Anti-Mouse PD-1 [Clone RMP1-30] — Purified in vivo GOLD™ Functional Grade

Clone RMP1-30 is a rat monoclonal antibody against mouse PD-1 (CD279) that is routinely used in in vivo mouse studies primarily for the detection and characterization of PD-1 expression by flow cytometry and immunophenotyping, but not for functional blocking of PD-1 signaling in live animals.

Key uses and details in in vivo mouse studies:

• Detection of PD-1 expression: RMP1-30 is used to identify and quantify PD-1 (CD279) on the surface of mouse T cells, B cells, and certain tumor cells via flow cytometry. It is effective for distinguishing PD-1+ versus PD-1– populations in mouse tissues and tumors. For example, RMP1-30 showed specific binding to PD-1 on both T cells and B16-F10 melanoma cells and is used to sort and analyze these cells.
• Assessment of PD-1 specificity: RMP1-30 is routinely validated for specificity by demonstrating no reactivity in PD-1 knockout mice, confirming that any signal is due to PD-1 recognition.
• Combination with other anti-PD-1 antibodies: Studies may use RMP1-30 alongside other anti-PD-1 clones (such as 29F.1A12) to corroborate findings, as these clones recognize overlapping populations of PD-1+ cells. This is important for validating staining specificity and reproducibility.

Crucially:

• RMP1-30 is not used for PD-1 blockade in vivo: While some anti-PD-1 clones (e.g., 29F.1A12 or RMP1-14) are used therapeutically to block PD-1 signaling in mouse models of cancer or autoimmune disease, RMP1-30 does not block ligand (PD-L1/PD-L2) binding to PD-1 and is therefore not suitable for PD-1 functional blocking studies in vivo. Its function is limited to detection and phenotyping.
• Immunogen and isotype: RMP1-30 was raised by immunizing rats with PD-1–transfected BHK cells and is an IgG2b isotype.

Limitations:

• Since RMP1-30 does not block the binding of PD-1 ligands, it cannot substitute for blocking antibodies in studies requiring PD-1/PD-L1 or PD-1/PD-L2 pathway inhibition.
• For in vivo studies where PD-1 function needs to be manipulated, blocking clones like 29F.1A12 or 4C11 (engineered for reduced immunogenicity and effector function) are preferred.

In summary, RMP1-30 is used in mouse studies to detect and quantify PD-1 in immunophenotyping and flow cytometry, but is not appropriate for functional PD-1 blockade in vivo.

Commonly Used Antibodies and Proteins with RMP1-30

RMP1-30 is a rat monoclonal antibody that specifically targets murine programmed cell death protein 1 (PD-1, CD279), widely used in immunology and cancer immunotherapy research. In the literature, it is frequently used alongside other anti-PD-1 antibodies and related checkpoint proteins to validate specificity, investigate epitope overlap, or assess functional blockade in experimental systems.

Antibodies Co-Used with RMP1-30

• 29F.1A12 (also called 1A12): This is another anti-mouse PD-1 monoclonal antibody often used in parallel with RMP1-30 to confirm PD-1 surface expression on live cells, especially in tumor models such as B16-F10 melanoma and primary T cells. Co-staining experiments with both antibodies show nearly complete overlap in positive cell populations, supporting the specificity of both clones for PD-1.
• RMP1-14: A closely related anti-PD-1 antibody, frequently compared to RMP1-30 for epitope mapping and blocking studies. Epitope blocking assays reveal that RMP1-14 and RMP1-30 can partially block each other’s binding, suggesting they recognize distinct but partially overlapping epitopes on PD-1. However, RMP1-30 can still be used for staining in the presence of RMP1-14, albeit with some reduction in fluorescence intensity.
• 10F.9G2: This clone also targets PD-1 and is sometimes included in comparative studies evaluating the functional blockade of PD-1 signaling. While not always co-used in the same experiment as RMP1-30, it is part of the same panel of anti-PD-1 antibodies used to dissect PD-1 biology in mice.

Proteins and Target Molecules

• PD-1 (CD279): The primary target of RMP1-30, PD-1 is a cell surface protein involved in immune checkpoint regulation. RMP1-30 is used to detect and quantify PD-1 expression on immune cells and in tumor models.
• PD-L1 (Programmed Death-Ligand 1): Although RMP1-30 does not bind PD-L1 directly, PD-L1 is frequently studied in conjunction with PD-1 to understand immune evasion mechanisms in cancer and infectious diseases. Anti-PD-L1 antibodies (such as 10F.9G2 and others) are often used alongside RMP1-30 in co-culture or functional assays to model PD-1/PD-L1 axis blockade.
• CD28 and CTLA-4: These are related members of the CD28/CTLA-4 family and are sometimes investigated in studies alongside PD-1 to understand broader immune checkpoint modulation. However, RMP1-30 itself is specific for PD-1, not these other proteins.

Functional and Staining Applications

• Flow Cytometry: RMP1-30 is commonly used in flow cytometry to detect PD-1 on live and dead cells, often in combination with 29F.1A12 to confirm specificity or for multi-parameter analysis.
• Immunotherapy Experiments: In preclinical immunotherapy studies, RMP1-30 may be used alongside 29F.1A12 or RMP1-14 to assess PD-1 blockade efficacy, especially when different antibody clones are being evaluated for therapeutic potential.
• Epitope Mapping: Comparative binding and blocking studies using RMP1-30, 29F.1A12, and RMP1-14 help define the epitope landscape of murine PD-1 and inform the design of bispecific or combination therapies.

Summary Table: Key Antibodies and Proteins Used with RMP1-30

Conclusion

RMP1-30 is most commonly used alongside other anti-PD-1 antibodies such as 29F.1A12 and RMP1-14 for validation, epitope mapping, and functional studies in murine models. PD-L1 and related checkpoint proteins are also frequently investigated in the same experimental systems, though RMP1-30 itself is specific for PD-1. These combinations are critical for dissecting the roles of immune checkpoints in health and disease.

Clone RMP1-30 is a monoclonal antibody widely used to detect and study PD-1 (Programmed cell Death 1) expression, especially in murine models. The key scientific findings from literature citations of RMP1-30 are:

• Specificity for PD-1 Detection: RMP1-30 reliably binds to surface PD-1 protein on live and dead murine cells—including B16-F10 melanoma cells and T-cells—with no reactivity in PD-1 knockout cells, confirming its high specificity for PD-1.

• Overlap with Other PD-1 Clones: RMP1-30 and the 29F.1A12 anti-PD-1 clones recognize largely overlapping cell populations. Dual staining experiments show nearly all PD-1 antibody-reactive cells are positive for both clones, reinforcing specificity and reliability in flow cytometric analyses.

• Culture Conditions Impact Detection: Reactivity of RMP1-30 (and 29F.1A12) to PD-1 on B16-F10 cells is over threefold higher in three-dimensional tumor spheroid cultures compared to standard two-dimensional cultures, indicating detection sensitivity varies with cell context.

• Non-blocking Functional Property: RMP1-30 does not block the PD-1/PD-L1 interaction. Unlike some anti-PD-1 antibodies (e.g., 1A12), RMP1-30 does not reverse PD-1–mediated T cell inhibition or increase luciferase activity in functional assays, making it suitable as a staining reagent rather than a blocking or agonist antibody.

• Epitope and Staining Characteristics: RMP1-30 can fully block its own binding but only partially blocks 29F.1A12. Conversely, when 1A12 or RMP1-14 are bound to PD-1, RMP1-30 staining intensity drops by about 25%, suggesting partial epitope overlap but also cooperative binding effects. These properties make RMP1-30 useful for staining PD-1, even in the presence of therapeutic antibodies, with minor reductions in staining signal.

• Compatibility in Immunotherapy Experiments: Because RMP1-30 does not interfere with PD-1/PD-L1 blockade, it can be used alongside blocking antibodies like 1A12 or RMP1-14 for flow cytometric analysis of PD-1 expression post-treatment.

In summary, RMP1-30 is best used as a PD-1 detection reagent rather than a functional blocking antibody. It is validated for specificity and co-recognition of PD-1 by various antibody clones, and its staining properties make it a standard tool in cancer immunology and immunotherapy research.

Dosing regimens of clone RMP1-30 (rat anti-mouse PD-1) are not standardized and can vary considerably across different mouse models depending on the research application. RMP1-30 is primarily used for flow cytometry and PD-1 detection rather than in vivo functional blockade, and published dosing regimens are typically not as well established as for anti-PD-1 clones like 29F.1A12 or RMP1-14. Here are the key points based on available research and manufacturer data:

• Flow Cytometry Applications:
  For flow cytometry, the recommended use is a dilution of 1:100 to 1:200, using 10??L of the working dilution to label 1 million cells in 100??L of buffer. No specific reference to administration in live animals for therapy or depletion is provided in manufacturer data.

• In Vivo Functional Use:
  Unlike clones such as 29F.1A12 or RMP1-14, which are commonly used for in vivo PD-1 blockade at doses of 200–500 ?g per mouse via intraperitoneal injection every 3–4 days in tumor or infection models, there is limited evidence that RMP1-30 is routinely used for such blockade studies.

  Existing publications primarily describe RMP1-30 for detecting PD-1 expression in situ or ex vivo, not for manipulating PD-1 signaling in live mice. When used for flow cytometry in tumor models (e.g., B16-F10 melanoma), the focus is on surface detection rather than immunomodulation.

• Comparison with Other Clones:
  | Clone | Typical Use | Common Dose (in vivo) | Route/Regimen ||---------------|------------------------|----------------------------------|---------------------------------------|| RMP1-30 | PD-1 detection (FACS) | 1:100–1:200 dilution (ex vivo) | NA for in vivo functional blockade || 29F.1A12 | PD-1 blockade (in vivo)| 100–200??g per mouse (5?mg/kg) | i.p. every 3–4 days (usually 3 doses) || RMP1-14 | PD-1 blockade (in vivo)| 200–500??g per mouse | i.p. every 3–4 days |

• Alternate Models and Regimens:
  If RMP1-30 were to be used in vivo for checkpoint blockade (not common), it would be reasonable—by analogy to similar anti-PD-1 clones—to begin with 200–500??g per mouse i.p. every 3–4 days, with adjustment based on pilot outcomes. However, this approach should be validated, as RMP1-30 may not block PD-1/PD-L1 binding as effectively as other clones.

• Model and Disease Context:
  Dosing may also vary depending on tumor type (e.g., B16 melanoma vs. MC38 colon carcinoma) and whether the model requires chronic vs. acute dosing.

Summary:
RMP1-30 is primarily a detection antibody, with dosing regimens (1:100–1:200 dilutions) established for flow cytometry. Its use and dosing for in vivo functional blockade are not standard and are less documented compared to other anti-PD-1 clones, which are injected at 100–500??g per mouse every 3–4 days in immune-oncology models.