IL12B Antibody

What is IL12B and what role does it play in immunological pathways?

IL12B encodes the 40 kDa subunit (p40) of interleukin-12, a heterodimeric cytokine composed of two subunits: p35 (IL12A) and p40 (IL12B). The p40 subunit is particularly significant as it can also pair with p19 to form IL-23. IL12B has multiple alternative names including CLMF p40, cytotoxic lymphocyte maturation factor 40 kDa subunit, natural killer cell stimulatory factor 40 kD subunit, and NK cell stimulatory factor chain 2 . The protein is approximately 37-46 kDa depending on post-translational modifications .

IL12B plays a crucial role in antimycobacterial immunity, as demonstrated by studies of patients with IL12B mutations who display increased susceptibility to mycobacterial infections. Experimental studies with mouse models carrying disrupted IL12B genes show high susceptibility to BCG (Bacillus Calmette-Guérin), Mycobacterium avium, and Mycobacterium tuberculosis, highlighting the essential role this protein plays in protective immunity against mycobacteria .

How do I select the appropriate IL12B antibody for my research?

When selecting an IL12B antibody, researchers should consider several key factors:

• Target epitope: Different antibodies recognize distinct regions of IL12B. For example, some antibodies target the C-terminal region (AA 271-298) , while others may target other epitopes. The choice should depend on the accessibility of the epitope in your experimental conditions.

• Host species and clonality: IL12B antibodies are available as polyclonal (e.g., rabbit-derived ) or monoclonal antibodies. Polyclonal antibodies recognize multiple epitopes and may provide stronger signals, while monoclonal antibodies offer higher specificity.

• Species reactivity: Verify the antibody's reactivity with your species of interest. Some IL12B antibodies react only with human samples , while others cross-react with mouse samples .

• Validated applications: Ensure the antibody has been validated for your specific application, such as Western Blotting (WB), Immunohistochemistry (IHC), Flow Cytometry (FACS), or Enzyme Immunoassay (EIA) .

• Purification method: Consider antibodies that have undergone rigorous purification processes, such as protein A column purification followed by peptide affinity purification .

What controls should I include when using IL12B antibodies?

For rigorous research with IL12B antibodies, the following controls are essential:

• Positive controls: Include samples known to express IL12B, such as PDBu-activated EBV-B cell lines, which secrete detectable levels of IL12p40 .

• Negative controls: Use samples from IL12B-deficient sources. For instance, EBV-B cells from individuals with homozygous loss-of-function deletions in IL12B can serve as negative controls .

• Isotype controls: Include an irrelevant antibody of the same isotype and host species to detect non-specific binding.

• Blocking peptide controls: Use the immunizing peptide to confirm specificity, particularly with peptide-derived antibodies.

• Secondary antibody controls: Include samples treated only with secondary antibodies to identify background signals.

• Technical validation: For Western blotting, verify that the detected band appears at the expected molecular weight (approximately 37-46 kDa for IL12B) .

How can IL12B antibodies be used to investigate IL12B deficiency disorders?

IL12B antibodies are valuable tools for investigating IL12B deficiency disorders through several approaches:

• Protein expression analysis: Western blotting with IL12B antibodies can detect the presence or absence of IL12B protein in patient samples, helping to confirm suspected cases of IL12B deficiency at the protein level .

• Functional assays: Combining IL12B antibodies with ELISA techniques allows researchers to measure IL12p40 and IL12p70 secretion by EBV-B cells or whole blood cells stimulated with appropriate activators (e.g., PDBu for EBV-B cells or BCG plus IFNγ for blood cells) .

• Complementation studies: IL12B antibodies can confirm successful protein expression in complementation experiments where exogenous IL12B is introduced to restore function in deficient cells.

• Tissue immunohistochemistry: IL12B antibodies can be used to examine IL12B expression patterns in tissue sections from patients with suspected IL12B-related disorders .

• Carrier identification: In families with known IL12B mutations, antibody-based protein detection can help identify carriers who may have intermediate levels of protein expression.

Studies have demonstrated that patients with IL12B deficiency typically present with mycobacterial infections (particularly BCG or NTM) and sometimes salmonellosis. Research using IL12B antibodies has helped establish that IL12 deficiency, while rare, is not limited to isolated cases but represents a genuine immunodeficiency disorder with clinical significance .

What methodological approaches can resolve contradictory IL12B detection results?

When facing contradictory IL12B detection results, researchers should consider the following systematic troubleshooting approaches:

• Multi-method validation: Confirm results using complementary techniques. For example, if Western blot results are ambiguous, validate with ELISA, flow cytometry, or immunohistochemistry .

• Cell stimulation optimization: IL12B expression often requires cell activation. For EBV-B cells, PDBu stimulation is effective, while whole blood cells respond to BCG plus IFNγ . Suboptimal stimulation can lead to false negatives.

• Antibody epitope considerations: If one antibody fails to detect IL12B, try antibodies targeting different epitopes. For instance, if a C-terminal antibody (AA 271-298) yields negative results, an antibody targeting internal or N-terminal regions might detect truncated variants .

• Control cytokine measurements: Include measurement of other cytokines like TNFα to confirm that cells are properly activated and capable of cytokine production .

• Alternative sample preparation: Modify extraction methods if standard protocols fail. For membrane-associated proteins like IL12B, different detergents or extraction buffers might be necessary.

• Species-specific considerations: Ensure the antibody's species reactivity matches your samples. Some IL12B antibodies react only with human samples, while others cross-react with mouse or other species .

• Quantification standardization: Use recombinant IL12B standards for calibration across experiments and establish clear detection thresholds.

How do experimental conditions affect IL12B antibody performance in complex samples?

The performance of IL12B antibodies in complex samples can be significantly influenced by several experimental conditions:

• Sample preparation impact:

  • For cellular samples, the choice between whole cell lysates, subcellular fractions, or secreted protein analysis affects detection sensitivity

  • Fixation methods for immunohistochemistry can alter epitope accessibility, particularly for conformational epitopes of IL12B

• Buffer systems and additives:

  • pH variations can dramatically affect antibody-antigen interactions

  • Presence of detergents (e.g., Triton X-100, SDS) may improve solubilization but potentially denature conformational epitopes

  • Protease inhibitors are essential to prevent IL12B degradation during sample processing

• Detection system optimization:

  • Signal amplification methods (e.g., TSA, ABC) can improve sensitivity for low-abundance IL12B detection

  • Fluorescent vs. chromogenic detection systems offer different sensitivity and multiplexing capabilities

• Blocking strategies:

  • Optimization of blocking solutions (BSA, normal serum, commercial blockers) to minimize background while preserving specific signals

  • Pre-absorption of antibodies with related proteins can improve specificity in complex samples

• Incubation parameters:

  • Temperature effects: room temperature vs. 4°C incubations alter binding kinetics and specificity

  • Incubation time optimization based on antibody affinity and sample complexity

• Cross-reactivity considerations:

  • IL12B shares structural similarities with other cytokine subunits, requiring validation in samples with varying cytokine compositions

  • Heterodimer formation with IL12A affects epitope accessibility in native conditions

Researchers should systematically optimize these parameters when working with complex samples such as tissue homogenates, serum, or mixed cell populations.

What are the optimized protocols for IL12B detection by Western blotting?

Optimized Western Blotting Protocol for IL12B Detection:

• Sample preparation:

  • Collect cells of interest (e.g., PDBu-activated EBV-B cells )

  • Lyse cells in RIPA buffer supplemented with protease inhibitors

  • Sonicate briefly to shear DNA and reduce sample viscosity

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

  • Measure protein concentration using BCA or Bradford assay

• Gel electrophoresis:

  • Load 20-50 μg of protein per lane on a 10-12% SDS-PAGE gel

  • Include molecular weight markers spanning 25-50 kDa range

  • Run at 100V until dye front reaches bottom of gel

• Transfer:

  • Transfer proteins to PVDF membrane (preferred over nitrocellulose for IL12B)

  • Use wet transfer system at 100V for 1 hour or 30V overnight at 4°C

  • Verify transfer efficiency with Ponceau S staining

• Blocking:

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

  • Alternative: 3% BSA in TBS-T if phospho-specific detection is needed

• Primary antibody incubation:

  • Dilute IL12B antibody to optimal concentration (typically 1:1000 to 1:2000)

  • Incubate overnight at 4°C with gentle rocking

  • Expected band size: 37-46 kDa

• Washing:

  • Wash 4 times with TBS-T, 5 minutes each

• Secondary antibody incubation:

  • Use HRP-conjugated anti-rabbit secondary antibody (1:5000 to 1:10000)

  • Incubate for 1 hour at room temperature

• Detection:

  • Develop using enhanced chemiluminescence substrate

  • Exposure time: start with 30 seconds and adjust as needed

• Controls:

  • Positive control: PDBu-activated EBV-B cells

  • Negative control: IL12B-deficient cells (e.g., cells with known IL12B mutation)

  • Loading control: β-actin or GAPDH

How should samples be prepared for optimal IL12B detection in immunohistochemistry?

Optimized Immunohistochemistry Protocol for IL12B Detection:

• Tissue fixation and processing:

  • Fix tissues in 10% neutral buffered formalin for 24-48 hours

  • Process and embed in paraffin following standard procedures

  • Section at 4-5 μm thickness onto positively charged slides

• Deparaffinization and rehydration:

  • Xylene: 3 changes, 5 minutes each

  • 100% ethanol: 2 changes, 3 minutes each

  • 95%, 80%, 70% ethanol: 3 minutes each

  • Distilled water: 5 minutes

• Antigen retrieval (critical for IL12B detection):

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Pressure cooker method: 121°C for 5 minutes, then cooling for 20 minutes

  • Alternative: 10mM EDTA buffer (pH 8.0) if citrate buffer yields weak signals

• Endogenous peroxidase blocking:

  • 3% hydrogen peroxide in methanol for 10 minutes at room temperature

  • Wash in PBS, 3 changes, 5 minutes each

• Protein blocking:

  • Apply 5% normal goat serum in PBS for 1 hour at room temperature

  • Do not wash off blocking solution before applying primary antibody

• Primary antibody incubation:

  • Dilute IL12B antibody in antibody diluent (typically 1:100 to 1:500)

  • Incubate in humidified chamber overnight at 4°C

  • For rabbit polyclonal IL12B antibodies, optimization of dilution is essential

• Washing:

  • PBS: 3 changes, 5 minutes each

• Detection system:

  • Apply HRP-polymer detection system compatible with rabbit primary antibodies

  • Follow manufacturer's recommended incubation time (typically 30 minutes)

  • Wash thoroughly with PBS, 3 changes, 5 minutes each

• Visualization:

  • Apply DAB substrate and monitor for signal development (usually 2-5 minutes)

  • Counterstain with Mayer's hematoxylin for 30 seconds

  • Blueing in running tap water for 5 minutes

• Dehydration and mounting:

  • 70%, 80%, 95% ethanol: 2 minutes each

  • 100% ethanol: 2 changes, 3 minutes each

  • Xylene: 3 changes, 5 minutes each

  • Mount with permanent mounting medium

• Controls and validation:

  • Positive tissue control: lymphoid tissues, especially activated lymphocytes

  • Negative control: omission of primary antibody

  • Blocking peptide control: pre-incubation of antibody with immunizing peptide

What are effective troubleshooting strategies for IL12B flow cytometry experiments?

Troubleshooting Guide for IL12B Flow Cytometry:

• No signal or weak signal detection:

  • Possible causes:

    • Insufficient stimulation of cells (IL12B often requires activation)

    • Low antibody concentration

    • Improper fixation affecting epitope accessibility

    • Degradation of IL12B protein during processing

  • Solutions:

    • Optimize cell stimulation (e.g., PDBu for EBV-B cells, BCG plus IFNγ for blood cells)

    • Titrate antibody to determine optimal concentration

    • Try alternative fixation/permeabilization protocols (particularly for intracellular IL12B)

    • Add protease inhibitors during sample preparation

    • Consider using a protein transport inhibitor (e.g., Brefeldin A) to accumulate intracellular cytokines

• High background or non-specific staining:

  • Possible causes:

    • Insufficient blocking

    • Fc receptor binding

    • Autofluorescence

    • Non-specific antibody binding

  • Solutions:

    • Increase blocking time/concentration (10% serum from secondary antibody species)

    • Add Fc receptor blocking reagent before antibody staining

    • Include unstained and FMO (fluorescence minus one) controls

    • Try alternative clone or format of IL12B antibody

    • Include dead cell discriminator to exclude non-specific binding to dead cells

• Inconsistent results across experiments:

  • Possible causes:

    • Variations in cell activation status

    • Inconsistent antibody handling

    • Instrument variability

  • Solutions:

    • Standardize activation protocols and timing

    • Use antibody aliquots and avoid freeze-thaw cycles

    • Include calibration beads for instrument standardization

    • Establish a standard operating procedure with fixed acquisition settings

    • Use biological reference controls across experiments

• Poor separation between positive and negative populations:

  • Possible causes:

    • Suboptimal antibody concentration

    • Inappropriate fluorophore choice

    • Biological heterogeneity in IL12B expression

  • Solutions:

    • Optimize antibody concentration through titration

    • Use brighter fluorophores (e.g., PE, APC instead of FITC)

    • Consider cell sorting for more homogeneous populations

    • Analyze data using appropriate statistical methods for heterogeneous populations

• Unexpected staining patterns:

  • Possible causes:

    • Cross-reactivity with related proteins

    • Detection of variant isoforms

    • Complex formation affecting epitope accessibility

  • Solutions:

    • Validate with alternative detection methods (e.g., Western blot)

    • Use antibodies recognizing different epitopes

    • Compare results with genetic knockout controls if available

    • Consider the biological context (e.g., IL12B can form heterodimers with IL12A)

How should researchers design experiments to distinguish between free IL12B and IL12 heterodimers?

Distinguishing between free IL12B (p40) and IL12 heterodimers (p70, consisting of p40+p35) requires careful experimental design:

• Sequential Immunoprecipitation Strategy:

  • First IP: Use antibodies against IL12B to pull down both free p40 and p70

  • Second IP: Use antibodies against IL12A to selectively capture p70 from the remaining supernatant

  • Western blot analysis with IL12B antibodies on both precipitates will reveal the distribution between free and complexed forms

• Differential ELISA Approach:

  • Use a sandwich ELISA with capture antibody against IL12B and detection antibody against IL12B to measure total IL12B

  • Use a sandwich ELISA with capture antibody against IL12B and detection antibody against IL12A to measure only heterodimeric p70

  • The difference between these measurements represents free IL12B

• Size Exclusion Chromatography Combined with Immunodetection:

  • Fractionate samples based on molecular weight (p40 at ~40 kDa, p70 at ~70 kDa)

  • Analyze fractions using IL12B antibodies to identify the distribution between monomeric, homodimeric (p40:p40), and heterodimeric (p40:p35) forms

• Flow Cytometry Dual Staining:

  • Simultaneously stain with differently labeled antibodies against IL12B and IL12A

  • Single-positive cells (IL12B+/IL12A-) indicate free p40 production

  • Double-positive cells (IL12B+/IL12A+) suggest heterodimer production

• Functional Discrimination:

  • IL12B knockout models (or patient cells with IL12B deficiency) allow validation of antibody specificity

  • Comparison of biological activities (e.g., IFNγ induction) can help distinguish functional p70 from p40, which may act as an antagonist

When interpreting results, researchers should recognize that free IL12B can exist as monomers or homodimers, and it may have biological activities distinct from those of the IL12 heterodimer.

What considerations are important when using IL12B antibodies for comparative studies across different species?

When conducting comparative studies of IL12B across species, researchers should carefully consider the following factors:

• Sequence homology and antibody cross-reactivity:

  • Human IL12B shares ~70% amino acid identity with mouse IL12B

  • Antibody selection should be guided by epitope conservation; some antibodies are specifically validated for both human and mouse reactivity

  • Prediction models based on immunogen sequence alignment can help estimate cross-reactivity potential, though experimental validation remains essential

• Epitope accessibility variations:

  • Post-translational modifications differ across species and may affect antibody binding

  • Glycosylation patterns in particular can mask epitopes in species-specific ways

  • C-terminal antibodies may offer better cross-species reactivity due to higher conservation in this region

• Experimental validation strategies:

  • Use positive controls from each species being studied

  • Include recombinant IL12B proteins from each species as standards

  • Validate antibody performance in each species using cells from genetic knockout animals if available

• Application-specific considerations:

  • For Western blotting: Be aware of potential molecular weight differences between species

  • For IHC: Optimize antigen retrieval separately for each species' tissues

  • For ELISA: Establish separate standard curves for each species

• Quantitative comparison limitations:

  • Different antibody affinities across species may prohibit direct quantitative comparisons

  • Consider developing conversion factors based on recombinant protein standards

  • Use relative rather than absolute quantification when comparing across species

• Alternative approaches:

  • Species-specific antibodies used in parallel may provide more reliable comparisons than a single cross-reactive antibody

  • Genetic approaches (e.g., qPCR) may complement protein-level studies for cross-species comparisons

The search results indicate that some IL12B antibodies have predicted cross-reactivity with dog samples, though with lower confidence than for human and mouse samples . Researchers should explicitly validate any predicted cross-reactivity before proceeding with multi-species comparisons.

How might IL12B antibodies contribute to understanding IL12B's role in emerging immunotherapies?

IL12B antibodies represent valuable tools for advancing our understanding of IL12B's role in novel immunotherapeutic approaches:

• Biomarker development for immunotherapy response:

  • IL12B antibodies can enable quantification of p40 levels before, during, and after immunotherapy

  • Correlation of IL12B expression patterns with treatment outcomes may reveal predictive biomarkers

  • Flow cytometric analysis using IL12B antibodies can identify specific immune cell populations responding to treatment

• Therapeutic antibody development pipeline:

  • Neutralizing vs. non-neutralizing IL12B antibodies can help distinguish the roles of free p40 vs. IL12/IL23 heterodimers in disease models

  • Antibody-dependent cellular cytotoxicity (ADCC) against IL12B-producing cells could represent a novel therapeutic approach

  • Bispecific antibodies incorporating anti-IL12B domains may allow targeted modulation of specific immune pathways

• Monitoring of IL12B-targeting biologics:

  • IL12B antibodies recognizing different epitopes from therapeutic antibodies can be used to monitor target engagement

  • Competitive binding assays with IL12B antibodies can assess therapeutic antibody biodistribution and tissue penetration

  • Immunohistochemistry with IL12B antibodies can evaluate changes in tissue expression patterns following treatment

• Precision medicine applications:

  • IL12B antibodies may help stratify patients with IL12B-related immunodeficiencies for gene therapy approaches

  • Identification of specific IL12B variants in patient samples could guide personalized therapeutic strategies

  • Monitoring IL12B expression during treatment can provide pharmacodynamic endpoints

• Novel delivery systems assessment:

  • IL12B antibodies can evaluate the expression and localization of IL12B following gene therapy or mRNA delivery

  • Assessment of IL12B production by engineered cells (e.g., CAR-T cells with IL12B payloads)

  • Verification of IL12B expression from viral vector-based delivery systems

These applications highlight the continuing importance of well-characterized IL12B antibodies in translational research connecting basic immunology with clinical applications.

What techniques combine IL12B antibodies with emerging single-cell technologies?

The integration of IL12B antibodies with cutting-edge single-cell technologies offers powerful new approaches to understanding IL12B biology:

• Single-cell proteogenomic analysis:

  • CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) can be adapted to include IL12B antibodies, allowing simultaneous analysis of IL12B protein expression and transcriptome-wide gene expression at single-cell resolution

  • This approach can reveal discrepancies between IL12B mRNA and protein levels, providing insights into post-transcriptional regulation

• Mass cytometry (CyTOF) applications:

  • Metal-conjugated IL12B antibodies can be incorporated into CyTOF panels with 30+ additional markers

  • This enables comprehensive immune phenotyping while simultaneously assessing IL12B production across diverse immune cell populations

  • Particularly valuable for analyzing rare IL12B-producing cells within heterogeneous samples

• Spatial transcriptomics with protein detection:

  • Technologies like Visium (10x Genomics) combined with immunofluorescence using IL12B antibodies

  • Provides spatial context for IL12B expression within tissue microenvironments

  • Can reveal cell-cell interactions between IL12B-producing cells and responding cells

• Microfluidic single-cell secretion analysis:

  • Droplet-based systems combining single-cell encapsulation with IL12B antibody-based detection

  • Enables quantification of IL12B secretion rates from individual cells

  • Can be combined with cell sorting to isolate high IL12B-producing cells for further characterization

• Proximity ligation assays at single-cell level:

  • In situ detection of IL12B interactions with binding partners (e.g., IL12RB1, IL12RB2)

  • Visualization of protein-protein interactions in individual cells and subcellular compartments

  • Can distinguish between intracellular IL12B and cell surface-associated IL12B

• Live-cell imaging with IL12B antibody fragments:

  • Non-disruptive IL12B detection using fluorescently labeled Fab fragments

  • Allows temporal monitoring of IL12B production and trafficking in living cells

  • Can be combined with optogenetic systems to simultaneously perturb and monitor IL12B-related pathways

These integrated approaches promise to deliver unprecedented insights into the cellular and molecular dynamics of IL12B in health and disease states, potentially revealing new therapeutic targets and biomarkers.