P4H9 Antibody

What is P4H9 Antibody and what does it target?

P4H9 is a monoclonal mouse IgG3 antibody that primarily targets ITGB2 (CD18, integrin beta-2), which functions as the beta subunit of CD11a, b, and c integrins. It was originally developed using human lymphokine-activated T-cells (LAK) as the immunogen and recognizes the full-length CD18 protein . The antibody is also known by its alternate name, 60.3, and has been deposited to the Developmental Studies Hybridoma Bank (DSHB) by Wayner and Carter from the Fred Hutchinson Cancer Research Center . CD18 serves as a general marker for leukocytes, making this antibody valuable for immunological research. Interestingly, P4H9 has also been found to detect molecules on fibroblasts in response to CEACAM1-expressing cancer cells, with these P4H9-detected molecules (PDM) showing association with myofibroblast differentiation and cancer progression .

What are the recommended applications for P4H9 antibody?

P4H9 antibody has been validated for multiple research applications:

The antibody specifically binds to hematopoietic cells and has been demonstrated to inhibit leukocyte binding to ICAM . For immunostaining applications, methanol fixation is recommended for optimal results . When using this antibody for research publications, it is important to acknowledge both the hybridoma contributor and the DSHB in the Materials and Methods section .

What is the difference between P4H9 antibody and P4HB antibodies?

This distinction is crucial for researchers to understand:

P4H9 targets a cell surface adhesion molecule involved in immune cell interactions, while P4HB antibodies target a multifunctional enzyme that catalyzes the formation, breakage, and rearrangement of disulfide bonds . P4HB (Protein disulfide-isomerase) has been found to interact with LGALS9 (galectin-9) and is involved in cell migration and T cell inhibition . This difference highlights the importance of careful antibody selection based on the specific research question being addressed.

How does P4H9 antibody inhibit leukocyte binding to ICAM, and what are the implications for immunological research?

P4H9 antibody functions as a blocking antibody by binding to CD18 (integrin β2), which prevents its interaction with intercellular adhesion molecules (ICAMs). This blocking mechanism has significant research applications:

• Mechanism of inhibition: P4H9 binds to the epitope on CD18 that is critical for ICAM recognition, thereby physically preventing the CD18-ICAM interaction that mediates leukocyte adhesion to endothelial cells and other cellular substrates .

• Research applications in inflammation: By blocking this interaction, researchers can study the specific contribution of CD18-ICAM binding in various inflammatory processes. This approach helps distinguish the role of this particular adhesion pathway from other mechanisms of leukocyte recruitment.

• Experimental design considerations: When using P4H9 as a blocking antibody, researchers should include appropriate controls such as isotype-matched non-blocking antibodies to confirm specificity. Concentration titration is essential to determine the optimal blocking conditions without non-specific effects.

• Implications for disease models: This blocking function makes P4H9 valuable for studying diseases characterized by excessive leukocyte recruitment, such as inflammatory disorders, autoimmune diseases, and ischemia-reperfusion injury. Researchers can use P4H9 to determine whether therapeutic targeting of CD18-ICAM interactions might be beneficial in these conditions.

What is the relationship between P4H9-detected molecule (PDM) expression and cancer progression?

Research has revealed a significant relationship between PDM expression on fibroblasts and cancer progression:

• Association with myofibroblast differentiation: PDM expression appears to be associated with myofibroblast differentiation, suggesting its involvement in the activation of cancer-associated fibroblasts (CAFs) . This finding links PDM to the tumor microenvironment remodeling process.

• Correlation with metastasis: Multivariate analysis has shown that PDM-expressing spindle-shaped fibroblasts are an independent risk factor for both lymph node metastasis and hematogenous metastasis in colorectal cancer patients . This suggests that PDM may play a role in facilitating cancer cell invasion and metastatic spread.

• Impact on patient survival: A Kaplan-Meier survival analysis indicated that patients with PDM-expressing spindle-shaped fibroblasts had significantly shorter survival times (P<0.0001) . This makes PDM expression a potential prognostic biomarker in cancer evaluation.

• Experimental evidence: Immunofluorescence studies have demonstrated PDM expression on CCD-18Co fibroblasts and colorectal cancer cell lines (HCT116 and HCT-15) , indicating that PDM is present on both stromal and cancer cells in the tumor microenvironment.

These findings suggest that P4H9 antibody could be utilized not only as a research tool but potentially as a prognostic marker in cancer pathology, helping to identify patients at higher risk of metastasis and poor outcomes.

How can researchers optimize P4H9 antibody for immunohistochemistry applications?

For optimal immunohistochemistry results with P4H9 antibody, researchers should consider the following protocol optimizations:

• Fixation protocol: According to depositor notes, methanol fixation is specifically recommended for immunostaining with P4H9 . This differs from common formalin fixation and may require protocol adjustments. The methanol fixation helps preserve the epitope recognized by P4H9 while maintaining tissue morphology.

• Antigen retrieval considerations: If using formalin-fixed tissues despite the recommendation for methanol fixation, researchers may need to employ specific antigen retrieval methods to expose the epitope. Heat-induced epitope retrieval in citrate buffer (pH 6.0) can be tested as a starting point.

• Blocking and antibody dilution: Given that P4H9 is a mouse IgG3 isotype antibody, researchers should use appropriate blocking agents to prevent non-specific binding. A titration experiment should be performed to determine the optimal antibody concentration that provides specific staining with minimal background.

• Detection system selection: For mouse primary antibodies on human tissues, specialized detection systems that reduce endogenous human anti-mouse antibody (HAMA) interference may be beneficial. Polymer-based detection systems can provide enhanced sensitivity with reduced background.

• Positive and negative controls: Include known CD18-positive tissues (e.g., lymphoid tissues) as positive controls and CD18-negative tissues as negative controls. An isotype-matched non-specific antibody control should also be included to confirm staining specificity.

What experimental approaches can be used to study the functional significance of PDM in cancer progression?

Several experimental approaches can be employed to investigate the role of PDM in cancer progression:

• Co-culture systems: Researchers can establish co-culture systems of cancer cells with fibroblasts to study how PDM-expressing fibroblasts influence cancer cell behavior. Parameters to measure include:

  • Cancer cell proliferation

  • Migration and invasion

  • Expression of epithelial-mesenchymal transition markers

  • Resistance to apoptosis

• 3D organoid models: Creating 3D organoid models incorporating PDM-expressing fibroblasts can provide insights into the three-dimensional interactions within the tumor microenvironment. This approach more closely mimics in vivo conditions compared to traditional 2D cultures.

• Functional blocking experiments: Utilizing P4H9 as a blocking antibody in both in vitro and in vivo models to determine whether interfering with PDM function affects cancer progression. Key endpoints include:

  • Tumor growth rate

  • Metastatic potential

  • Survival in animal models

• Molecular identification of PDM: Advanced techniques such as immunoprecipitation followed by mass spectrometry can help identify the molecular nature of PDM, which remains incompletely characterized. This would enable more targeted studies of its function.

• Gene expression analysis: Comparing gene expression profiles between PDM-positive and PDM-negative fibroblasts to identify associated pathways and potential therapeutic targets. RNA sequencing of isolated populations can provide comprehensive insights.

These approaches would provide mechanistic understanding of how PDM contributes to cancer progression and might identify new therapeutic strategies targeting the cancer-fibroblast interaction.

How does P4H9 antibody compare with other antibodies targeting CD18 in terms of specificity and applications?

When selecting an antibody for CD18 research, it's important to understand how P4H9 compares with alternatives:

What is the relationship between PDM detected by P4H9 and other markers of cancer-associated fibroblasts?

Understanding how PDM relates to established CAF markers can provide insights into its role in the tumor microenvironment:

Understanding these relationships would not only advance our knowledge of tumor biology but could also identify new therapeutic targets within the tumor microenvironment.