PSG11 Antibody

What is PSG11 and what biological functions does it serve?

PSG11 (Pregnancy-specific beta-1-glycoprotein 11) belongs to the immunoglobulin superfamily and is a member of the CEA (carcinoembryonic antigen) family. It is primarily produced by placental syncytiotrophoblasts during pregnancy and serves critical immunomodulatory functions. PSG11 acts by inducing dose-dependent secretion of anti-inflammatory cytokines by human monocytes, thereby promoting maternal-fetal tolerance. This protein plays a significant role in regulating immune responses during pregnancy, making it a potential target for research into pregnancy complications including pregnancy loss, preeclampsia, and fetal growth restriction .

How does PSG11 differ from other pregnancy-specific glycoproteins?

While PSG11 shares structural similarities with other PSG family members, it has distinct characteristics. PSG11 is also known by multiple synonyms including PSG13, PSG14, PSBG-11, and PSBG-13. Its unique function in immune modulation during pregnancy distinguishes it from other family members. PSG11 contains specific domain structures, with epitope mapping studies showing that monoclonal antibodies like mAb PSG.11 specifically recognize the A1 domain of the protein. Cross-reactivity studies have demonstrated that antibodies raised against PSG1 may detect PSG11 due to sequence homology, but with different patterns of recognition compared to other PSG family members .

What are the key structural features of PSG11 that researchers should be aware of?

PSG11 belongs to the immunoglobulin superfamily with characteristic domain organization. It contains multiple immunoglobulin-like domains that contribute to its function. The protein has a molecular weight of approximately 37,146 Da. PSG11 is a secreted protein containing a signal peptide for secretion, and is primarily localized to the extracellular region. The protein's structural integrity is essential for its biological activity, including its ability to induce cytokine secretion. When designing experiments, researchers should consider that the A1 domain appears to be particularly important for antibody recognition, as demonstrated by epitope mapping studies with monoclonal antibodies .

What are the validated applications for PSG11 antibodies in research?

PSG11 antibodies have been validated for multiple research applications with specific methodological considerations for each technique:

• Western Blotting (WB): PSG11 antibodies like PACO11520 can detect the protein at approximately 37-60 kDa. Recommended dilutions should be followed according to manufacturer specifications, typically in the range of 1:500-1:1000.

• Immunohistochemistry (IHC): These antibodies have been validated for detection of PSG11 in fixed tissue sections, particularly for placental tissues and potentially in tumor samples.

• ELISA: Both as primary antibodies in sandwich ELISA kits and for developing custom assays for detection of PSG11 in various biological samples.

Researchers should note that PSG11 antibodies have been particularly useful in studying pregnancy-related conditions and potentially as tumor biomarkers .

How should researchers optimize sandwich ELISA protocols for PSG11 detection?

For optimal PSG11 detection using sandwich ELISA, researchers should follow these methodological considerations:

• Sample preparation: Proper dilution of samples is critical, with recovery tests showing optimal performance in the range of 85-102% for serum and plasma samples.

• Incubation conditions: The standard protocol requires 90 minutes of static incubation at 37°C for the sample, followed by 60 minutes for biotin-antibody working solution, and 30 minutes for HRP-Streptavidin Conjugate.

• Washing steps: Thorough washing between steps is essential, with three wash cycles recommended after the addition of biotin-antibody.

• Linearity considerations: Sample dilutions of 1:2, 1:4, and 1:8 have demonstrated good linearity in the range of 80-105% for serum and plasma samples.

• Quality control: Intra-assay precision typically shows CV% of approximately 4.68-5.26%, while inter-assay precision ranges from 3.15-6.24%, indicating good reproducibility of results .

What sample types are suitable for PSG11 detection, and how should they be prepared?

PSG11 can be detected in various biological samples, each requiring specific preparation protocols:

• Serum: Collection in standard serum separator tubes followed by clotting for 2 hours at room temperature or overnight at 4°C, then centrifugation. Recovery rates average around 96% for serum samples.

• EDTA plasma: Collection in EDTA-treated tubes followed by centrifugation within 30 minutes. Recovery rates average around 96% for EDTA plasma.

• Heparin plasma: Collection in heparin-treated tubes followed by centrifugation. Recovery rates average around 94% for heparin plasma.

For all sample types, proper storage at -80°C is recommended to maintain protein integrity. Avoid repeated freeze-thaw cycles as this may affect PSG11 stability. Hemolyzed or lipemic samples should be avoided as they may interfere with assay performance .

How can PSG11 antibodies be used to investigate the relationship between PSG11 and inflammatory pathways?

PSG11 antibodies can be valuable tools for studying inflammatory pathways through several methodological approaches:

• Co-culture experiments: Researchers can use neutralizing PSG11 antibodies in co-culture systems of trophoblasts with immune cells to assess the direct impact of PSG11 blockade on cytokine production.

• Immunoprecipitation followed by mass spectrometry: This approach can identify binding partners of PSG11 in the inflammation cascade.

• ChIP assays: For investigating transcriptional regulation of inflammatory genes in response to PSG11 signaling.

• Cytokine profiling: Using antibody-based depletion of PSG11 followed by multiplex cytokine assays to determine the specific cytokine signature influenced by PSG11.

These approaches can help elucidate the molecular mechanisms through which PSG11 induces dose-dependent secretion of anti-inflammatory cytokines from monocytes .

How can researchers distinguish between the functions of different PSG family members when antibody cross-reactivity exists?

Addressing the challenge of PSG family cross-reactivity requires sophisticated experimental design:

• RNA interference approach: Use siRNA or shRNA specifically targeting PSG11 mRNA, followed by antibody detection of remaining protein levels.

• CRISPR/Cas9 gene editing: Generate cell lines with specific PSG11 knockout while leaving other family members intact, then validate with antibodies.

• Domain-specific competitive inhibition: Utilize recombinant fragments of PSG11-specific domains to competitively block antibody binding in functional assays.

• Mass spectrometry validation: Combine immunoprecipitation with PSG11 antibodies followed by mass spectrometry to verify the specific PSG variant being detected.

• Combinatorial antibody approach: Use multiple antibodies targeting different epitopes, creating a detection profile that can distinguish between family members based on domain composition differences .

What are the important considerations when using PSG11 antibodies to investigate pregnancy complications?

When investigating pregnancy complications using PSG11 antibodies, researchers should consider:

• Gestational age-specific reference ranges: PSG11 expression varies throughout pregnancy, necessitating appropriate timing controls.

• Maternal-fetal interface sampling strategy: Consider the spatial heterogeneity of placental tissue when collecting samples for immunohistochemistry.

• Pathology-specific modifications: PSG11 may undergo post-translational modifications in pathological conditions that could affect antibody binding.

• Concurrent analysis of multiple PSG family members: Use a panel approach to detect shifts in the relative abundance of different PSG proteins in complicated pregnancies.

• Integration with clinical parameters: Correlate antibody-based PSG11 measurements with clinical outcomes and biomarkers of placental function.

This comprehensive approach can provide insights into how altered PSG11 expression contributes to conditions such as preeclampsia and fetal growth restriction .

What methodological approaches can be used to study the role of PSG11 in tumor biology?

To investigate PSG11's potential role in tumor biology, researchers can employ:

• Tissue microarray analysis: Use PSG11 antibodies on multi-tumor arrays to establish expression patterns across cancer types with appropriate controls to distinguish from CEACAMs.

• Cell line models: Transfect cancer cell lines with PSG11 expression constructs and use antibodies to confirm expression before functional studies.

• Xenograft models: Analyze PSG11 expression in tumor xenografts using immunohistochemistry, correlating with tumor progression metrics.

• Secretome analysis: Use antibody-based capture methods to isolate PSG11 from tumor cell secretomes for proteomic characterization.

• Circulating PSG11 detection: Develop sensitive ELISA protocols to quantify PSG11 in patient serum as a potential biomarker.

These approaches can help determine whether ectopic PSG11 expression in tumors serves as a biomarker or contributes functionally to tumor progression .

How can researchers investigate post-translational modifications of PSG11 using available antibodies?

To study post-translational modifications (PTMs) of PSG11, researchers should consider:

• Combined immunoprecipitation and PTM-specific detection: Use PSG11 antibodies for immunoprecipitation followed by western blotting with antibodies specific for glycosylation, phosphorylation, or other modifications.

• Enzymatic deglycosylation studies: Treat samples with PNGase F or other glycosidases before antibody detection to assess the contribution of glycans to epitope recognition.

• Mass spectrometry after immunopurification: Purify PSG11 using antibody-based methods followed by mass spectrometry to map specific modification sites.

• Site-directed mutagenesis: Generate PSG11 variants with mutations at potential modification sites and assess antibody binding to determine the impact of specific PTMs.

• Comparison across physiological states: Compare PTM patterns using antibody-based methods in normal pregnancy versus pathological conditions to identify clinically relevant modifications .

What are common issues with Western blot detection of PSG11 and how can they be resolved?

Researchers often encounter several challenges when detecting PSG11 by Western blot:

• Multiple bands or smearing: This may indicate glycosylation heterogeneity of PSG11. Treatment with deglycosylation enzymes before Western blotting can help resolve this issue.

• Cross-reactivity with other PSG family members: Use blocking peptides specific to PSG11 epitopes to confirm specificity. Additionally, include recombinant PSG proteins as controls.

• Weak signal: Optimize antibody concentration, increase protein loading, or enhance detection with signal amplification systems. Extended exposure times may also help for low abundance samples.

• High background: Increase blocking agent concentration, optimize washing steps, and dilute primary antibody appropriately. Pre-absorption of antibody with non-specific proteins may reduce background.

• Variability between experiments: Implement standardized loading controls and consider using automated Western blot systems for better reproducibility .

What control samples should be included when using PSG11 antibodies in experimental design?

For robust experimental design with PSG11 antibodies, the following controls should be included:

• Positive controls:

  • Placental tissue extracts from different trimesters

  • Recombinant PSG11 protein at known concentrations

  • Cell lines transfected to overexpress PSG11

• Negative controls:

  • Tissues known not to express PSGs in normal conditions

  • Isotype-matched control antibodies

  • Pre-immune serum (for polyclonal antibodies)

• Specificity controls:

  • Samples containing other PSG family members to assess cross-reactivity

  • CEACAM-expressing cell lines to confirm lack of cross-reactivity

  • Competitive inhibition with recombinant PSG11

• Technical controls:

  • Loading controls appropriate for the sample type

  • Internal reference standards for quantitative assays

  • Replicate samples to assess assay precision .

How can researchers validate the specificity of PSG11 antibody staining in immunohistochemistry applications?

To validate PSG11 antibody specificity in immunohistochemistry:

• Peptide competition: Pre-incubate the antibody with excess PSG11 recombinant protein or immunizing peptide before application to serial tissue sections. Specific staining should be abolished.

• Multiple antibody approach: Use different antibodies targeting distinct epitopes of PSG11. Concordant staining patterns increase confidence in specificity.

• RNAscope or RNA in situ hybridization correlation: Compare protein detection with mRNA localization to confirm expression patterns.

• Knockout/knockdown validation: Use tissues from models with PSG11 gene manipulation or siRNA-treated samples as negative controls.

• Comparison with normal expression patterns: Include placental tissues (known to express PSG11) as positive controls and non-pregnant tissues as negative controls.

• Serial dilution testing: Perform antibody titration to identify the optimal concentration that maximizes specific staining while minimizing background .

What are the stability considerations for PSG11 in different sample storage conditions?

PSG11 stability varies across storage conditions, with important implications for experimental design:

To maximize PSG11 stability:

• Add protease inhibitors to samples immediately after collection.

• Store in buffer containing stabilizing agents (e.g., glycerol).

• Aliquot samples to avoid repeated freeze-thaw cycles.

• For long-term storage, use -80°C freezers rather than -20°C.

• For ELISA kit components, follow manufacturer guidelines for storage temperature and reconstitution protocols .

How do different commercially available PSG11 antibodies compare in terms of sensitivity and specificity?

When comparing PSG11 antibodies from different sources, researchers should consider these performance characteristics:

The choice between these options should be guided by the specific research question. For general detection, polyclonal antibodies offer greater sensitivity across applications. For discriminating between closely related PSG family members, domain-specific monoclonal antibodies provide higher specificity but potentially lower sensitivity .

What emerging research areas might benefit from PSG11 antibody-based investigations?

Several cutting-edge research areas could benefit from PSG11 antibody applications:

• Extracellular vesicle (EV) biology: Investigating PSG11 packaging in placenta-derived EVs and their immunomodulatory effects using antibody-based capture and characterization.

• Single-cell protein analysis: Combining PSG11 antibodies with mass cytometry or imaging mass cytometry to map PSG11 expression at the single-cell level within the maternal-fetal interface.

• Spatial transcriptomics correlation: Integrating PSG11 antibody-based protein detection with spatial transcriptomics to create comprehensive maps of expression and function.

• Liquid biopsy development: Using highly sensitive PSG11 antibodies to detect circulating PSG11 as a non-invasive biomarker for pregnancy complications or certain cancers.

• Therapeutic antibody development: Engineering antibodies against specific PSG11 epitopes for potential therapeutic applications in pregnancy complications or cancer .

How can researchers interpret discrepancies between PSG11 antibody-based protein detection and mRNA expression data?

When faced with discordant protein and mRNA data for PSG11, researchers should consider:

• Post-transcriptional regulation: PSG11 may be subject to microRNA regulation or RNA binding protein interactions affecting translation efficiency. Validate with ribosome profiling or polysome analysis.

• Protein stability differences: PSG11 protein half-life may vary across cellular contexts. Pulse-chase experiments using antibody detection can quantify these differences.

• Secretion dynamics: As a secreted protein, intracellular levels of PSG11 may not correlate with mRNA if secretion rates vary. Compare intracellular and secreted fractions using antibody-based methods.

• Alternative splicing: PSG11 has multiple isoforms that may not all be detected by a single antibody. Use isoform-specific primers for RT-PCR and compare with protein detection patterns.

• Technical considerations: Different sensitivities between RT-PCR and antibody-based methods may explain apparent discrepancies. Perform absolute quantification of both mRNA and protein using calibrated standards .