CD44 Antibody, Biotin

What is CD44 and why is it targeted with biotinylated antibodies?

CD44 is an 80-95 kDa glycosylated type I transmembrane protein expressed on leukocytes, erythrocytes, epithelial cells, and weakly on platelets. It functions as a receptor for hyaluronic acid and is involved in cell-cell interactions, adhesion, migration, and lymphocyte activation and homing . CD44 is also known as phagocytic glycoprotein-1 (Pgp-1) and extracellular matrix receptor type III .

Biotinylated anti-CD44 antibodies provide significant advantages for research applications due to the exceptionally high affinity between biotin and avidin/streptavidin. This system enables signal amplification through multiple layers of detection, allowing researchers to achieve enhanced sensitivity when studying CD44 expression and function in various cell types and tissues . Additionally, the biotin-avidin system provides flexibility in experimental design, as researchers can choose from various streptavidin-conjugated detection molecules depending on their application needs.

How do different clones of biotinylated anti-CD44 antibodies vary in their applications?

Multiple biotinylated anti-CD44 antibody clones are available for research purposes, each with specific characteristics and optimal applications:

Clone selection should be based on the specific research question, target species, and intended application. For example, if studying CD44 in human samples via flow cytometry, G44-26 or F10-44-2 might be appropriate options, whereas for mouse studies, IM7 would be the preferred choice .

What is the optimal protocol for using biotinylated anti-CD44 antibodies in flow cytometry?

For optimal flow cytometric analysis with biotinylated anti-CD44 antibodies:

• Sample preparation: Prepare single-cell suspension (1×10^6 cells in 100 μl buffer).

• Titration: Determine optimal antibody concentration experimentally. Recommended starting concentrations are:

  • ≤0.125 μg per test for IM7 clone

  • 10 μL per 10^6 cells for some clones

• Staining procedure:

  • Incubate cells with biotinylated anti-CD44 at 4°C for 20-30 minutes

  • Wash cells 2-3 times with buffer

  • Add streptavidin-conjugated fluorophore

  • Incubate 15-20 minutes at 4°C

  • Wash and analyze

• Controls: Include isotype control at the same concentration as the antibody of interest . Include unstained and compensation controls for multicolor experiments.

• Instrument settings: Optimize based on the specific streptavidin-conjugate used. Refer to fluorochrome spectra resources for appropriate instrument settings .

Proper titration is crucial as both under- and over-staining can lead to false results. The two-step nature of the biotin-streptavidin system requires careful optimization of both the primary antibody and the streptavidin-conjugate concentrations.

How can biotinylated anti-CD44 antibodies be utilized for immunofluorescence and immunohistochemistry?

For immunofluorescence and immunohistochemistry applications:

• Sample preparation:

  • For paraffin sections: Deparaffinize, rehydrate, and perform antigen retrieval

  • For cell lines: Fix cells (e.g., 4% paraformaldehyde or methanol) and permeabilize if detecting intracellular epitopes

• Blocking:

  • Block endogenous biotin when present in tissues

  • Block non-specific binding with appropriate serum or protein solution

• Primary antibody:

  • Apply biotinylated anti-CD44 antibody at experimentally determined concentration

  • Typical concentrations range from 2-5 μg/mL

  • Incubate overnight at 4°C or 1-2 hours at room temperature

• Detection:

  • For fluorescence: Apply streptavidin conjugated to fluorophore (e.g., DyLight 550)

  • For chromogenic detection: Apply streptavidin-HRP and develop with appropriate substrate

• Counterstaining:

  • For fluorescence: DAPI for nuclear visualization

  • For chromogenic: Hematoxylin for nuclear detail

Research has demonstrated successful application of biotinylated anti-CD44 (8E2F3) in detecting CD44 in human cell lines such as A431 skin carcinoma and U-2 OS osteosarcoma, with clear membrane localization pattern visualized using streptavidin-DyLight 550 .

What considerations are important when using biotinylated anti-CD44 antibodies for Western blotting?

When using biotinylated anti-CD44 antibodies for Western blotting, consider these methodological aspects:

• Sample preparation:

  • CD44 is glycosylated, so molecular weight may vary (typically 80-95 kDa)

  • Consider deglycosylation treatments if comparing across samples

  • Use appropriate lysis buffers with protease inhibitors

• Gel selection and transfer:

  • 8-10% polyacrylamide gels typically work well for CD44's molecular weight range

  • Consider longer transfer times for complete transfer of glycoproteins

• Blocking:

  • Block endogenous biotin in tissue samples

  • Use biotin-free blocking reagents (avoid avidin or streptavidin in blocking buffer)

• Detection:

  • Apply biotinylated anti-CD44 antibody (e.g., clone 156-3C11)

  • Incubate with streptavidin-HRP

  • Develop using enhanced chemiluminescence

• Controls:

  • Include positive control (cell line with known CD44 expression)

  • Consider using recombinant CD44 as standard

  • Include loading control for normalization

• Interpretation:

  • CD44 often appears as multiple bands representing different isoforms or glycosylation states

  • Standard CD44 (CD44s) typically appears around 85-95 kDa

  • Variant isoforms (CD44v) may appear at higher molecular weights

Careful optimization of antibody concentration and detection system is essential for obtaining clean, specific signal with minimal background.

How can biotinylated anti-CD44 antibodies enhance cell adhesion in tissue engineering applications?

Biotinylated anti-CD44 antibodies have demonstrated significant utility in tissue engineering applications, particularly for improving cell adhesion to scaffolds in cartilage repair:

• Biotin-conjugated anti-CD44 antibody-avidin binding system:
  This approach uses a multi-step process:

  • Scaffold materials (e.g., chitosan membranes or 3D porous scaffolds) are functionalized with avidin

  • Biotinylated anti-CD44 antibodies bind to the avidin on the scaffold

  • Cells expressing CD44 (e.g., chondrocytes) then bind to the antibodies via their surface CD44, enhancing adhesion to the scaffold

• Performance benefits:
  Research has demonstrated that this binding system significantly improves:

  • Cell spreading area

  • Reduced cell exfoliation rates (better retention)

  • Enhanced cell proliferation

  • Improved glycosaminoglycan (GAG) content

  • Better histological outcomes

• Comparative advantage:
  When comparing constructs with and without the biotin-conjugated anti-CD44 antibody-avidin binding system, those utilizing the system showed markedly improved cell adhesion to scaffolds in cartilage tissue engineering applications .

This innovative application leverages the specificity of CD44 antibodies and the strength of biotin-avidin interactions to create more effective tissue-engineered constructs, potentially improving clinical outcomes for cartilage repair and regeneration .

What experimental methods can assess the effectiveness of biotin-conjugated anti-CD44 antibody-avidin systems?

To evaluate the effectiveness of biotin-conjugated anti-CD44 antibody-avidin systems in tissue engineering applications, researchers can employ several quantitative and qualitative assessment methods:

• Cell adhesion and retention metrics:

  • Cell spreading area measurement via microscopy and image analysis

  • Cell exfoliation rate determination under mechanical stress

  • Quantification of cell retention on scaffolds over time

• Proliferation and viability assessments:

  • Cell proliferation rates via metabolic assays (MTT, XTT)

  • Live/dead staining to assess viability

  • DNA content quantification as an indicator of cell number

• Functional tissue assessment:

  • Histological analysis using appropriate stains for tissue-specific components

  • Glycosaminoglycan (GAG) content measurement for cartilage applications

  • Mechanical testing of engineered constructs to assess functional properties

• Molecular analysis:

  • mRNA expression of tissue-specific markers

  • Protein analysis via immunohistochemistry

  • Secretome analysis of engineered tissues

• In vivo evaluation:

  • Implantation of engineered constructs in animal models

  • Assessment of integration with host tissue

  • Functional recovery measurement in the target tissue

These comprehensive evaluation methods provide robust evidence for the effectiveness of biotin-conjugated anti-CD44 antibody-avidin systems in improving cell adhesion, retention, and functional outcomes in tissue engineering applications.

What are common technical challenges when using biotinylated anti-CD44 antibodies and how can they be resolved?

When working with biotinylated anti-CD44 antibodies, researchers may encounter several technical challenges:

Additionally, when using the biotin-streptavidin system:

• Store biotinylated antibodies with preservatives (e.g., 0.05% sodium azide) at 4°C in the dark

• Avoid buffers containing biotin (including some culture media supplements)

• Consider direct fluorochrome-conjugated antibodies if biotin-related issues persist

How should researchers address potential cross-reactivity when using anti-CD44 antibodies across different species?

When using anti-CD44 antibodies across species, researchers must carefully consider cross-reactivity issues:

• Verify species reactivity:

  • Review validated species reactivity data from manufacturers

  • For example, clone 8E2F3 reacts with human and mouse CD44 but is non-reactive with rabbit CD44

  • Clone IM7 reacts with mouse CD44 but cross-reactivity with other species should be verified

• Perform validation experiments:

  • Test antibodies on positive and negative control samples from the target species

  • Include appropriate isotype controls

  • When possible, use knockout/knockdown samples as definitive negative controls

• Sequence homology analysis:

  • Compare epitope sequences across species (when known)

  • Higher homology suggests greater likelihood of cross-reactivity

  • For example, CD44 shows high sequence conservation in mammals (baboon 94%, canine 93%, equine 93%)

• Titration for each species:

  • Optimal concentrations may differ between species

  • Perform separate titration experiments for each species

• Alternative approaches:

  • If cross-reactivity is problematic, consider species-specific antibodies

  • Use orthogonal methods to confirm findings (e.g., mRNA analysis)

  • For novel species applications, validate with multiple detection methods

Careful validation is particularly important when the antibody has not been explicitly tested in your species of interest, even when sequence homology suggests potential cross-reactivity.

How can biotinylated anti-CD44 antibodies be utilized in multiplex immunofluorescence studies?

Biotinylated anti-CD44 antibodies offer valuable advantages in multiplex immunofluorescence studies when used with appropriate considerations:

• Strategic panel design:

  • Reserve the biotin-streptavidin system for targets requiring signal amplification

  • Plan fluorophore combinations to minimize spectral overlap with the streptavidin-conjugate

  • Consider the brightness of streptavidin-conjugated fluorophores when designing panels

• Sequential staining approaches:

  • When using multiple biotinylated antibodies, employ sequential staining with blocking steps

  • Apply the biotinylated anti-CD44 antibody first, followed by streptavidin-conjugate

  • Block remaining biotin binding sites before applying additional biotinylated antibodies

• Visualization techniques:

  • For co-localization studies, choose spectrally distinct fluorophores

  • CD44 typically shows membrane localization that can be visualized clearly with streptavidin-DyLight 550

  • Include nuclear counterstain (e.g., DAPI) for cellular context

• Controls for multiplex studies:

  • Single-stained controls are essential for assessing spectral overlap

  • Include unstained and isotype controls

  • Consider fluorescence-minus-one (FMO) controls for accurate gating

• Image acquisition and analysis:

  • Adjust exposure settings to prevent oversaturation from amplified signals

  • Consider spectral unmixing for overlapping fluorescence signals

  • Use appropriate analysis software for co-localization quantification

Successful multiplex immunofluorescence has been demonstrated using biotinylated anti-CD44 (8E2F3) in combination with other markers in cancer cell lines, allowing simultaneous assessment of CD44 expression and localization alongside other proteins of interest .

What are important considerations when using biotinylated anti-CD44 antibodies for isolating CD44-positive cells?

When using biotinylated anti-CD44 antibodies for cellular isolation:

• Selection of appropriate clone:

  • Choose a clone that recognizes extracellular epitopes of CD44

  • Consider epitope accessibility in your target cell population

  • Verify the clone doesn't interfere with critical CD44 functions if cells will be used functionally

• Isolation methodologies:

  • Magnetic separation: Use streptavidin-conjugated magnetic beads following biotinylated antibody labeling

  • FACS-based isolation: Use streptavidin-conjugated fluorophores for flow cytometric sorting

  • Column-based separation: Apply biotinylated antibody followed by streptavidin-conjugated beads

• Optimization for cell viability and function:

  • Titrate antibody to minimize potential signaling effects through CD44

  • Optimize incubation times and temperatures

  • Use buffers that maintain cell viability throughout the isolation process

• Validation of isolated populations:

  • Confirm CD44 expression in isolated fraction

  • Assess purity using flow cytometry or immunofluorescence

  • Verify cell viability post-isolation

• Functional considerations:

  • Be aware that antibody binding may trigger CD44 signaling

  • For functional studies, consider removing bound antibodies or using antigen-free systems

  • Include appropriate controls when assessing functional properties

These approaches have been successfully applied in research settings to isolate CD44-positive populations for various downstream applications, including tissue engineering and cancer stem cell research .