CD45 Antibody

What is CD45 and why is it an important target for immunological research?

CD45, also known as leukocyte common antigen (LCA) or protein tyrosine phosphatase receptor type C (PTPRC), is a transmembrane-type protein tyrosine phosphatase expressed on all nucleated hematopoietic cells. It exists in multiple isoforms resulting from alternative splicing of exons 4, 5, and 6 (also called A, B, and C) . CD45 plays critical roles in immune cell function, particularly in T-cell receptor signaling thresholds . Its ubiquitous expression on hematopoietic cells makes it an excellent pan-leukocyte marker for identifying immune cells in various experimental contexts.

The importance of CD45 stems from several key characteristics:

• Universal expression on all nucleated cells of hematopoietic origin

• Variable expression patterns that correlate with cell type and activation status

• Critical role in immune cell signaling pathways

• Potential as a therapeutic target for hematological malignancies

How do the different isoforms of CD45 affect antibody selection for research applications?

CD45 exists in multiple isoforms due to alternative splicing of exons 4-6, with the major isoforms including:

When selecting antibodies, researchers must consider whether they need:

• Pan-CD45 antibodies: These recognize epitopes common to all isoforms (e.g., clones AP4, DN11, SHL-1, and P6 described in research)

• Isoform-specific antibodies: These recognize specific exon-encoded regions (e.g., clones P1 and P14 specifically bind to isoforms containing exon A-encoded sequences)

The choice depends on your experimental goals—whether you need to identify all leukocytes or discriminate between specific subpopulations based on CD45 isoform expression.

What is the difference between conventional CD45 antibodies and CD45RA antibodies?

The distinction between conventional CD45 antibodies and CD45RA antibodies lies in their epitope specificity:

Conventional CD45 antibodies recognize epitopes present in all CD45 isoforms, making them suitable for pan-leukocyte identification. Research has characterized several monoclonal antibodies (e.g., AP4, DN11, SHL-1, YG27, and P6) that bind to all five CD45 isoforms .

CD45RA antibodies specifically recognize epitopes encoded by exon A, which is only present in certain isoforms (CD45RABC and CD45RAB). These antibodies (e.g., P1 and P14) only bind to isoforms that include exon A-encoded sequences . CD45RA antibodies are particularly useful for identifying naive T cells and B cells.

This distinction is critical when designing experiments to differentiate between immune cell subsets based on their CD45 isoform expression patterns.

What are the optimal protocols for using CD45 antibodies in flow cytometry?

Effective flow cytometry with CD45 antibodies requires careful optimization:

• Sample preparation:

  • Use freshly isolated cells when possible

  • For whole blood, use appropriate RBC lysis buffers

  • Include viability dyes to exclude dead cells

• Staining protocol:

  • Titrate antibodies to determine optimal concentration

  • Include appropriate isotype controls

  • Block Fc receptors to reduce non-specific binding

  • Incubate at appropriate temperature (typically 4°C for 30 minutes)

• Panel design considerations:

  • CD45 is highly expressed on lymphocytes, so lower-brightness fluorophores may be sufficient

  • For multi-color panels, consider spectral overlap with other markers

Research demonstrates successful detection of CD45 in human blood lymphocytes using Mouse Anti-Human CD45 Alexa Fluor® 488-conjugated Monoclonal Antibody (Clone 2D1) compared against appropriate isotype controls .

How should CD45 antibodies be used for immunohistochemistry on fixed tissue samples?

For optimal immunohistochemistry results with CD45 antibodies:

• Tissue preparation:

  • Use appropriate fixation (typically 10% neutral buffered formalin)

  • Process and embed in paraffin following standard protocols

  • Section at 4-6 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) with pH 9 buffer is often effective for CD45

  • Complete dewaxing before retrieval

• Staining protocol:

  • Block endogenous peroxidase activity

  • Use protein blocking to reduce background

  • Apply optimized antibody concentration (typically 5 μg/mL)

  • Incubate at room temperature for 1 hour or at 4°C overnight

  • Use appropriate detection system (e.g., HRP-polymer)

  • Develop with DAB or other chromogen

  • Counterstain, dehydrate, and mount

Recent protocols have successfully detected CD45 in human tonsil sections using monoclonal antibodies at 5 μg/mL for 1 hour at room temperature followed by HRP-polymer detection systems .

What approaches can be used to validate the specificity of CD45 antibodies?

Rigorous validation of CD45 antibody specificity involves multiple complementary approaches:

• Positive and negative control samples:

  • Test on cell lines known to express CD45 (e.g., Jurkat) and those that lack CD45 (e.g., MCF-7)

  • Include appropriate tissue controls (lymphoid tissues are excellent positive controls)

• Recombinant expression systems:

  • Transfect cells (e.g., COS-7) with constructs expressing specific CD45 isoforms

  • Test antibody binding to each isoform to determine specificity

• Multiple detection methods:

  • Compare results across different techniques (flow cytometry, Western blot, IHC)

  • Ensure consistent staining patterns across methods

• Blocking experiments:

  • Pre-incubate with immunizing peptide to block specific binding

  • Compare staining with and without blocking

Research has demonstrated comprehensive validation by transiently transfecting COS-7 cells with plasmids expressing individual CD45 isoforms and testing antibody binding specificity through flow cytometric analysis .

How can CD45 antibodies be utilized in multiplex immunofluorescence studies?

Multiplex immunofluorescence with CD45 antibodies requires careful planning:

• Panel design considerations:

  • Select primary antibodies from different host species or isotypes

  • Choose fluorophores with minimal spectral overlap

  • Consider CD45 expression levels when selecting fluorophore brightness

• Staining approach:

  • Sequential staining with blocking steps between antibodies

  • Automated staining platforms can improve consistency

  • Appropriate antigen retrieval is critical (typically pH 9 HIER)

• Analysis considerations:

  • Include single-stained controls for spectral unmixing

  • Use spectral imaging systems for crowded panels

  • Apply automated image analysis for quantification

Successful multiplex detection of CD45 has been reported in human lymph node sections using Mouse Anti-Human CD45 Monoclonal Antibody (5 μg/mL) visualized with Alexa Fluor 555-conjugated secondary antibody and counterstained with DAPI, showing specific membrane localization .

What are the considerations for using CD45 antibodies in antibody-radionuclide conjugate therapy?

Developing CD45-targeted antibody-radionuclide conjugates (ARCs) involves several critical considerations:

• Antibody selection:

  • High specificity for CD45

  • Appropriate binding kinetics and internalization properties

  • Stability under conjugation conditions

• Radioisotope selection:

  • Half-life appropriate for the biological half-life of the antibody

  • Emission characteristics suitable for the treatment goal

  • Chemistry compatible with antibody conjugation

• Biodistribution optimization:

  • Target-to-non-target ratios are critical

  • Pre-clinical studies show yttrium-90-labeled anti-CD45 antibodies deliver 2.5- and 3.7-fold more radiation to bone marrow than to liver and lungs, respectively

  • Similar ratios observed with iodine-131 conjugates

• Clinical applications:

  • Conditioning regimens for hematopoietic cell transplantation

  • Treatment of lymphomas (B-NHL, T-NHL, and Hodgkin lymphoma)

  • Potential for treating minimal residual disease

Clinical trials have evaluated CD45-targeted ARCs based on per-patient dosimetry using the BC8 antibody labeled with iodine-131 followed by autologous stem cell support in adults with various lymphomas .

How do CD45 antibodies contribute to understanding T-cell signaling mechanisms?

CD45 antibodies provide valuable tools for investigating T-cell signaling pathways:

• Functional studies:

  • Antibodies can modulate CD45 phosphatase activity

  • Different epitope-binding antibodies may have distinct functional effects

  • Crosslinking CD45 can alter its association with signaling complexes

• Signaling pathway analysis:

  • CD45 dephosphorylates and regulates Src family kinases like Lck and Fyn

  • Upon T-cell activation, CD45 recruits and dephosphorylates SKAP1 and FYN

  • CD45 also modulates LYN activity through dephosphorylation

• Structure-function relationships:

  • The first phosphatase domain (D1) has enzymatic activity

  • The second domain (D2) affects substrate specificity

  • Antibodies targeting different domains may reveal distinct functions

• Co-receptor interactions:

  • CD45 acts as a positive regulator of T-cell coactivation upon binding to DPP4

  • Antibodies can be used to study these interactions

Research using specific CD45 antibodies has revealed critical roles in T-cell receptor signaling thresholds and interaction with various signaling molecules.

What are common pitfalls in CD45 antibody-based experiments and how can they be avoided?

Several common challenges arise in CD45 antibody experiments:

• Epitope masking in fixed tissues:

  • Challenge: Formalin fixation can mask CD45 epitopes

  • Solution: Optimize antigen retrieval (pH 9 HIER buffers often work well)

• Isoform specificity misinterpretation:

  • Challenge: Using an isoform-specific antibody when pan-CD45 detection is needed

  • Solution: Confirm antibody specificity through validation with recombinant isoforms

• Background in immunohistochemistry:

  • Challenge: Non-specific staining, particularly in tissues with high endogenous peroxidase

  • Solution: Thorough blocking steps and optimization of antibody concentration

• Signal variability in flow cytometry:

  • Challenge: Inconsistent staining intensity across samples

  • Solution: Standardize sample preparation, antibody concentration, and staining conditions

• Interference in multiplex applications:

  • Challenge: Cross-reactivity between detection systems

  • Solution: Careful selection of antibody combinations and appropriate controls

Proper experimental design with appropriate controls and thorough validation of antibody specificity are essential for avoiding these common pitfalls.

How should CD45 expression data be interpreted in heterogeneous tissue samples?

Interpreting CD45 staining in complex tissues requires careful consideration:

• Expression level variations:

  • CD45 is expressed at different levels across immune cell types

  • Lymphocytes typically show higher expression than myeloid cells

  • Quantitative analysis should account for these variations

• Isoform distribution:

  • Different regions may contain cells expressing different CD45 isoforms

  • Pan-CD45 vs. isoform-specific antibodies will give different patterns

• Spatial distribution analysis:

  • Assess clustering patterns of CD45+ cells

  • Proximity to other cell types or tissue structures

  • Correlation with pathological features

• Quantification approaches:

  • Cell counting in defined regions

  • Percent positive area measurement

  • Digital image analysis for precise quantification

Research has successfully detected CD45 in tissues like human tonsil and lymph node, which contain diverse immune cell populations with varying CD45 expression patterns .

How are CD45 antibodies being used in the development of novel immunotherapies?

CD45 antibodies are finding increasing applications in immunotherapy development:

• Antibody-radionuclide conjugates (ARCs):

  • Delivery of targeted radiation to hematopoietic tissues

  • Treatment of lymphomas (B-NHL, T-NHL, and Hodgkin lymphoma)

  • Conditioning regimens for stem cell transplantation

• T-cell modulation:

  • CD45 antibodies can modulate T-cell activation thresholds

  • Potential applications in autoimmunity and cancer immunotherapy

  • Combination with checkpoint inhibitors

• Targeting mechanisms:

  • CD45 targeting can overcome blockade of other targets (e.g., CD20)

  • Bystander effect for treating cells with low or absent CD45 expression

  • Panhematopoietic expression allows targeting of minimal residual disease

Clinical studies have demonstrated the potential of CD45-targeted ARCs using antibodies like BC8 labeled with iodine-131 or yttrium-90 .

What role do CD45 antibodies play in studying the tumor microenvironment?

CD45 antibodies serve as essential tools for tumor microenvironment research:

• Immune infiltrate characterization:

  • Quantifying total leukocyte infiltration using pan-CD45 antibodies

  • Distinguishing infiltrating immune cells from tumor cells

  • Spatial distribution analysis of immune cells within tumors

• Combined phenotypic analysis:

  • CD45 as a backbone marker in multiparameter panels

  • Identification of specific immune subsets within CD45+ population

  • Correlation with tumor progression and treatment response

• Functional status assessment:

  • Combining CD45 isoform analysis with activation markers

  • Evaluating immune cell exhaustion in the tumor context

  • Studying immune cell-tumor cell interactions

• Therapeutic target evaluation:

  • CD45-targeted therapies for hematological malignancies

  • Potential for targeting tumor-associated macrophages and other CD45+ cells

  • Bystander effect for eliminating tumor cells in close proximity to CD45+ cells

Research demonstrates that CD45 targeting with antibody-radionuclide conjugates can overcome limitations of other targeted therapies in lymphomas and potentially other cancers .

How are technical advances in antibody engineering impacting CD45 antibody development?

Recent technical advances are transforming CD45 antibody development:

• Bispecific antibody platforms:

  • Combining CD45 targeting with engagement of effector cells

  • Dual-targeting strategies (e.g., CD45 + tumor-specific antigens)

  • Enhanced specificity through avidity effects

• Antibody fragment engineering:

  • Smaller formats for improved tissue penetration

  • Modified pharmacokinetics for optimized biodistribution

  • Site-specific conjugation for consistent product quality

• Novel conjugation technologies:

  • Site-specific conjugation of radionuclides or other payloads

  • Stable linker chemistry for in vivo applications

  • Optimized drug-antibody ratios

• Humanization and deimmunization:

  • Reduced immunogenicity for therapeutic applications

  • Maintained specificity and affinity

  • Extended in vivo half-life

These advances are particularly relevant for developing CD45-targeted therapies like the antibody-radionuclide conjugates being evaluated in clinical trials for lymphoma treatment .