C17orf99 Antibody

What is C17orf99 and why is it significant to immunology researchers?

C17orf99 (chromosome 17 open reading frame 99) encodes IL-40 (Interleukin-40), a recently discovered B cell-associated cytokine that plays a significant role in humoral immune responses. Identified in 2017, this small secreted protein (~27 kDa) is expressed primarily in bone marrow, fetal liver, and activated B cells . IL-40 is evolutionarily unique and only present in mammalian genomes, suggesting specialized functions in mammalian immune responses . Its significance lies in its involvement in B cell development and immunoglobulin production, particularly IgA . Research interest has increased due to its potential role in autoimmune conditions such as rheumatoid arthritis, systemic lupus erythematosus, and primary Sjögren's syndrome .

What are the key applications for C17orf99/IL-40 antibodies in research?

C17orf99/IL-40 antibodies are valuable tools for multiple research applications:

• Immunohistochemistry (IHC): Typically used at dilutions of 1:50-1:200 to visualize expression patterns in tissue sections

• Immunocytochemistry/Immunofluorescence (ICC/IF): For cellular localization studies, showing positivity in the nucleus but not nucleoli in certain cell lines

• Western Blotting: For detecting the protein at its expected molecular weight of approximately 27 kDa

• ELISA: For quantifying IL-40 levels in serum and other biological fluids, especially in autoimmune disease studies

When selecting antibodies for these applications, researchers should consider validation data showing specificity and sensitivity in the intended experimental system.

How is C17orf99/IL-40 expression regulated in B cells?

C17orf99/IL-40 expression in B cells is regulated through several mechanisms:

• Activation-dependent expression: Naive B cells express minimal IL-40, but expression increases significantly upon activation

• Receptor-mediated stimulation: CD40 ligation combined with IL-4 strongly induces C17orf99 expression

• BCR signaling: Anti-IgM stimulation enhances expression

• Cytokine influence: Multiple cytokines modulate expression, with TGF-β1 particularly potentiating C17orf99 expression

• Tissue-specific regulation: Expression is highest in bone marrow and fetal liver, suggesting developmental regulation

Expression analysis using qPCR shows that C17orf99 expression decreases rapidly (becoming undetectable by 48 hours) when isolated B cells are cultured in vitro without appropriate stimulation .

What controls should be included when using C17orf99 antibodies?

When using C17orf99 antibodies, include the following controls:

• Positive tissue controls: Human liver tissue has been validated for IHC applications

• Positive cell line controls: U-251MG cell line shows nuclear positivity and is recommended for ICC/IF applications

• Negative controls: Include isotype controls and samples known to lack C17orf99 expression

• Blocking peptide controls: Use specific antigen controls when available to confirm specificity

• Cross-reactivity controls: Test species with varying homology (human: 100%, dog: 85%, rat: 77%) to establish specificity

Always validate antibody performance in your specific experimental system before proceeding with larger studies.

How can C17orf99/IL-40 antibodies be used to investigate autoimmune diseases?

For autoimmune disease research, C17orf99/IL-40 antibodies can be employed with these methodological approaches:

• Serum biomarker analysis: Measure IL-40 levels in patients with rheumatoid arthritis or other autoimmune conditions using validated ELISA protocols

• Tissue expression studies: Compare IL-40 expression patterns in affected tissues using IHC:

  • Recommended dilution: 1:50-1:200

  • Antigen retrieval: Heat-mediated in citrate buffer

  • Detection system: HRP-conjugated secondary antibodies with DAB visualization

• Cellular source identification: Use dual immunofluorescence staining with CD19 (B cell marker) and C17orf99 antibodies to identify specific cell populations producing IL-40 in disease states

Research has shown elevated IL-40 levels in rheumatoid arthritis patients compared to healthy controls, with levels decreasing following B-cell depletion therapy .

What are the optimal experimental conditions for detecting C17orf99 in B cell subpopulations?

To effectively detect C17orf99 in B cell subpopulations:

• B cell isolation and activation protocol:

  • Isolate B cells using negative selection to avoid pre-activation

  • Activate with optimal stimuli: anti-CD40 (1μg/mL) + IL-4 (20ng/mL)

  • Include TGF-β1 (5ng/mL) for enhanced expression

  • Culture for 24-36 hours (expression decreases after 48 hours)

• qPCR detection strategy:

  • RNA isolation: QIAGEN RNeasy Kit

  • cDNA synthesis: QuantiTect Reverse Transcription

  • Reference genes: GAPDH (housekeeping gene)

  • Target: C17orf99 with validated primers

• Gating strategy for flow cytometry:
  Follow established protocols for B cell subset identification as described in references 17 and 18 from the original study .

How can researchers validate the specificity of their C17orf99 antibodies?

Comprehensive validation of C17orf99 antibodies should include:

• Cross-platform validation:

  • Western blot: Confirm single band at expected molecular weight (~27 kDa)

  • IHC: Compare staining patterns across multiple tissues with known expression profiles

  • ICC/IF: Verify subcellular localization matches expected pattern (nuclear but not nucleolar)

• Genetic validation approaches:

  • Overexpression system: Transfect HEK293 cells with C17orf99 expression constructs

  • siRNA knockdown: Verify reduced antibody signal with target knockdown

  • CRISPR knockout: Generate C17orf99 knockout cell lines as negative controls

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide (sequence: FSFLPSQTSDWFWCQAANNANVQHSALTVVPPGGDQKMEDWQGPLESPILALPLYRSTRRLSEEEFGGFRIGNGEVRGRKAAAM)

  • Compare staining with and without peptide competition

• Cross-species reactivity assessment:
  Test antibodies against samples from species with varying homology:

  • Human (100%)

  • Dog (85%)

  • Rat (77%)

What approaches can be used to study C17orf99 function in B cell development and IgA production?

To investigate C17orf99 function in B cell development and IgA production:

• In vivo knockout models:

  • Analyze C17orf99-/- mice for:

    • B cell development in bone marrow and peripheral lymphoid organs

    • IgA levels in serum, gut, feces, and lactating mammary gland

    • Size and number of Peyer's patches

    • Composition of gut microbiome

• In vitro functional assays:

  • B cell differentiation in the presence/absence of recombinant IL-40

  • IgA class switching frequency measurement

  • Antibody secretion quantification

  • B cell proliferation and survival assays

• Rescue experiments:

  • Add recombinant IL-40 to cultures of C17orf99-/- B cells to assess functional complementation

  • Measure restoration of IgA production and B cell development markers

How can researchers reconcile contradictory data regarding C17orf99 expression or function?

When confronting contradictory data in C17orf99 research:

• Technical variance analysis:

  • Antibody clone comparison: Different antibodies may recognize different epitopes or isoforms

  • Detection method sensitivity: qPCR may detect transcripts while protein levels remain below detection threshold

  • Sample preparation differences: Expression decreases rapidly in culture without stimulation

• Biological context considerations:

  Context	Expression Level	Regulation Factors	Detection Methods
  Naive B cells	Low/Undetectable	Requires activation	qPCR, Flow cytometry
  Activated B cells	High	CD40L, IL-4, TGF-β1	qPCR, Flow cytometry, Western blot
  Bone marrow	Moderate	Developmental stage	IHC, qPCR
  Autoimmune disease	Elevated	Disease activity	ELISA, IHC

• Experimental design reconciliation:

  • Timing differences: C17orf99 expression peaks at different timepoints post-stimulation

  • Cell subset heterogeneity: Expression may vary between B cell subpopulations

  • Disease state influence: Expression patterns differ between healthy and disease states

• Validation through orthogonal approaches:

  • Combine transcriptomic (RNA-seq) with proteomic (mass spectrometry) analyses

  • Verify with functional assays using recombinant protein

  • Confirm with genetic models (knockout/knockdown)

How might C17orf99/IL-40 connect to other cytokine signaling pathways?

Current evidence suggests multiple connections between IL-40 and other cytokine networks:

• Cytokine induction relationships:

  • IL-4 induces C17orf99 expression in B cells

  • TGF-β1 potentiates C17orf99 expression

  • IL-38 (anti-inflammatory) downregulates C17orf99 in co-cultures of respiratory epithelial cells with macrophages

• Potential signaling pathway intersections:

  • JAK/STAT pathway: Given IL-40's classification as an interleukin

  • TGF-β signaling: Based on TGF-β1's potentiation effect

  • NF-κB pathway: Common in B cell activation processes

• Methodological approaches to investigate pathway connections:

  • Phosphoproteomic analysis following IL-40 stimulation

  • Inhibitor studies targeting specific nodes in potential signaling pathways

  • Co-immunoprecipitation to identify binding partners

  • Reporter assays for pathway activation

Research examining IL-40 in the context of other cytokines will provide valuable insights into its place within the immune signaling network.

What methodologies are optimal for studying C17orf99/IL-40 in human autoimmune conditions?

For human autoimmune disease research on C17orf99/IL-40:

• Multiparameter analysis techniques:

  • Multiplex cytokine assays including IL-40 alongside established biomarkers

  • Single-cell transcriptomics to identify IL-40-producing cell populations

  • Mass cytometry (CyTOF) with IL-40 antibodies for high-dimensional profiling

• Longitudinal monitoring strategies:

  • Serial sampling before and after treatment interventions

  • Correlation with disease activity scores (e.g., DAS28 for RA)

  • Integration with patient-reported outcomes

• Experimental design considerations:

  Disease	Sample Type	Key Measurements	Controls
  Rheumatoid Arthritis	Serum, Synovial fluid	IL-40, RF, anti-CCP	Age/sex-matched healthy, OA patients
  SLE	Serum, PBMCs	IL-40, dsDNA antibodies	Age/sex-matched healthy
  Sjögren's syndrome	Serum, Salivary gland	IL-40, SSA/SSB antibodies	Sicca without autoimmunity

Recent research demonstrates elevated IL-40 levels in RA that decrease following B-cell depletion therapy, suggesting IL-40 as a potential biomarker for treatment response .

What strategies should researchers employ when developing new detection methods for C17orf99/IL-40?

When developing new C17orf99/IL-40 detection methods:

• Epitope mapping and antibody development:

  • Target multiple epitopes across the protein sequence

  • Evaluate antibody pairs that recognize different regions for sandwich ELISA development

  • Develop monoclonal antibodies with high specificity and sensitivity

• Assay optimization parameters:

  • Detection range: Optimize to cover physiological and pathological ranges

  • Sample matrix effects: Validate in serum, plasma, cell culture supernatants

  • Stability: Assess analyte stability under various storage conditions

  • Cross-reactivity: Test against related cytokines and proteins

• Novel detection technologies:

  • Digital ELISA platforms for ultrasensitive detection

  • Aptamer-based biosensors for real-time monitoring

  • Mass spectrometry-based targeted proteomics approaches

How can researchers effectively study the relationship between C17orf99/IL-40 and gut microbiome alterations?

To investigate C17orf99/IL-40 and gut microbiome relationships:

• Integrated sampling approach:

  • Paired collection of fecal samples and serum for IL-40 quantification

  • IgA-bound bacteria isolation from fecal samples

  • Intestinal tissue biopsies for local IL-40 expression analysis

  • Luminal content collection for secretory IgA measurement

• Microbiome analysis techniques:

  • 16S rRNA sequencing for bacterial composition

  • Metagenomics for functional pathway analysis

  • Metabolomics to identify microbial metabolites affected by IL-40/IgA changes

  • FISH combined with flow cytometry for targeted bacterial group analysis

• Experimental models:

  • Gnotobiotic mice with defined microbial communities

  • Fecal microbiota transplantation between C17orf99-/- and wild-type mice

  • Ex vivo intestinal organoid co-culture with microbiota

Research with C17orf99-/- mice has demonstrated altered microbiome composition, likely resulting from reduced IgA levels in the gut, highlighting the functional importance of this cytokine in host-microbiome interactions .

What are the optimal sample preparation methods for C17orf99/IL-40 detection in different tissues?

For optimal C17orf99/IL-40 detection across tissue types:

• Tissue-specific preparation protocols:

  Tissue Type	Fixation	Antigen Retrieval	Blocking	Antibody Dilution
  Liver tissue	10% formalin, 24h	Heat-mediated, citrate buffer pH 6.0	5% normal serum	1:150
  Lymphoid tissue	10% formalin, 12-24h	Heat-mediated, EDTA buffer pH 9.0	10% normal serum	1:50-1:100
  Cell lines	4% PFA, 10 min	0.1% Triton X-100, 10 min	3% BSA	1-4 μg/ml
  Bone marrow	Fresh-frozen	Acetone, -20°C, 10 min	5% BSA	1:100

• Sample collection considerations:

  • Process tissues rapidly to preserve labile cytokines

  • For serum/plasma, use protease inhibitors and process within 2 hours

  • For cell preparations, maintain at 4°C throughout processing

  • For bone marrow samples, analyze immediately as C17orf99 expression decreases rapidly in culture

• Preservation of phosphorylation states:

  • Use phosphatase inhibitors in lysis buffers

  • Rapid fixation for phospho-flow cytometry analysis

  • Flash freezing for proteomics applications

How can researchers design multiplex assays that include C17orf99/IL-40 detection?

For designing multiplex assays incorporating C17orf99/IL-40:

• Antibody compatibility assessment:

  • Screen for cross-reactivity between detection antibodies

  • Optimize antibody concentrations to prevent interference

  • Validate detection in mixed standard curves

• Assay format selection:

  • Bead-based multiplex systems (Luminex) for serum/plasma samples

  • Multiplex ELISA arrays for limited sample volume

  • Digital ELISA platforms for ultrasensitive detection

  • Flow cytometry-based cytokine detection for cellular sources

• Validation requirements:

  • Single-analyte standard curves vs. multiplex format

  • Spike-recovery in complex matrices

  • Dilution linearity assessment

  • Comparison with established single-analyte methods

What approaches can resolve contradictory findings between C17orf99 transcript and protein expression data?

To reconcile transcript-protein expression discrepancies:

• Post-transcriptional regulation assessment:

  • miRNA prediction and validation for C17orf99 transcript

  • RNA stability assays with transcription inhibitors

  • Polysome profiling to assess translation efficiency

  • Protein half-life determination with cycloheximide chase

• Technical considerations:

  Issue	Investigation Approach	Potential Resolution
  Transcript detected, protein absent	Protein degradation during extraction	Modify buffer conditions, add protease inhibitors
  Protein detected, low transcript	Stable protein with high turnover transcript	Measure transcript stability, adjust timing
  Inconsistent ratios between samples	Cell type-specific post-transcriptional regulation	Compare transcript:protein ratios across cell types
  Antibody specificity issues	Multiple antibody validation	Epitope mapping, use multiple antibodies

• Single-cell approaches:

  • CITE-seq for simultaneous protein and transcript detection

  • Imaging mass cytometry with RNA probes and protein antibodies

  • Single-cell western blotting compared to scRNA-seq

What quality control parameters should be established for C17orf99 recombinant protein production?

For quality control of C17orf99/IL-40 recombinant protein:

• Production verification checklist:

  • Expression vector confirmation: Verify correct C17orf99 sequence

  • Protein size validation: Western blot showing ~27 kDa band

  • Purity assessment: SDS-PAGE showing ≥90% purity

  • Endotoxin testing: Limulus amebocyte lysate (LAL) assay (<1 EU/μg protein)

  • Host cell protein contamination: Host cell protein ELISA

• Stability parameters:

  • Thermal stability: Differential scanning fluorimetry

  • Freeze-thaw stability: Activity retention after cycles

  • Long-term storage: Activity at various timepoints

  • Aggregation assessment: Size exclusion chromatography

• Tag influence assessment:

  • Compare activity of differently tagged versions (His-tag, Myc-tag)

  • Assess tag cleavage effect on biological activity

  • Compare glycosylation patterns between expression systems

What experimental approaches might identify the receptor or binding partners for C17orf99/IL-40?

To identify C17orf99/IL-40 receptors and binding partners:

• Protein interaction screening methods:

  • BioID or APEX2 proximity labeling with IL-40 as bait

  • Co-immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening against immune cell cDNA libraries

  • Protein microarray screening with recombinant IL-40

• Signaling pathway analysis:

  • Phosphoproteomic analysis following IL-40 stimulation

  • Transcriptomic profiling of IL-40-treated cells

  • CRISPR screening for genes affecting IL-40 responsiveness

  • Reporter cell lines expressing different candidate receptors

• Structural biology approaches:

  • In silico modeling based on related cytokines

  • Epitope mapping using antibody binding interference

  • Hydrogen-deuterium exchange mass spectrometry

  • X-ray crystallography or cryo-EM of IL-40 complexes

How might researchers develop conditional knockout models to better understand tissue-specific C17orf99 functions?

For developing conditional C17orf99 knockout models:

• Strategic targeting approaches:

  • Floxed C17orf99 allele design targeting critical exons

  • Cell type-specific Cre lines for B cell subtypes:

    • CD19-Cre for general B cell deletion

    • Mb1-Cre for early B cell progenitor deletion

    • AID-Cre for germinal center B cell deletion

  • Inducible systems (tamoxifen-inducible CreERT2)

  • Tissue-specific promoters for mammary gland or intestinal epithelium

• Experimental design for phenotypic analysis:

  Tissue/Cell Type	Cre Driver	Key Phenotypes to Assess	Controls
  B cells	CD19-Cre	IgA production, Peyer's patches, microbiome	Cre-only, floxed-only
  Mammary epithelium	MMTV-Cre	Lactation-associated IgA	Heterozygous deletion
  Intestinal cells	Villin-Cre	Gut IgA, microbiome composition	Littermate controls
  Inducible deletion	Rosa26-CreERT2	Temporal requirements for IL-40	Vehicle-treated floxed

• Rescue approaches:

  • AAV-mediated expression of C17orf99 in knockout tissues

  • Bone marrow chimeras with wild-type and knockout cells

  • Recombinant IL-40 administration studies

What methodological approaches can identify the role of C17orf99/IL-40 in pathogen response and mucosal immunity?

To investigate C17orf99/IL-40 in pathogen response and mucosal immunity:

• Infection model design:

  • Bacterial challenges: Citrobacter rodentium, Salmonella

  • Viral challenges: Influenza, rotavirus

  • Parasitic models: Giardia, Cryptosporidium

  • Compare WT vs. C17orf99-/- mice for pathogen clearance, tissue damage, and immune response

• Human translational approaches:

  • IL-40 levels in mucosal secretions during infection

  • SNP analysis of C17orf99 in cohorts with recurrent infections

  • Ex vivo stimulation of human mucosal tissues with pathogens

  • Correlation between IL-40 levels and secretory IgA in various conditions

• Mechanistic investigations:

  Mechanism	Experimental Approach	Readouts
  IgA class switching	In vitro B cell cultures with IL-40	Flow cytometry for surface IgA, ELISPOT
  Plasma cell differentiation	Transcription factor analysis	qPCR for Blimp-1, XBP1
  Epithelial interaction	Co-culture systems	Transcriptomics, barrier function
  Antigen-specific responses	Immunization models	Antigen-specific IgA ELISA