IL12RB1 Antibody

What is IL12RB1 and why is it significant in immunological research?

IL12RB1 encodes IL12Rβ1, a type I transmembrane receptor that forms essential components of both IL12 and IL23 signaling complexes. The extracellular portion contains the cytokine-binding region that physically associates with IL12/IL23, while the cytoplasmic portion collaborates with IL12Rβ2 or IL23R to transmit intracellular signals via pre-associated kinases TYK2 and JAK2 .

IL12RB1 has significant clinical relevance as individuals homozygous for IL12RB1 null alleles show increased susceptibility to persistent forms of tuberculosis, salmonellosis, and candidiasis . Importantly, IL12RB1 plays dual roles in human health - promoting both protective delayed type hypersensitivity (DTH) responses against pathogens and potentially harmful autoimmune reactions. This duality makes IL12RB1 a critical target for immunological research focused on balancing immune protection against pathogens while preventing autoimmunity.

For researchers investigating IL12RB1, antibodies provide essential tools for detecting expression patterns, understanding receptor localization, and studying signaling pathway activation across different cell types and disease states.

What are the different isoforms of IL12RB1 and how do they impact antibody selection?

Human IL12RB1 undergoes alternative mRNA processing that generates two distinct protein isoforms with different functional properties:

• Isoform 1 (IL12Rβ1): The canonical transmembrane receptor that binds the IL12p40 domain of IL12/IL23 and cooperates with co-receptors IL12Rβ2 or IL23R to initiate STAT signaling .

• Isoform 2: Retains the IL12p40-binding domains but lacks the transmembrane domain of Isoform 1. Initially predicted to be non-functional, recent evidence suggests Isoform 2 actually promotes IL12 responses and T helper 1 (TH1) development .

These isoforms result from intragenic competition between IL12RB1 exon 9-10 splicing and IL12RB1 exon 9b splicing/polyadenylation . The production of Isoform 2 is regulated by an IL12RB1 exon 9b polyadenylation site upstream of heterogeneous nuclear ribonucleoprotein H (hnRNP H) binding .

When selecting antibodies for IL12RB1 research, investigators must consider:

• Epitope location relative to isoform differences

• Whether the study aims to detect both isoforms or discriminate between them

• Domain-specific antibodies that recognize extracellular versus intracellular portions

Researchers should carefully verify which epitopes antibodies recognize to ensure they're appropriate for the specific isoforms being studied.

How does IL12RB1 expression vary across different immune cell populations?

IL12RB1 expression demonstrates significant variation across immune cell populations and contexts, which impacts experimental design when using antibodies. T cells show particularly regulated expression patterns of IL12RB1. In primary human tissues and peripheral blood mononuclear cells (PBMCs), IL12RB1 expression is allele-biased, meaning cells preferentially express one allele over the other .

This allele-biased expression is maintained even after T cell activation, suggesting it's a stable epigenetic characteristic rather than an activation-dependent phenomenon . CD4+ and CD8+ T cells purified from PBMCs both exhibit this allele-biased expression pattern .

When designing IL12RB1 antibody experiments, researchers should:

• Include appropriate cell type controls that reflect physiological expression levels

• Consider tissue-specific expression patterns (notably different in lung tissue compared to peripheral blood)

• Account for potential allele-biased expression when interpreting quantitative results

• Examine both CD4+ and CD8+ T cell populations separately rather than assuming identical expression patterns

What are the recommended protocols for detecting IL12RB1 in primary human T cells?

When investigating IL12RB1 in primary human T cells, the following methodological approach is recommended based on published research protocols:

T Cell Isolation Protocol:

• Obtain peripheral blood mononuclear cells (PBMCs) from blood units donated by healthy adults

• Exclude samples from donors taking immunosuppressant medications including antineoplastic agents, antivirals, corticosteroids, disease-modifying anti-rheumatic drugs, or immunosuppressive mAb drugs

• For CD4+ and CD8+ T cell purification, suspend PBMCs in magnetic bead-Ab conjugates specific to CD4 (clone L200) or CD8 (clone SK1)

• After positive selection and washing in PBS, count cells and immediately lyse for RNA and DNA extraction

Western Blot Detection:

• Use cell lysates prepared in RIPA buffer with protease inhibitors

• Separate proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF membranes

• Block with 5% non-fat milk in TBST

• Incubate with primary IL12RB1 antibodies (targeting either N-terminal or C-terminal regions depending on which isoform you want to detect)

• Wash and incubate with appropriate HRP-conjugated secondary antibodies

• Develop using ECL substrate

Flow Cytometry Protocol:

• Harvest cells and wash in PBS with 1% BSA

• Block Fc receptors to prevent non-specific binding

• Stain with fluorochrome-conjugated IL12RB1 antibody targeting extracellular domains

• For intracellular detection (especially for Isoform 2), use appropriate fixation and permeabilization solutions

• Include isotype controls and known positive/negative cell populations

Using these protocols allows researchers to reliably detect IL12RB1 expression while accounting for the nuances of its isoform expression and allele-biased nature.

How can researchers distinguish between IL12RB1 isoforms using antibodies?

Distinguishing between IL12RB1 isoforms requires careful antibody selection and experimental design. The key structural difference between the isoforms is that Isoform 2 lacks the transmembrane domain present in Isoform 1 and has an altered C-terminal amino acid sequence .

Recommended Approach for Isoform Discrimination:

• Epitope-Specific Antibodies:

  • Use antibodies targeting the C-terminal region unique to each isoform

  • For Isoform 1: Select antibodies recognizing epitopes in the transmembrane or cytoplasmic domain

  • For Isoform 2: Use antibodies specific to the unique C-terminal sequence resulting from exon 9b inclusion

• Subcellular Localization Analysis:

  • Isoform 1 (IL12Rβ1) localizes to the cell surface as a type 1 transmembrane protein

  • Isoform 2 has a distinct intracellular localization pattern in the reticulum

  • Use confocal microscopy with differentially labeled antibodies to visualize spatial distribution

• Combined RNA and Protein Analysis:

  • Perform RT-PCR using primers spanning exon junctions to distinguish isoform-specific mRNAs

  • Correlate mRNA findings with protein detection using isoform-specific antibodies

  • Use siRNA knockdown of specific isoforms to validate antibody specificity

• Functional Validation:

  • Assess IL12-dependent IFNγ secretion in cells with confirmed expression of each isoform

  • Perform microRNA-mediated knockdown experiments targeting specific isoforms to validate antibody detection

Using this multifaceted approach allows researchers to reliably distinguish between the functionally distinct IL12RB1 isoforms and accurately interpret experimental findings.

What controls should be included when using IL12RB1 antibodies in research?

Robust experimental design for IL12RB1 antibody research requires comprehensive controls to ensure valid interpretation:

Positive Controls:

• Jurkat T cell line (confirmed to express IL12RB1 with allele-biased pattern)

• Activated primary human T cells (known to express IL12RB1)

• BSC40 Iso2 cells (stably express Isoform 2 mRNA) for Isoform 2-specific antibodies

Negative Controls:

• Cell lines with confirmed absence of IL12RB1 expression

• Isotype-matched control antibodies to assess non-specific binding

• Pre-absorption of antibody with recombinant IL12RB1 protein to confirm specificity

Validation Controls:

• siRNA or shRNA knockdown of IL12RB1 to confirm antibody specificity

• Comparison with mRNA expression data using RT-PCR

• Parallel detection with multiple antibodies targeting different epitopes

• IL12RB1-deficient cells from patients with IL12RB1 genetic deficiencies (if available)

Experimental Design Controls:

• Test antibody performance across multiple detection methods (Western blot, flow cytometry, immunohistochemistry)

• Include unstimulated and stimulated conditions (e.g., PHA stimulation for T cells)

• Compare antibody performance across different tissue types (lung tissue vs. PBMCs)

• Consider allele-specific detection if studying genetic variants

These comprehensive controls ensure that results obtained with IL12RB1 antibodies accurately reflect biological reality rather than technical artifacts.

How does allele-biased expression impact IL12RB1 antibody studies?

Allele-biased expression of IL12RB1, wherein one allele is preferentially expressed over the other, presents unique considerations for antibody-based studies. Research has demonstrated that in primary human lung tissue and T cells, IL12RB1 is preferentially expressed from one allele, and this bias persists even after T cell activation .

Methodological Considerations for Allele-Biased Expression:

• Genotype-Phenotype Correlation:

  • Researchers should genotype samples for IL12RB1 polymorphisms to identify potential allelic variants

  • Correlation between genotype and protein expression levels may be nonlinear due to allele bias

  • Consider using allele-specific PCR to quantify relative expression from each allele

• Population Heterogeneity:

  • Cell populations may contain a mixture of cells expressing different alleles

  • Single-cell analysis techniques may reveal expression patterns obscured in bulk analysis

  • Flow cytometric sorting based on IL12RB1 expression followed by genotyping can identify allele-specific patterns

• Antibody Epitope Considerations:

  • Polymorphisms near antibody epitopes may affect binding efficiency

  • Validate antibodies using cells with known allelic variants

  • Use multiple antibodies targeting different epitopes to ensure comprehensive detection

• Functional Implications:

  • Allele-biased expression may impact cellular responses to IL12/IL23

  • Compare functional readouts (e.g., STAT phosphorylation, IFNγ production) with antibody-based detection

  • Consider how allele bias might affect disease susceptibility or treatment responses

This understanding of allele-biased expression is essential for accurate interpretation of IL12RB1 antibody studies, particularly in heterogeneous human samples where genetic variation may influence detection and functional outcomes.

What factors influence alternative processing of IL12RB1 pre-mRNA and how can researchers account for these?

The alternative processing of IL12RB1 pre-mRNA into either Isoform 1 or Isoform 2 is regulated by complex mechanisms that researchers must consider when designing antibody-based experiments:

Key Regulatory Mechanisms:

• Intragenic Competition:

  • Competition between IL12RB1 exon 9-10 splicing and IL12RB1 exon 9b splicing/polyadenylation determines isoform production

  • The exon 9b-associated polyadenylation site plays a critical role in this regulatory process

• RNA-Binding Proteins:

  • Heterogeneous nuclear ribonucleoprotein H (hnRNP H) binds near the regulated polyadenylation site

  • While hnRNP H binding is observed, it is not required for exon 9b polyadenylation

  • Other RNA-binding proteins may influence splicing decisions

• Cell Type and Activation State:

  • Different cell types may preferentially produce one isoform over the other

  • T cell activation may alter the ratio of isoforms produced

Methodological Recommendations:

Understanding these factors allows researchers to design more nuanced experiments that account for the complexity of IL12RB1 pre-mRNA processing and its functional consequences.

How do IL12RB1 deficiencies manifest and what are the implications for antibody-based diagnostics?

IL12RB1 deficiencies have significant clinical implications and present unique challenges for antibody-based diagnostics and research:

Clinical Manifestations of IL12RB1 Deficiency:

• Infectious Disease Susceptibility:

  • Individuals homozygous for IL12RB1 null alleles develop disseminated and recurrent diseases following exposure to mycobacterial and salmonella pathogens

  • Despite being asymptomatic for the majority of their lives, these individuals show clear susceptibility to specific infections

• Immunological Phenotype:

  • T helper cells from IL12RB1-deficient individuals exhibit defective production of IFNγ

  • This phenotype mirrors observations in il12rb1-/- mice, supporting conserved function between species

• Disease Spectrum:

  • Studies across multiple ethnicities have established IL12RB1's essential role in resistance to several intracellular pathogens, including bacterial, fungal, and mycobacterial pathogens

Implications for Antibody-Based Research and Diagnostics:

• Detection Challenges:

  • Null mutations may result in absence of protein expression or production of truncated proteins

  • Antibodies targeting different epitopes may yield discrepant results depending on the specific mutation

  • Flow cytometric analysis may show reduced or absent surface expression of IL12Rβ1

• Functional Assessment:

  • Combine antibody detection with functional assays measuring STAT phosphorylation or IFNγ production

  • Use IL12-stimulation protocols to assess downstream signaling capacity

  • Compare results with genetic testing to establish genotype-phenotype correlations

• Methodological Recommendations:

  • Use multiple antibodies targeting different domains of IL12RB1

  • Include controls from known IL12RB1-deficient patients when available

  • Correlate protein expression with genetic analysis and functional outcomes

  • Consider both Isoform 1 and Isoform 2 when interpreting results

• Research Applications:

  • IL12RB1-deficient cells provide valuable negative controls for antibody validation

  • Studying partial deficiencies can reveal nuanced relationships between expression levels and function

  • Investigating compensatory mechanisms in IL12RB1-deficient individuals may reveal new therapeutic targets

Understanding these manifestations and implications is essential for accurate interpretation of antibody-based studies in both research and diagnostic contexts.

How do IL12RB1 isoforms differentially regulate IL12 responses and what are the implications for experimental design?

Recent research has revealed that both IL12RB1 isoforms contribute to IL12 responses but through distinct mechanisms, which has significant implications for experimental design and data interpretation:

Functional Differences Between Isoforms:

• Isoform 1 (IL12Rβ1):

  • Functions as a cell surface receptor binding the IL12p40 domain of IL12/IL23

  • Cooperates with co-receptors IL12Rβ2 or IL23R to initiate intracellular STAT signaling

  • Leads to increased secretion of IFNγ, which limits mycobacteria survival

• Isoform 2:

  • Retains the IL12p40-binding domains but lacks the transmembrane domain of Isoform 1

  • Localizes to an intracellular reticulum rather than the cell surface

  • Initially predicted to be non-functional, but recent evidence shows it promotes IL12 responses and TH1 development

  • Microarray-mediated knockdown experiments demonstrate that Isoform 2 promotes T cell IL12 responses

Experimental Design Implications:

This nuanced understanding of IL12RB1 isoform functionality allows researchers to design more comprehensive experiments that capture the complexity of IL12 signaling regulation.

What are the most reliable quantitative methods for analyzing IL12RB1 expression data?

Accurate quantification of IL12RB1 expression presents unique challenges due to its allele-biased expression and multiple isoforms. Researchers should consider the following methodological approaches:

Recommended Quantitative Methods:

• qRT-PCR Analysis:

  • Design primers spanning exon junctions specific to each isoform

  • Use allele-specific primers when analyzing samples with known polymorphisms

  • Include multiple reference genes for normalization

  • Consider digital PCR for absolute quantification, especially useful for allele-biased genes

• Flow Cytometric Analysis:

  • Use fluorophore-conjugated antibodies with known epitope specificity

  • Include appropriate isotype controls

  • Quantify using mean fluorescence intensity (MFI) rather than percent positive cells

  • Consider using standardized beads to normalize across experiments

  • Combine with intracellular staining protocols to detect internal Isoform 2

• Western Blot Quantification:

  • Use infrared or chemiluminescent detection systems with linear dynamic range

  • Include titration curves of recombinant protein standards

  • Normalize to multiple housekeeping proteins

  • Use antibodies that can distinguish between isoforms based on molecular weight differences

• Mass Spectrometry-Based Approaches:

  • Targeted proteomics using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM)

  • Identify isoform-specific peptides for quantification

  • Include isotopically labeled standards for absolute quantification

  • Particularly useful for distinguishing closely related isoforms

Analytical Considerations:

• Accounting for Allele Bias:

  • Genotype samples to identify polymorphisms that might affect quantification

  • Consider allele-specific expression when interpreting population-level data

  • Single-cell analysis may reveal heterogeneity masked in bulk measurements

• Isoform Ratio Analysis:

  • Calculate the ratio of Isoform 1 to Isoform 2 as a potential biomarker

  • Correlate isoform ratios with functional outcomes

  • Consider how experimental conditions might alter this ratio

• Statistical Approaches:

  • Use appropriate statistical methods that account for non-normal distributions

  • Consider mixed effects models when analyzing samples from multiple individuals

  • Perform power calculations based on expected effect sizes given known variability in IL12RB1 expression

These methodological approaches provide a framework for reliable quantification of IL12RB1 expression while accounting for its unique biological characteristics.

How should researchers interpret IL12RB1 antibody results in the context of genetic variation?

Genetic variation in IL12RB1 adds complexity to antibody-based studies and requires careful interpretation:

Types of Genetic Variation:

• Null Mutations:

  • Homozygous IL12RB1 null mutations result in complete absence of functional protein

  • Individuals with these mutations show susceptibility to mycobacterial and other infections

  • Antibody-based methods may show absence of protein or expression of truncated forms

• Polymorphisms Affecting Expression:

  • Certain polymorphisms may influence the degree of allele-biased expression

  • Epigenetic factors may interact with genetic variation to determine expression levels

  • Quantitative variation in protein levels may correlate with susceptibility to certain diseases

• Splice-Affecting Variants:

  • Variants near splice sites may alter the ratio of Isoform 1 to Isoform 2

  • Such variants could influence the balance between membrane and intracellular receptor forms

  • Changes in isoform ratios may impact cellular responsiveness to IL12/IL23

Interpretative Framework:

• Genotype-Phenotype Correlation:

  • Always interpret antibody results in the context of known genotype when possible

  • Consider how specific variants might affect antibody binding epitopes

  • Use family-based studies to understand inheritance patterns of expression variation

• Population Considerations:

  • Different ethnic groups may have varying frequencies of IL12RB1 variants

  • Population-specific reference ranges may be necessary for accurate interpretation

  • Consider admixture when studying ethnically diverse populations

• Functional Correlation:

  • Correlate antibody-detected expression levels with functional readouts like STAT phosphorylation

  • Assess IL12-dependent IFNγ production in relation to IL12RB1 variant expression

  • Consider how genetic variants might affect either surface expression or signaling capacity

• Clinical Correlation:

  • Interpret results in the context of clinical phenotypes such as infection susceptibility

  • Consider how variants of uncertain significance might relate to borderline clinical phenotypes

  • Use longitudinal studies to assess how expression changes over time or with disease progression

This comprehensive approach to interpreting IL12RB1 antibody results in the context of genetic variation enables more accurate assessment of biological significance and clinical implications.