoimmune conditions.

• Genetic contributions: Polymorphisms in CD247 have been associated with autoimmune disease susceptibility, highlighting the importance of genetic factors in disease development . These findings suggest that genetic screening for CD247 variants could help identify individuals at higher risk for developing autoimmune conditions.

This research is advancing our understanding of how T-cell signaling abnormalities contribute to autoimmune pathogenesis and may lead to the development of novel therapeutic approaches targeting the restoration of normal CD247 expression and function.

What are emerging applications of CD247 antibodies in cancer immunotherapy research?

Emerging applications of CD247 antibodies in cancer immunotherapy research include:

• CAR-T cell optimization: CD247 antibodies are crucial for evaluating novel CAR designs that incorporate modified CD3ζ signaling domains. Research has shown that intraperitoneal immunotherapy with T cells expressing anti-EpCAM CAR can be effective against peritoneal carcinomatosis models . CD247 antibodies help assess CAR expression, signaling capacity, and persistence in these studies.

• Tumor microenvironment analysis: CD247 downregulation in tumor-infiltrating lymphocytes (TILs) often indicates T-cell dysfunction or exhaustion. Immunohistochemistry with CD247 antibodies (using dilutions of 1:50-1:500) can map functional and dysfunctional T-cell populations within the tumor microenvironment .

• Checkpoint inhibitor response prediction: Analysis of CD247 expression and phosphorylation in patient samples before and during immune checkpoint blockade therapy is being investigated as a potential predictive biomarker for treatment response.

• Combination therapy development: CD247 expression analysis helps evaluate whether novel immunotherapeutic approaches can restore TCR signaling capacity in dysfunctional TILs, informing the development of combination strategies with checkpoint inhibitors.

• Ex vivo expansion protocols: CD247 antibodies are used to monitor TCR complex integrity during ex vivo expansion of TILs for adoptive cell therapy, ensuring that expanded cells maintain proper signaling capacity.

• Monitoring treatment-related adverse events: Changes in peripheral T-cell CD247 expression may correlate with the development of immune-related adverse events during immunotherapy, potentially serving as an early warning biomarker.

These applications highlight how CD247 antibodies contribute to advancing cancer immunotherapy by providing critical insights into T-cell signaling, dysfunction, and therapeutic manipulation in the context of cancer.

How should researchers design experiments to investigate the relationship between CD247 polymorphisms and immune responses to vaccination?

To investigate the relationship between CD247 polymorphisms and immune responses to vaccination, researchers should design experiments that account for genetic, immunological, and clinical variables:

• Study cohort selection:

  • Include diverse populations to capture genetic variation

  • Consider stratifying by age groups, as immune responses can vary with age

  • Account for body mass index (BMI), as it has been shown to influence vaccination responses in relation to CD247 polymorphisms

  • Document pre-existing immunity through baseline antibody titers

• Genetic analysis approach:

  • Perform targeted genotyping of known CD247 polymorphisms associated with immune function

  • Consider whole gene sequencing to identify novel variants

  • Include analysis of regulatory regions that might affect CD247 expression

  • Analyze haplotypes rather than individual SNPs when possible

• Immunological assessments:

  • Measure multiple parameters of vaccine response:

    • Antibody titers at multiple time points (baseline, peak, and long-term)

    • Antibody functionality (neutralization, affinity)

    • T-cell responses (proliferation, cytokine production)

    • Memory B and T-cell generation

  • Quantify CD247 expression and phosphorylation in relevant cell populations using flow cytometry

• Experimental design considerations:

  • Use a longitudinal design with multiple sampling time points

  • Include sufficient sample size based on power calculations to detect genotype-dependent differences

  • Control for confounding factors (previous exposure, concurrent medications, comorbidities)

  • Consider challenge studies where ethically appropriate

• Technical approach:

  • For CD247 expression analysis: Use flow cytometry with appropriate antibody dilutions (0.40 μg per 10^6 cells)

  • For functional assays: Isolate T-cells before and after vaccination to assess CD247-dependent signaling

• Data analysis strategy:

  • Employ multivariate analysis to account for confounding variables

  • Consider gene-environment interactions, particularly BMI effects

  • Use systems biology approaches to integrate genetic, transcriptomic, and functional data

  • Apply machine learning to identify complex patterns in response data

• Validation approaches:

  • Include independent validation cohorts

  • Perform in vitro mechanistic studies to confirm functional impact of identified polymorphisms

  • Consider animal models with corresponding genetic variations when appropriate

This comprehensive approach will provide insights into how CD247 genetic variation influences vaccine responses, potentially leading to more personalized vaccination strategies.

What quality control measures should be implemented when working with CD247 recombinant monoclonal antibodies?

Implementing comprehensive quality control measures when working with CD247 recombinant monoclonal antibodies ensures reliable and reproducible results:

• Initial validation upon receipt:

  • Perform Western blot with positive control lysates (Jurkat cells, human spleen tissue) to confirm expected 16 kDa band size

  • Run immunocytochemistry on known positive cells (Jurkat) to verify proper staining pattern

  • Compare lot-specific data to manufacturer's certificate of analysis for consistency

• Regular performance checks:

  • Implement a schedule of periodic validation using positive control samples

  • Document antibody performance metrics (signal-to-noise ratio, background levels)

  • Track antibody performance across different lots and over time

• Storage and handling:

  • Store antibodies according to manufacturer recommendations (typically -20°C for long-term storage)

  • Avoid repeated freeze-thaw cycles by preparing small working aliquots

  • Monitor storage conditions (temperature logs for freezers)

  • Check for signs of precipitation or contamination before each use

• Application-specific controls:

  • For Western blot: Include molecular weight markers and positive/negative control lysates

  • For IHC/ICC: Run parallel positive control tissues (tonsil, spleen) and negative controls (primary antibody omission)

  • For flow cytometry: Use isotype controls and known positive/negative cell populations

• Cross-platform validation:

  • Confirm CD247 detection using multiple techniques (e.g., Western blot, IHC, flow cytometry)

  • Correlate protein detection with mRNA expression when possible

  • Consider orthogonal validation approaches as used in enhanced validation protocols

• Documentation and reporting:

  • Maintain detailed records of antibody performance in a laboratory information management system

  • Document lot numbers, dilutions, incubation conditions, and results

  • Include quality control data in research publications and reports

• Functional validation:

  • For critical experiments, confirm antibody binding corresponds to functional readouts of T-cell activity

  • Consider affinity binding assays to confirm specific KD values (reported as 5.0 x 10-7 for some CD247 antibodies)

These systematic quality control measures help minimize experimental variability and ensure reliable detection of CD247 across different experimental conditions and applications.

How can researchers optimize antibody dilutions for different applications and sample types?

Optimizing antibody dilutions for CD247 detection across different applications requires a systematic approach tailored to each specific technique and sample type:

• Western Blot optimization:

  • Starting point: Begin with manufacturer-recommended dilutions (1:500-1:3000 for polyclonal; 1:10,000 for monoclonal)

  • Titration approach: Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000, 1:10,000)

  • Sample considerations: For tissues with high CD247 expression (spleen, lymph nodes), use higher dilutions; for samples with lower expression, use lower dilutions

  • Optimization metric: Select dilution that gives strongest specific signal (16 kDa band) with minimal background

  • Verification: Confirm optimal dilution with both positive controls (Jurkat cells) and experimental samples

• Immunohistochemistry optimization:

  • Starting point: Begin with 1:100 for polyclonal or 1:1,000 for monoclonal antibodies

  • Tissue-specific considerations:

    • Lymphoid tissues (tonsil, spleen): May require higher dilutions due to abundant target

    • Non-lymphoid tissues: May require lower dilutions to detect infiltrating T-cells

  • Antigen retrieval effects: TE buffer (pH 9.0) typically yields optimal results but may affect optimal dilution

  • Detection system effects: More sensitive detection systems allow higher dilutions

  • Optimization metric: Select dilution with clear membrane/cytoplasmic staining of T-cells with minimal background

• Immunofluorescence/ICC optimization:

  • Starting point: Begin with 1:100 dilution and adjust based on results

  • Cell type considerations: Primary T-cells may require different dilutions than cell lines

  • Fixation effects: Paraformaldehyde versus methanol fixation may affect epitope accessibility

  • Optimization approach: Test 3-5 dilutions (e.g., 1:50, 1:100, 1:200, 1:500) while keeping all other variables constant

  • Optimization metric: Select dilution with best signal-to-noise ratio in positive control cells (Jurkat)

• Flow cytometry optimization:

  • Starting point: Begin with 0.40 μg per 10^6 cells for intracellular staining

  • Titration approach: Test at least 5 concentrations (e.g., 0.1, 0.2, 0.4, 0.8, 1.6 μg per 10^6 cells)

  • Optimization metric: Calculate staining index (difference between positive and negative populations divided by 2× SD of negative population)

  • Considerations for intracellular staining: Permeabilization method affects optimal antibody concentration

• Immunoprecipitation optimization:

  • Starting point: Begin with 2 μg for 2 mg of total protein lysate

  • Sample-specific adjustments: Increase antibody amount for samples with lower CD247 expression

  • Optimization metric: Clean pull-down of CD247 with minimal non-specific binding

This systematic optimization approach ensures optimal CD247 detection while minimizing reagent waste and non-specific background across all experimental applications.