Anti-Mouse CD3ε [Clone 500A2] — Purified in vivo GOLD™ Functional Grade

Clone 500A2, a monoclonal antibody targeting the mouse CD3ε chain, is commonly used in in vivo mouse studies for several applications:

1. T Cell Activation and Modulation: The antibody can engage or modulate T cells, affecting immune responses. It is used to activate T cells or influence their functions in various immunological conditions.

2. Immunophenotyping and Functional Assays: Clone 500A2 is utilized to identify and study T cells, including thymocytes and mature T lymphocytes, in mouse models. This is crucial for understanding T cell biology and immune system dynamics.

3. Depletion of T Cells: The antibody can be used to deplete T cells in vivo, which helps in studying T cell roles in disease models and immune responses.

4. Immunohistochemistry: Clone 500A2 is applied in immunohistochemistry to study T cell distribution and localization in frozen tissue samples.

In summary, these applications highlight the versatility of clone 500A2 in mouse immunological research, offering insights into T cell biology and immune functions.

In the literature, the 500A2 antibody, which targets the CD3ε chain on mouse T cells, is often used alongside other antibodies that recognize different surface markers. Some commonly used antibodies or proteins in conjunction with 500A2 include:

• Other anti-CD3 clones: Such as 17A2 and 145-2C11, which are also used to target the CD3 complex on T cells.
• Anti-CD4 and anti-CD8 antibodies: These are used to differentiate between T helper (CD4+) and cytotoxic T (CD8+) cells in mouse immune cell populations.
• Anti-human or anti-mouse CD14, CD20, and CD34 antibodies: These are used to identify and study various immune and hematopoietic cell types. For example, CD14 is a marker for monocytes/macrophages, CD20 for B cells, and CD34 for hematopoietic progenitor cells.
• Secondary antibodies: These are used in conjunction with primary antibodies like 500A2 for indirect detection methods in assays such as flow cytometry, immunohistochemistry, and Western blotting. Secondary antibodies can be conjugated to various labels (e.g., fluorescent dyes or enzymes) to enhance detection sensitivity and specificity.

Clone 500A2 is a monoclonal antibody that specifically targets the CD3ε (epsilon) chain of the T-cell receptor complex in mice, and scientific literature has revealed several important characteristics and applications of this antibody.

Epitope Recognition and Binding Characteristics

Clone 500A2 recognizes a distinct epitope on the 25-kDa ε chain of the T-cell receptor-associated CD3 complex. The antibody demonstrates partial cross-blocking ability with other anti-CD3 antibodies, specifically 17A2 and 145-2C11, indicating that these three antibodies recognize distinct but overlapping epitopes on CD3. Importantly, studies suggest that 500A2 may bind an epitope on CD3ε close to a site where CD4 associates with the T-cell receptor, as activation of mouse T-cell clones by this antibody can be blocked by Fab fragments of the GK1.5 anti-CD4 antibody.

Target Cell Expression

The antibody binds to CD3ε expressed on mouse thymocytes, mature T lymphocytes, and NKT cells. This CD3ε subunit is a transmembrane glycoprotein and critical component of the TCR complex, essential for TCR signaling, T-cell enumeration, and antigen recognition.

Functional Applications

Clone 500A2 demonstrates significant functional activity in T-cell research. Both plate-bound and soluble forms of the antibody can activate T cells in vitro. The antibody has been successfully utilized in various experimental approaches including flow cytometric analysis, immunohistochemistry of frozen tissue, and in vitro activation of mouse T cells. Researchers have employed this antibody for engaging or depleting T cells in in vivo mouse studies.

Specificity

The antibody shows species-specific reactivity, binding to mouse T cells while reportedly not cross-reacting with rat leukocytes. This specificity makes it particularly valuable for mouse immunology research.

Dosing regimens for clone 500A2, an anti-mouse CD3ε monoclonal antibody, vary considerably across different mouse models and applications, with no universal protocol established. The variation in dosing strategies reflects differences in experimental objectives, mouse strains, and specific research applications.

In Vitro and Flow Cytometry Applications

For flow cytometric staining, clone 500A2 is typically used at concentrations of ≤1.0 µg per million cells in 100 µl volume. For immunohistochemical staining on frozen tissue sections, the recommended concentration ranges from 2.5 to 10.0 µg per ml. These applications demonstrate the antibody's utility in identifying mouse T cells in multicolor flow cytometry panels.

In Vivo Functional Studies

In vivo applications show more substantial variation in dosing protocols. For T lymphocyte activation studies, researchers have used intraperitoneal injections of 50 µg in C3H/HeJ mice, with analysis performed 18 hours post-injection. However, other studies have employed different dose ranges. Research on anti-drug antibody formation in hemophilia A mice utilized both 0.5 µg and 5 µg doses of anti-mouse CD3, administered orally, demonstrating a 10-fold difference in dosing depending on the therapeutic goal.

Importance of Empirical Optimization

The available evidence indicates that no universal regimen exists for clone 500A2 in mouse models, and all protocols should be empirically optimized for each specific system and application. This recommendation underscores the critical importance of titrating the antibody for optimal performance based on the particular experimental context, mouse strain, route of administration, and desired immunological outcome. Factors such as the specific mouse model being used, the experimental timeframe, and whether the goal is T cell activation, depletion, or identification all influence the appropriate dosing strategy.