RF45 Antibody

What exactly is RF45 antibody and why is there confusion in the literature?

There appears to be terminological overlap in scientific literature regarding "RF45 antibody," which can refer to two distinct entities:

CD45 Antibodies: The majority of research publications use RF45 to refer to antibodies targeting CD45, a protein tyrosine phosphatase expressed on all nucleated hematopoietic cells. This glycoprotein comprises approximately 10% of all surface proteins in lymphocytes and is crucial for lymphocyte development and antigen signaling .

Plant RF45 Antibodies: In plant science contexts, RF45 refers to antibodies targeting a probable disease resistance protein in Arabidopsis thaliana (UniProt: F4IBE4). These are primarily used in plant pathology research .

This distinction is critical when searching literature or designing experiments, as methodologies differ significantly between these research domains.

What are the different isoforms of CD45 that RF45 antibodies might target?

CD45 exists in multiple isoforms created through alternative splicing:

When selecting an RF45/CD45 antibody, researchers must consider which isoform they intend to target, as this significantly impacts experimental outcomes and interpretation .

How do I select the appropriate RF45 antibody clone for my experimental needs?

Selection should be guided by:

• Target specificity: Determine whether you need pan-CD45 recognition (e.g., clone HI30 for all human CD45 isoforms) or isoform-specific targeting (e.g., anti-CD45RC)

• Species reactivity: Verify compatibility with your model system (e.g., 30-F11 clone for mouse CD45)

• Application suitability: Confirm validation for your intended application:

  • Flow cytometry: Pre-titrated antibodies like HI30 work at 5 µL (0.015 µg) per test for human samples

  • Immunohistochemistry: MAB114 has been validated at 5 µg/mL for paraffin-embedded sections

  • Western blotting: Several clones are validated at 1:1000-1:2000 dilutions

• Fluorophore selection: For multicolor flow panels, consider spectral compatibility (e.g., Alexa Fluor 405, PE-Cy5)

Cross-reference manufacturer datasheets with recent literature to ensure optimal performance in your specific experimental system.

What are best practices for using CD45 antibodies in multiparameter flow cytometry?

Optimizing multiparameter flow cytometry with CD45 antibodies requires:

• Panel design considerations:

  • CD45 is typically used as a lineage marker to identify hematopoietic cells

  • The bright expression of CD45 makes it suitable for less powerful fluorophores

  • Use antibody combinations like CD45RA/CD45RO to distinguish naïve from memory T cells

• Technical optimization:

  • Titrate antibodies to determine optimal concentration (typically 0.015 µg per test for human samples)

  • Protect tandem dyes like PE-Cyanine5 from light exposure to prevent photo-induced oxidation

  • For fixation, samples can be stored in IC Fixation Buffer for up to 3 days with minimal impact on brightness

• Controls and validation:

  • Include FMO (Fluorescence Minus One) controls

  • Use positive controls like splenocytes and negative controls like Neuro-2A cells for mouse CD45 staining

  • Validate gating strategies with known CD45+ and CD45- populations

• Data interpretation:

  • CD45 expression levels can vary between leukocyte subsets

  • Consider how activation state affects CD45 isoform expression patterns

This approach ensures reliable identification and characterization of leukocyte populations in complex samples.

How can I effectively use RF45 antibodies for therapeutic target validation studies?

Target validation studies using RF45/CD45 antibodies require a systematic approach:

• Expression profiling:

  • Use flow cytometry with anti-CD45 antibodies to quantify target expression on malignant cells

  • Perform immunohistochemistry on tissue sections to evaluate spatial distribution

  • CD45 expression is confirmed in 85-90% of acute myeloid leukemia and acute lymphoblastic leukemia cases

• Functional validation:

  • Employ CD45 antibodies to modulate signaling pathways

  • CD45 dephosphorylates and negatively regulates the src family kinase LCK within the immunological synapse

  • Short-course anti-CD45RC mAb treatment in animal models induces preferential depletion of CD45RC high T cells, leading to donor-specific tolerance

• In vivo biodistribution:

  • Radiolabeled CD45 antibodies can be used to assess targeting in preclinical models

  • Studies show enrichment of radiolabeled antibody in immunodeficient mice xenotransplanted with human CD45+ leukemia cells

• Therapeutic delivery approaches:

  • Antibody-drug conjugates (ADCs)

  • Antibody-radionuclide conjugates (ARCs) using isotopes like yttrium-90 or iodine-131

  • Native antibodies for immune modulation

These methodologies have been validated in multiple preclinical models and early clinical studies for hematologic malignancies .

Why might my CD45 antibody staining show unexpected variation between samples?

Troubleshooting variable CD45 staining requires systematic investigation:

• Biological variables affecting CD45 expression:

  • Activation state changes isoform distribution (CD45RA on naïve cells vs. CD45RO on memory cells)

  • CD45 can be moved into or out of the immunological synapse membrane microdomain depending on interactions with the extracellular galectin lattice or the intracellular actin cytoskeleton

  • Sialylation of N-linked carbohydrates can affect detection by some antibody clones

• Technical considerations:

  • Antibody internalization: CD45 antibodies can be internalized, affecting surface detection

  • Epitope masking: Ensure fixation/permeabilization protocols preserve epitope recognition

  • For paraffin sections, heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic may be necessary

• Protocol optimization:

  • For flow cytometry, follow validated staining conditions (e.g., 3 μg/mL for 3 hours at room temperature for MAB114)

  • For IHC, evaluate different antibody concentrations and incubation times (e.g., 5 μg/mL for 1 hour)

  • Consider dual staining approaches to verify specificity (e.g., splenocytes show positive staining while Neuro-2A cells remain negative)

• Quality control measures:

  • Include proper positive and negative controls in each experiment

  • Verify antibody performance with a new lot on a well-characterized sample

This systematic approach helps identify whether variation stems from technical issues or reflects genuine biological differences.

How do I address potential cross-reactivity when using RF45 antibodies in plant research?

Working with RF45 antibodies in plant systems requires special considerations:

• Specificity verification:

  • The sequence homology of synthetic peptides used for immunization can lead to cross-reactivity

  • For example, some anti-RF45 antibodies show 100% homology with sequences in AT1G59124, AT1G59218, and AT1G58848

  • Western blotting against purified recombinant proteins can help determine specificity

• Pre-adsorption controls:

  • Pre-incubate antibody with the immunizing peptide

  • Compare staining patterns between pre-adsorbed and non-adsorbed antibodies

  • Specific staining should be eliminated by pre-adsorption

• Antibody validation strategies:

  • Use knockout/knockdown plant lines as negative controls

  • Compare multiple antibodies against the same target

  • Consider peptide affinity purification of antibodies for enhanced specificity

• Optimized protocols for plant tissues:

  • Western blotting: Use dilutions between 1:1000-1:2000

  • Store antibodies properly (−20°C to −70°C under sterile conditions)

  • Avoid repeated freeze-thaw cycles

These approaches minimize false positives and ensure reliable results in plant pathology research.

How are RF45/CD45 antibodies being used in novel therapeutic approaches for hematological malignancies?

CD45-targeted therapies represent an emerging approach for treating hematological malignancies, with several innovative strategies:

• Antibody-Radionuclide Conjugates (ARCs):

  • Clinical rationale: CD45 is ubiquitously expressed on hematopoietic cells, has infrequent antigen loss/blockade, and enables targeting of minimal residual disease

  • Phase I trials have evaluated escalating doses of CD45-targeted ARCs using the BC8 antibody labeled with iodine-131 (131I) followed by autologous stem cell support

  • 90Y-labeled anti-CD45 antibody shows benefits in reduced-intensity allogeneic hematopoietic cell transplantation for multiple myeloma

  • Unlike 131I, yttrium-90 has a shorter half-life (2.5 vs. 8 days) and lacks a gamma component, eliminating the need for patient radiation isolation

• Immunomodulatory approaches:

  • Anti-CD45RC mAb treatment:

    • Targets CD45RC high T cells but spares regulatory T cells (Tregs)

    • Prevents and treats acute graft-versus-host disease (GVHD) in rat and mouse models

    • Induces donor-specific tolerance in transplantation settings

    • Preserves beneficial immune responses against third-party alloantigens, tumors, and pathogens

• Humanized antibody development:

  • Chimeric and humanized anti-CD45 antibodies developed at Fred Hutch show promising targeting in xenotransplant models

  • Benefits include decreased infusion toxicities and limited anti-mouse antibody (HAMA) immunization

  • These can be deployed across multiple modalities (antibody-drug conjugates, radioimmunotherapy)

• Integration with stem cell transplantation:

  • Anti-CD45 antibodies are being investigated as conditioning regimens for hematopoietic stem cell transplantation

  • 90Y-conjugated anti-CD45 mAb has been integrated with reduced-intensity conditioning regimens

These approaches leverage CD45's stable expression on malignant cells while employing various effector mechanisms to achieve therapeutic outcomes.

Can AI-driven antibody design approaches be applied to develop new RF45/CD45 antibodies?

Recent advances in AI-driven protein design are transforming antibody development, with potential applications for RF45/CD45 targeting:

• RFdiffusion for antibody design:

  • The Baker Lab (February 2025) reported a fine-tuned version of RFdiffusion specifically for human-like antibody design

  • Key capabilities include:

    • Design of antibody loops—the intricate, flexible regions responsible for binding

    • Generation of complete single chain variable fragments (scFvs)

    • Computer-based development of entirely new functional antibodies

• Methodology and workflow:

  • The AI system produces antibody blueprints that:

    • Are unlike any seen during training

    • Can bind user-specified targets

    • Maintain human-like characteristics

  • The process involves:

    • Training on antibody complex structures

    • Specifying framework sequence and structure

    • Designing CDR loop conformations and dock positions between antibody and target

• Application to CD45 targeting:

  • Could generate novel anti-CD45 antibodies with:

    • Enhanced specificity for particular CD45 isoforms

    • Optimized binding kinetics

    • Reduced immunogenicity

    • Improved stability and manufacturability

• Experimental validation:

  • Research teams have validated AI-designed antibodies against disease-relevant targets

  • Cryo-EM reconstructions confirm binding modes predicted by the model

  • Success rates are improving but still require high-throughput screening

This represents a frontier in antibody engineering that could yield next-generation anti-CD45 therapeutics with precisely tuned properties.

What role do RF45/CD45 antibodies play in studying immunological synapse dynamics?

Anti-CD45 antibodies have become crucial tools for understanding the complex spatial and temporal dynamics of the immunological synapse:

• CD45 trafficking during T cell activation:

  • CD45 movement into or out of the immunological synapse (IS) is regulated by:

    • Interactions with the extracellular galectin lattice

    • Associations with the intracellular actin cytoskeleton

  • Within the IS, CD45 dephosphorylates and negatively regulates the src family kinase LCK

  • Clathrin mediates both internalization and vesicular release of T cell receptors at the immunological synapse

• Methodological approaches:

  • Live cell imaging with fluorescently labeled anti-CD45 antibodies

  • Super-resolution microscopy to track CD45 distribution during IS formation

  • Functional studies using antibodies to modulate CD45 activity

• CD45 isoform-specific functions:

  • CD45 isoform expression influences IS architecture

  • O-glycosylation in spliced regions and sialylation of N-linked carbohydrates fine-tune galectin interactions

  • Different usage of CD45 isoforms affects T cell receptor signaling thresholds

• Therapeutic implications:

  • Understanding CD45 dynamics has led to novel immunotherapeutic strategies

  • Targeting specific CD45 isoforms like CD45RC can modulate immune responses without global immunosuppression

  • These insights inform the development of targeted therapies for autoimmune diseases and transplantation

This research area represents the intersection of basic immunology and translational medicine, with significant implications for immunotherapy development.

How effective are anti-CD45RC antibodies in treating experimental autoimmune diseases?

Recent research demonstrates significant efficacy of anti-CD45RC antibodies in autoimmune disease models:

These findings suggest anti-CD45RC antibodies could represent a promising therapeutic approach for various autoimmune conditions with potential advantages over existing therapies.