Anti-Mouse/Human PNAd (Clone MECA-79) – Purified in vivo GOLD^TM Functional Grade

Use of MECA-79 in In Vivo Mouse Studies

The MECA-79 monoclonal antibody is a widely used tool in immunology research, especially for studying lymphocyte homing and the biology of high endothelial venules (HEVs) in mice.

Target and Specificity

• MECA-79 recognizes Peripheral Node Addressin (PNAd), a carbohydrate modification present on HEVs in secondary lymphoid organs (like lymph nodes) and on HEV-like vessels induced at sites of chronic inflammation in mice.
• It specifically stains HEVs in lymph nodes and detects functionally active L-selectin ligands, which are critical for lymphocyte homing to lymphoid tissues.

Functional Applications

• Blocking Lymphocyte Homing: In vivo, MECA-79 antibody has been shown to block the homing of lymphocytes to peripheral lymph nodes by inhibiting L-selectin-dependent adhesion—a key step in the extravasation of lymphocytes from blood into lymphoid tissues.
• Inflammation Models: MECA-79 staining is not limited to physiological HEVs; it also identifies HEV-like vessels that are induced at sites of chronic inflammation (e.g., in autoimmune diseases like rheumatoid arthritis or in Helicobacter pylori-induced gastritis). This allows researchers to study ectopic HEV formation and lymphocyte recruitment during inflammation.
• Gene Targeting: Transgenic mouse models (e.g., GlcNAc6ST-2-Cre mice) use MECA-79 reactivity as a marker for mature HEVs. Crossing these with reporter strains enables conditional gene manipulation specifically in MECA-79-positive HEVs, facilitating the study of gene function in these specialized vessels and in inflammation-induced HEV-like structures.

Validation and Controls

• Specificity: MECA-79 reactivity is a hallmark of functional HEVs, and its expression marks a developmental switch from immature to mature HEVs in neonatal lymph nodes.
• Negative Controls: Some tissues (e.g., colonic villi in certain studies) do not react with MECA-79, helping to distinguish HEV-specific processes from general vascular biology.

Summary Table: Key Uses of MECA-79 in Mouse Studies

Conclusion

MECA-79 is indispensable for identifying, characterizing, and functionally manipulating HEVs in mice. It is used in vivo to block lymphocyte homing, mark sites of inflammation-induced vascular remodeling, and enable precise genetic studies in mature HEVs. Its application spans basic immunology, inflammation research, and the development of targeted therapies.

The correct storage temperature for sterile packaged clone MECA-79 (an anti-PNAd antibody) is 2–8°C for standard use and short-term storage. For long-term storage (longer than a month), most suppliers recommend aliquoting aseptically and storing at –20°C or –80°C.

Key manufacturer recommendations:

• Leinco: Store sterile as received at 2–8°C for up to one month. For longer term, aliquot and store at –80°C.
• Merck Millipore: Stable for 1 year at 2–8°C from receipt.
• BioLegend: Store undiluted between 2–8°C. Do not freeze (for the biotin-conjugated version). The purified form is also handled at 4°C.
• Bio-Techne: Store at 4°C short term. Aliquot and store at –20°C long term, avoid freeze-thaw cycles.

Summary Table:
| Supplier | Short-Term Storage | Long-Term Storage | Notes ||------------------|-------------------|--------------------|-----------------------------|| Leinco | 2–8°C (?1 month) | –80°C (aliquots) | Avoid repeated freeze-thaw || Merck Millipore | 2–8°C (?1 year) | N/A | — || BioLegend | 2–8°C | Do not freeze | Biotinylated forms || Bio-Techne | 4°C | –20°C (aliquots) | Avoid repeated freeze-thaw |

For most standard use cases, store at 2–8°C and follow product-specific guidance on long-term storage and freeze-thaw cycle avoidance. Always check the datasheet accompanying your specific lot, as formulation and recommendations may vary by supplier and antibody conjugate.

Antibodies and Proteins Commonly Used with MECA-79 in Scientific Literature

MECA-79 is a monoclonal antibody that recognizes the peripheral node addressin (PNAd), a marker present on high endothelial venules (HEVs) of lymph nodes. It is widely used to study lymphocyte trafficking, lymphoid tissue organization, and targeted drug delivery. In research, MECA-79 is often paired with other antibodies or proteins to provide additional information about cellular markers, validate targeting, or investigate functional mechanisms.

Commonly Paired Antibodies

Anti-IgM is frequently used alongside MECA-79 to identify and distinguish B lymphocytes in tissues such as Peyer’s patches and the appendix. Immunofluorescence co-staining with anti-rabbit IgM and MECA-79 allows visualization of B cells expressing the MECA-79 epitope, distinguishing them from other cell types in both follicular and interfollicular regions.

Isotype control antibodies (e.g., rat IgM isotype control for MECA-79) are used to verify staining specificity and rule out nonspecific antibody binding, which is essential for interpreting results in histology and flow cytometry experiments.

Anti-CD3 and anti-CD28 are used in functional assays to stimulate T cell activation, where the immunosuppressive effect of MECA-79-targeted drug-loaded particles can be measured by monitoring T cell proliferation and cytokine production (e.g., IL-2, IFN-?, IL-6, IL-17).

Proteins and Enzymes

O-sialoglycoprotease is employed in biochemical studies to determine whether the MECA-79 epitope is sensitive to enzymatic cleavage, confirming that the antigen is an O-sialoglycoprotein and providing insight into its post-translational modifications.

Sodium chlorate is used as a metabolic inhibitor to inhibit protein sulfation, which is critical for PNAd structure. This helps confirm that the MECA-79 epitope is sulfation-dependent.

LSST-2 (HEC-GlcNAc6ST), a sulfotransferase enzyme, is studied in relation to MECA-79 because it is involved in the biosynthesis of the MECA-79 antigen. Antibodies against LSST-2 are used in parallel with MECA-79 to link enzyme expression to the generation of the MECA-79 epitope on HEVs.

Summary Table

Additional Notes

• MECA-79 is often used in animal models (e.g., heart or skin transplantation) to track targeted drug delivery to lymph nodes, and in these contexts is paired with markers specific to the organ or disease model (e.g., allograft tracking agents).
• Flow cytometry and immunofluorescence are common techniques where MECA-79 is combined with other lineage-specific or functional markers to provide a comprehensive view of cellular subsets and trafficking patterns.

In summary, MECA-79 is most commonly paired with anti-IgM for B cell identification, isotype controls for specificity, anti-CD3/CD28 for functional assays, O-sialoglycoprotease and sodium chlorate for biochemical characterization, and anti-LSST-2 for studies on the biosynthesis of the MECA-79 epitope.

Key Findings from Clone MECA-79 in Scientific Literature

The monoclonal antibody MECA-79 is widely recognized for its ability to detect and characterize vascular addressins, particularly peripheral node addressin (PNAd), a group of sialomucins expressed on high endothelial venules (HEVs) in lymph nodes and inflamed tissues. Below are the most significant findings associated with MECA-79 citations in the literature, organized by topic.

Molecular Basis of MECA-79 Recognition

• Epitope Specificity: MECA-79 recognizes a carbohydrate structure known as 6-sulfo N-acetyl-lactosamine (6-sulfo LacNAc) on the extended core-1 branch of O-glycans. This epitope is present on the PNAd family of glycoproteins, including the sialyl 6-sulfo Lewis X terminus, but binding specifically requires GlcNAc-6-sulfation and does not require sialylation or fucosylation.
• Enzyme Sensitivity: The MECA-79-reactive epitope is sensitive to O-sialoglycoprotease, indicating it is carried on O-glycosylated mucin-type glycoproteins. Enzyme treatment abolishes MECA-79 staining on B cells but not IgM staining, suggesting that the MECA-79 antigen is a distinct O-sialoglycoprotein.
• Sulfation Dependency: MECA-79 staining is abolished by treatment with sodium chlorate, a sulfation inhibitor, confirming the critical role of sulfation in forming its carbohydrate epitope.

Functional and Cellular Insights

• Lymphocyte Homing: MECA-79 is a powerful tool for studying lymphocyte migration, as it stains HEVs in lymph nodes and at sites of chronic inflammation. Importantly, MECA-79 antibody blocks L-selectin-dependent lymphocyte attachment both in vitro and in vivo, and inhibits normal lymphocyte homing to peripheral lymph nodes in mouse models.
• HEV Marker: MECA-79 is routinely used to identify HEVs in tissues, and its staining correlates with the presence of functional L-selectin ligands, which are critical for lymphocyte trafficking into lymphoid organs and sites of inflammation.
• Animal Models: The antibody was generated by immunizing rats with lymphocytes from mouse lymph nodes, making it a robust reagent for both mouse and human studies.

Biochemical and Proteomic Findings

• Protein Carriers: MECA-79 recognizes a glycoprotein of approximately 127 kDa on stromal cells and B lymphocytes, as shown by Western blotting. In both mouse and human tissues, endomucin and CD34—well-known HEV markers—are decorated with the MECA-79 carbohydrate epitope, indicating their roles as PNAd carriers.
• Comparative Expression: Endomucin and CD34, affinity-isolated from human tonsils, show similar MECA-79 reactivity, suggesting they are major protein scaffolds for the MECA-79-defined carbohydrate structure in HEVs.
• Transfection Studies: Expression of the MECA-79 epitope can be induced in cultured cells by transfection with 6-sulfo-sulfotransferase (6-Sul-T), confirming the critical role of this enzyme in the biosynthesis of the MECA-79 determinant.

Pathological and Clinical Relevance

• Chronic Inflammation: MECA-79 detects HEV-like vessels at sites of chronic inflammation, where PNAd+ vessels can be induced, linking MECA-79 staining to physiological and pathological lymphocyte recruitment.
• Tumor HEVs: MECA-79 is used to identify tumor-associated HEVs, which are implicated in immune cell recruitment to tumors, and to correlate HEV density with clinical outcomes in certain cancers (e.g., urothelial carcinoma).
• Molecular Signature: MECA-79-negative HEV-like vessels can still express other selectin ligands (e.g., sialyl Lewis X), suggesting diversity in the molecular composition of HEV-like structures in different tissues and disease states.

Summary Table: Key Characteristics of MECA-79

In summary, MECA-79 is a pivotal reagent for identifying and studying the molecular and functional characteristics of HEVs, particularly in the context of lymphocyte trafficking, inflammation, and tumor immunology. Its epitope is a sulfated carbohydrate carried on specific O-glycosylated mucins, and it defines a functional subset of endothelial cells critical for immune surveillance and pathology.