PTX3 Antibody

What is PTX3 and why is it significant for immunological research?

Pentraxin 3 (PTX3), also known as TNF-inducible gene 14 protein (TSG-14), is a member of the pentraxin superfamily characterized by a structural motif called the pentraxin domain. Unlike short pentraxins (CRP, SAP) produced primarily in the liver, PTX3 is a long pentraxin produced by various cell types including endothelial cells, smooth muscle cells, adipocytes, fibroblasts, mononuclear phagocytes, and dendritic cells . PTX3 functions as a soluble pattern recognition receptor in the humoral innate immune system with ancestral antibody-like properties, making it significant for studying the intersection between innate and adaptive immunity . It plays crucial roles in pathogen recognition, complement activation, inflammation regulation, and tissue repair processes .

PTX3 has a calculated molecular weight of 42 kDa (381 amino acids), but the observed molecular weight typically ranges between 40-45 kDa in Western blot applications . This variation may occur due to post-translational modifications, particularly glycosylation. When analyzing PTX3 by Western blot under reducing conditions, researchers should expect to see bands within this range. Under non-reducing conditions, higher molecular weight bands may be observed due to PTX3's ability to form multimeric structures through disulfide bonds .

What are the optimal conditions for using PTX3 antibodies in various experimental applications?

Based on published data, the following conditions are recommended for PTX3 antibody applications:

Note that optimal conditions may vary between antibody clones and should be titrated for each specific experimental system .

How should PTX3 antibodies be stored and handled to maintain optimal activity?

Most commercially available PTX3 antibodies are stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . These antibodies should be stored at -20°C where they remain stable for approximately one year after shipment. Aliquoting is generally unnecessary for -20°C storage with this buffer composition. Some antibody preparations may contain 0.1% BSA for stabilization.

When working with PTX3 antibodies, avoid repeated freeze-thaw cycles and exposure to strong light. For immunoprecipitation applications, crosslinking the antibody to protein G beads is recommended to prevent antibody co-elution with the target protein .

What controls should be included when using PTX3 antibodies in research?

To ensure experimental validity when using PTX3 antibodies, the following controls are essential:

• Positive controls: HUVEC cells, HeLa cells, or HepG2 cells (for Western blot); HepG2 cells (for immunofluorescence)

• Negative controls: PTX3 knockout/knockdown samples (available from publications showing successful PTX3 targeting)

• Specificity controls: Blocking peptide competition experiments or pre-adsorption with recombinant PTX3 protein

• Isotype controls: Matched rabbit or mouse IgG (depending on antibody host species)

For ELISA applications measuring PTX3 in patient samples, healthy donor samples should be included as reference controls, with normal plasma levels typically below 2 ng/mL .

How can I differentiate between membrane-bound, intracellular, and secreted PTX3 in my experiments?

PTX3 exists in multiple cellular compartments, requiring specific approaches to distinguish its various forms:

• Secreted PTX3: Measure in cell culture supernatants or biological fluids using ELISA. Neutrophils store preformed PTX3 in specific granules and rapidly release it upon stimulation . Collect supernatants at early time points (15-60 minutes) post-stimulation to capture this rapid release.

• Intracellular PTX3: Use permeabilizing agents (0.1% Triton X-100 or saponin) for flow cytometry or immunofluorescence. Confocal microscopy has shown PTX3 in the cytoplasm of CLL cells and neutrophil granules .

• Membrane-bound PTX3: For flow cytometry, compare permeabilized versus non-permeabilized samples. For distinguishing truly membrane-associated PTX3 from passively adsorbed protein, perform acid wash (pH 3.0 glycine buffer) before staining .

In neutrophils specifically, PTX3 has a unique localization pattern, being stored in neutrophil extracellular traps (NETs) along with other antimicrobial proteins like azurocidin 1 .

What are the known complexes and interacting partners of PTX3 that might affect antibody detection?

PTX3 forms various complexes that may influence antibody detection and experimental interpretation:

When designing experiments to detect PTX3-protein interactions, consider calcium dependence, as many PTX3 interactions require calcium ions. Using EDTA in buffers may disrupt certain complexes while preserving others .

How should I interpret contradictory results between PTX3 protein levels and gene expression data?

Discrepancies between PTX3 protein levels and gene expression may arise from several factors:

• Post-transcriptional regulation: PTX3 expression is regulated by microRNAs and RNA-binding proteins that affect mRNA stability and translation efficiency.

• Protein storage and release: Unlike most proteins, PTX3 can be stored preformed in neutrophil granules and rapidly released without new gene transcription . Thus, high protein levels may appear without corresponding mRNA increases.

• STAT3-dependent transcription: In certain cell types like CLL cells, PTX3 transcription is directly controlled by STAT3. Phosphorylated STAT3 binds to the PTX3 gene promoter to activate transcription . Differences in STAT3 activation may explain discrepancies.

• Protein half-life considerations: PTX3 has a plasma half-life of approximately 6-7 hours . Sample timing relative to stimulation may show declining protein levels while mRNA has already returned to baseline.

When encountering contradictory results, consider analyzing both intracellular and secreted PTX3, as well as examining multiple time points post-stimulation to capture the full dynamics of expression and release.

How can PTX3 antibodies be utilized to study the intersection between innate and adaptive immunity?

PTX3 represents a fascinating link between innate and adaptive immunity, and antibodies against PTX3 can help investigate this intersection:

• Marginal zone B cell interactions: PTX3 binds to splenic marginal zone B cells, promoting their differentiation and antibody production. Using fluorescently-labeled PTX3 with flow cytometry, researchers have shown that PTX3 binding to these cells increases upon exposure to neutrophils primed with GM-CSF and LPS or to CpG-rich DNA .

• Class switching mechanisms: PTX3 promotes class switching from IgM to IgG. This process can be tracked using PTX3 antibodies in combination with B cell markers to monitor the development of plasmablasts and plasma cells in response to PTX3 stimulation .

• Neutrophil-B cell crosstalk: A subset of neutrophils surrounding the splenic marginal zone express PTX3 with an immune activation gene signature distinct from circulating neutrophils. Co-immunoprecipitation and proximity ligation assays using PTX3 antibodies can help map these cellular interactions .

This research direction may provide insights into developing more effective vaccines against encapsulated pathogens by harnessing PTX3's endogenous adjuvant properties for marginal zone B cells .

What is the significance of anti-PTX3 autoantibodies in autoimmune diseases, and how are they detected?

Anti-PTX3 autoantibodies have been detected in several autoimmune diseases, with important clinical implications:

Interestingly, anti-PTX3 autoantibodies in SLE appear to have a protective effect against lupus nephritis. Studies have shown that patients with these autoantibodies have lower proteinuria, serum creatinine, and renal fibrosis compared to antibody-negative patients . Lupus-prone mice immunized with PTX3 produce anti-PTX3 antibodies and show delayed occurrence of nephritogenic antibodies, decreased proteinuria, and increased survival .

To study these autoantibodies, researchers typically use an in-house ELISA method where plates are coated with recombinant PTX3, followed by incubation with patient sera and detection with anti-human IgG secondary antibodies .

How can PTX3 antibodies be applied to cancer research and potential therapeutic development?

PTX3 plays complex roles in cancer biology, with both pro- and anti-tumorigenic effects depending on cancer type. PTX3 antibodies can be valuable tools in this research area:

For cancer studies, it's important to note that the STAT3 transcription factor directly binds to the PTX3 gene promoter in certain cancer cells (like CLL), activating its transcription. This mechanism may explain the elevated PTX3 levels observed in some cancers .

How can I optimize detection of PTX3 in clinical samples for biomarker studies?

Optimizing PTX3 detection in clinical samples requires addressing several technical challenges:

• Sample collection timing: PTX3 levels rise rapidly (within hours) in acute conditions like myocardial infarction, sepsis, or vasculitis, peaking earlier than traditional inflammatory markers like CRP. In acute myocardial infarction, PTX3 peaks at approximately 7.5 hours after CCU admission, while CRP peaks around 24 hours . Serial sampling at multiple time points is advised.

• Sample type considerations:

  • Plasma vs. Serum: Both are suitable, but EDTA plasma may preserve certain PTX3 complexes differently than serum or heparin plasma

  • Urinary PTX3: For kidney-related conditions like ANCA-associated vasculitis, urinary PTX3 correlates with disease activity and kidney involvement

• Reference ranges and cutoffs:

  • Healthy controls: Normal plasma PTX3 is typically <2 ng/mL

  • Disease states: Levels can increase to 200-800 ng/mL in severe sepsis

  • Cutoff determination: ROC curve analysis recommended for specific clinical applications

• Interfering factors: PTX3 forms complexes with numerous plasma proteins that may mask epitopes. Pre-treatment of samples with mild detergents (0.1% Nonidet P-40) may improve detection of complexed PTX3 .

For longitudinal studies, standardization of sample collection, processing time, and storage conditions is critical to minimize pre-analytical variability.

What factors should I consider when comparing results from different PTX3 antibody clones?

When comparing results obtained with different PTX3 antibody clones, consider these factors:

• Epitope specificity: PTX3 has distinct N-terminal and C-terminal (pentraxin) domains with different functions. Antibodies targeting different domains may yield varying results:

  • N-terminal domain antibodies: May preferentially detect PTX3 involved in FGF interactions or matrix organization

  • C-terminal domain antibodies: May better detect complement-associated functions

• Detection of oligomeric forms: PTX3 naturally forms octamers through disulfide bonds. Some antibodies may preferentially recognize monomeric versus oligomeric forms.

• Cross-reactivity profiles: Check the documented species reactivity. While many PTX3 antibodies cross-react with human, mouse, and rat PTX3, the affinity may vary substantially .

• Validation methods: Review how each antibody was validated (Western blot, knockout controls, peptide competition, etc.) and under what conditions optimal performance was demonstrated .

• Batch-to-batch variation: For polyclonal antibodies particularly, significant variation can occur between production lots. Include standardized positive controls when switching antibody batches.

When publishing research using PTX3 antibodies, report the clone, catalog number, dilution, and validation performed to enable proper comparison across studies.

How do I distinguish between PTX3 and other pentraxin family members in my experiments?

Distinguishing PTX3 from other pentraxin family members requires careful experimental design:

• Sequence homology considerations: The C-terminal pentraxin domain of PTX3 shares approximately 57% amino acid identity with short pentraxins (CRP and SAP), while the N-terminal domain is unique to long pentraxins. Antibodies targeting the N-terminal domain offer greater specificity .

• Molecular weight differentiation: On Western blots, PTX3 appears at 40-45 kDa, whereas CRP and SAP are detected at approximately 25 kDa, making them easily distinguishable by size .

• Expression pattern analysis: Unlike short pentraxins predominantly produced by hepatocytes, PTX3 is produced by various cell types including endothelial cells, fibroblasts, and myeloid cells. Cell-type specific expression can help differentiate pentraxins .

• Immunodepletion strategy: For complex samples, consider sequential immunodepletion using antibodies against one pentraxin family member before testing for another.

• Gene expression verification: Complement protein detection with RT-PCR or RNA-seq targeting specific pentraxin transcripts, as their expression is regulated differently (PTX3 is induced by inflammatory cytokines like IL-1β and TNF-α, while CRP and SAP are primarily induced by IL-6) .

For multiplex analysis of pentraxin family members, carefully validated antibody panels with minimal cross-reactivity are essential, particularly in conditions like sepsis where multiple pentraxins may be elevated simultaneously .