Anti-Human CD20 [Clone 2H7] — Purified in vivo GOLD™ Functional Grade

The most common in vivo application of clone 2H7 in mice is the depletion or tracking of human B cells in humanized mouse models, because 2H7 is a mouse monoclonal antibody specific for the human CD20 antigen, which is present on human B cells. This application is primarily utilized in mice that have been engrafted with human immune components (humanized mice), as 2H7 does not recognize mouse CD20.

Key in vivo applications include:

• B cell depletion: 2H7 is used to selectively deplete human B cells in humanized mouse models for studies on immune function, lymphoma therapy, or autoimmunity.
• Tracking and targeting human B cells: By binding to human CD20, 2H7 enables the tracking or immunohistochemical identification of human B cells within mouse tissues.
• Preclinical modeling of B cell-targeted therapies: 2H7 is employed experimentally to evaluate mechanisms and efficacy of CD20-targeted therapies (such as examining the effectiveness of B cell depletion protocols in humanized immune systems or lymphoproliferative disease models).
• Studying human immune cell behavior: Researchers use 2H7 to manipulate or observe human B cell dynamics in vivo, supporting studies in fields such as infectious disease, vaccine response, and immunopathology.

Important limitations and context:

• Species specificity: Clone 2H7 only binds to human (and in some cases rhesus monkey) CD20; it does not react with endogenous mouse B cells. Its use in regular (non-humanized) mice will not affect mouse B cells.
• Humanized mouse requirement: Since 2H7 targets human CD20, in vivo work must be performed in mice engrafted with human B cells or immune systems, such as NOD scid gamma (NSG) mice or proprietary models like THX mice.

In summary, clone 2H7 is primarily used in vivo to deplete or interrogate human B cells in humanized mouse models, facilitating a range of preclinical immunological and therapeutic studies.

Commonly used antibodies or proteins used with 2H7 (anti-CD20) in the literature include rituximab, F8, and various isotype control antibodies. Other reagents and targets frequently paired with 2H7 will depend on context, such as combination therapies, immunophenotyping, and functional assays.

Essential context and supporting details:

• Rituximab: This is another widely used anti-CD20 antibody and is often studied alongside 2H7 to compare binding affinity, epitope specificity, therapeutic efficacy, or for structure-function analyses because both antibodies bind overlapping or similar epitopes on CD20.

• F8: F8 is used with 2H7, particularly in recombinant or bispecific antibody engineering, for example, the construction of chelating recombinant antibodies for tumor-targeting studies.

• Isotype control antibodies: These serve as negative controls in flow cytometry, ELISA, or in vivo studies to ensure the specificity of 2H7's effects and rule out non-specific binding or immune activation.

Other context-dependent commonly used markers and proteins:

• Chemotherapy agents and BCR signaling inhibitors: In clinical and preclinical studies of B cell malignancies, 2H7 (or other anti-CD20 antibodies) is often evaluated in combination with chemotherapy drugs or inhibitors of the B cell receptor signaling pathway.

• Secondary detection antibodies and fluorescent conjugates: In flow cytometry or imaging, 2H7 is used with secondary antibodies or as a directly labeled conjugate (e.g., Alexa Fluor or HRP-labeled 2H7) alongside other lineage or activation markers.

• Other B cell markers: Depending on the application, panels may include anti-CD19, anti-CD45, or other B cell differentiation and activation markers when combined with 2H7 for immunophenotyping.

Summary Table of Commonly Used Antibodies/Proteins with 2H7

If you need details on a specific application (e.g., immunotherapy, flow cytometry panels, or in vivo depletion), let me know for more focused information.

The key findings from scientific literature citing clone 2H7 consistently establish it as a well-characterized monoclonal antibody for detecting human CD20, a surface antigen broadly expressed on B cells. Major conclusions from these citations include:

• Epitope Mapping: Clone 2H7 recognizes a specific sequence—YNCEPANPSEKNSPST—located in the large extracellular loop of human CD20. This mapping is foundational for its specificity and is repeatedly cited for distinguishing it from other anti-CD20 clones.

• Target Specificity: 2H7 binds to a 33–37 kDa non-glycosylated phosphoprotein (CD20) present during pre-B cell development, expressed on both resting and activated B cells, but lost during differentiation to plasma cells. The antibody also displays cross-reactivity with rhesus monkey CD20 but is specific to primate species.

• Native Oligomer: It is notable that 2H7 only recognizes CD20 in its native oligomeric (multimeric) conformation, which is important for applications like immunoprecipitation or certain structural studies.

• Applications:

  • Flow Cytometry (FC): 2H7 is a gold standard for surface staining in flow cytometry, distinguishing B cells without requiring cell permeabilization steps.
  • Immunoprecipitation (IP) and Immunohistochemistry (IHC): Published studies and vendors cite its use in IP and IHC, notably on acetone-fixed frozen tissue sections.
  • Cell Separation: Its verified suitability for cell separation and immune monitoring is cited in clinical and translational research.
  • Diagnostic/Reagent Use: Extensively used in identifying B cell lymphomas and leukemias and is routinely referenced in protocols for clinical flow cytometry.

• Comparisons with Other Clones: 2H7 is often referenced alongside or compared to other anti-CD20 clones (such as 1F5, L27, or 1412). Specifically, 2H7 recognizes an extracellular epitope accessible without permeabilization, while some other clones target different or cytoplasmic CD20 domains and require fixation or permeabilization. This makes 2H7 preferable for live-cell analyses.

• Functional Insights: Literature has built upon early structural studies, expanding to functional investigations into B cell activation, calcium flux, and immune response modulation. CD20 can form complexes with other cell surface molecules (including MHC I/II, CD53, CD81, and CD82), and is implicated in the function of calcium channels on the B cell membrane.

• Citation Legacy: Key foundational and methodologic citations are included in the definitive "Leucocyte Typing" series (III, IV, V) and multiple peer-reviewed articles on antibody validation and B cell characterization. Newer therapeutic antibody engineering papers (e.g., humanized CD20 antibodies) continue to reference 2H7 as a comparative tool for functional and binding studies.

In summary, clone 2H7 is considered a reference reagent for surface detection of human CD20 and is widely cited for its reliability, specificity, epitope definition, and compatibility with standard immunological assays, especially flow cytometry and immunohistochemistry.

Dosing regimens for the clone 2H7 anti-human CD20 antibody typically range from 100–250 µg per mouse every 3–4 days via intraperitoneal injection; however, these doses are adjusted according to the mouse model used to balance efficacy and safety.

Key model-dependent variations:

• Humanized mice (e.g., mice engrafted with human hematopoietic stem cells): Dosing is generally toward the higher end (up to 250 µg per mouse) due to the larger target cell population and the need for robust B cell depletion.
• Immunodeficient models: These may also tolerate and sometimes require higher dosing, but careful monitoring is necessary because of altered pharmacokinetics and potential toxicity.
• Disease-specific or genetically engineered models: Dosing frequency and amount may be modulated depending on disease burden, immune reconstitution, or presence of co-treatments to minimize side effects and optimize anti-CD20 activity.

Route and frequency are consistent (intraperitoneal, every 3–4 days), but the exact dose is refined based on:

• Mouse strain and body weight (standardizing per 20–25 g mouse)
• Engraftment level (in humanized models, higher dosing if B cell reconstitution is robust)
• Experimental endpoint (e.g., short-term depletion vs. long-term immune modulation)

Most published regimens avoid exceeding 250 µg per mouse to minimize non-specific binding and immune complex formation.

In summary, the primary variable is the dose per injection (100–250 µg/mouse), chosen by considering the immune status, human cell engraftment, and experimental aims of the mouse model. Other aspects (such as route and interval) are usually similar across regimens.