Recombinant Human CD23

Recombinant human CD23 (also known as FcεRII) is a valuable tool for immunological research due to its critical roles in immune regulation and its well-characterized functional properties.

Functional Significance

CD23 serves as a low-affinity Fc receptor for IgE, making it essential for studying IgE-mediated immune responses, allergic reactions, and parasite immunity. The protein exhibits a versatile ligand-binding repertoire that includes IgE, CD21, MHC class II, and integrins, enabling investigation of multiple immune regulatory pathways. Beyond its role as an IgE receptor, CD23 functions in B cell growth, differentiation, and IgE production, with both membrane-bound and soluble forms (sCD23) exhibiting pleiotropic cytokine-like activities that influence lymphocyte survival and myeloid lineage development.

Research Applications

Recombinant CD23 is particularly valuable for several experimental approaches:

Antibody Development and Validation — The protein serves as an ideal antigen for antibody screening, immunization studies, and as a blocking agent to confirm antibody specificity. It can be used in blocking experiments with corresponding antibodies, where a 100-fold molar excess is recommended for immunohistochemistry, immunocytochemistry, and Western blot applications.

Diagnostic and Clinical Research — CD23 functions as a reliable immunophenotypic marker to distinguish chronic lymphocytic leukemia (CLL), which is CD23-positive, from other B-cell malignancies such as mantle cell lymphoma. This makes it valuable for both research into B-cell-related diseases and diagnostic development.

Standardized Assays — Recombinant CD23 is suitable for use as an ELISA standard and capture reagent, particularly when formulated with carrier proteins for cell and tissue culture applications. The protein can be biotinylated or engineered with affinity tags for enhanced detection in binding assays and surface plasmon resonance studies.

Practical Advantages

Recombinant CD23 provides consistent, reproducible results across experiments due to standardized production from mammalian cell expression systems. The protein is typically available in multiple formats—carrier-free, with BSA, or with affinity tags—allowing researchers to select the optimal formulation for their specific experimental needs. Its well-characterized structure, including the C-type lectin domain and coiled-coil topology, enables predictable interactions in research applications.

Yes, recombinant human CD23 can generally be used as a standard for quantification or calibration in ELISA assays, provided it is well-characterized, matches the assay's requirements, and is compatible with the antibodies used in your specific ELISA format. This is a common practice in commercial ELISA kits for CD23 quantification, where recombinant human CD23 is supplied as the standard.

Key considerations and supporting details:

• Commercial ELISA kits for human CD23 routinely use recombinant human CD23 as the standard. These standards are typically the extracellular domain of CD23, produced in mammalian expression systems to ensure proper folding and post-translational modifications.
• The standard must be compatible with your assay antibodies. Most sandwich ELISAs for CD23 are designed to detect both natural and recombinant forms, as long as the recombinant protein contains the relevant epitopes recognized by the capture and detection antibodies.
• Standard curve generation: The recombinant CD23 standard is serially diluted to generate a standard curve, which is then used to interpolate the concentration of CD23 in unknown samples.
• Validation: It is important to confirm that your recombinant CD23 standard behaves similarly to native CD23 in your assay matrix (e.g., serum, plasma, cell culture supernatant). This ensures accurate quantification.
• Isoform and domain considerations: CD23 exists as different isoforms (CD23a, CD23b) and as both membrane-bound and soluble forms. Most ELISA kits use the soluble extracellular domain as the standard, which is suitable for quantifying soluble CD23 in biological samples.
• Research use only: Most recombinant standards and ELISA kits are for research use only and not for diagnostic purposes.

Best practices:

• Use a recombinant CD23 standard that matches the sequence and post-translational modifications of the CD23 you expect in your samples, if possible.
• Prepare and store the standard according to the manufacturer’s or protocol’s instructions to maintain stability and activity.
• Validate the standard curve in your specific assay context, especially if using a recombinant standard from a different source than the kit manufacturer.

Limitations:

• If your ELISA is designed to detect a specific isoform or post-translational modification, ensure your recombinant standard matches this specificity.
• Some recombinant proteins may not fully mimic the native protein’s conformation or glycosylation, which could affect antibody recognition in rare cases.

In summary: Recombinant human CD23 is widely used as a standard for ELISA quantification, but always confirm compatibility with your assay system and validate performance in your specific application.

Recombinant Human CD23 has been validated for several key applications in published research, primarily in immunology and allergy studies. The most commonly validated applications include:

• ELISA (Enzyme-Linked Immunosorbent Assay): Used as a capture antigen or standard for quantifying CD23 or IgE interactions in serum and other biological samples.
• Western Blot (WB): Utilized as a control protein to confirm antibody specificity and for detection of CD23 in cell lysates.
• Immunohistochemistry (IHC) and Immunocytochemistry (ICC): Applied as a blocking agent to validate antibody specificity in tissue or cell staining experiments.
• Bioassays: Employed to study functional interactions, such as IgE binding, B cell differentiation, and cytokine release.
• Structural and Binding Studies: Used in NMR spectroscopy and other biophysical assays to characterize CD23 structure and its interactions with IgE and CD21.
• Cell-based Assays: Incorporated in experiments investigating CD23-mediated signaling, antigen uptake, and presentation, as well as CAR-T cell detection and immunotherapy models.

Supporting details:

• In ELISA, recombinant CD23 is validated for use as a capture antigen to measure soluble CD23 or IgE in serum, and as a standard in allergy biomarker assays.
• In Western blot and IHC/ICC, recombinant CD23 serves as a blocking control to confirm antibody specificity, often by pre-incubation with the antibody to prevent non-specific binding.
• Functional bioassays utilize recombinant CD23 to assess its role in IgE binding, B cell activation, and modulation of immune responses, including inhibition of IgE synthesis and blocking of IgE binding to immune cells.
• Structural studies have used recombinant fragments of CD23 to elucidate its binding sites and interactions with IgE and CD21, providing insight into its mechanism of action.
• Cell-based assays and immunotherapy research have validated recombinant CD23 in models of chronic lymphocytic leukemia (CLL), CAR-T cell detection, and studies of CD23-mediated immune regulation.

Additional relevant information:

• Recombinant CD23 is also used in research on allergic diseases, autoimmune conditions, and B cell malignancies, reflecting its importance as a low-affinity IgE receptor and regulator of immune responses.
• Both membrane-bound and soluble forms of recombinant CD23 are studied, with soluble CD23 (sCD23) being particularly relevant for blocking experiments and as a disease biomarker.
• The protein is frequently validated in human, mouse, and rat systems due to its conserved function across species.

These applications are well-supported in the literature and are foundational for studies investigating B cell biology, IgE regulation, and therapeutic antibody development.

Reconstitution Protocol

Initial Preparation

Begin by centrifuging the vial before opening to collect any lyophilized powder that may have adhered to the tube walls or cap during transportation. This ensures you recover the full amount of protein.

Reconstitute the lyophilized CD23 protein to a concentration of 0.1–0.5 mg/mL in sterile distilled water or sterile PBS, depending on your specific product formulation. Allow 15–30 minutes for reconstitution at room temperature with gentle agitation. Avoid vigorous vortexing or pipetting, as this can cause foaming and protein denaturation.

Storage Conditions

Short-term Storage

After reconstitution, the protein solution remains stable at 2–8°C for up to one week, which is suitable for experiments with 5–7 day cycles. For immediate use in cell culture experiments, simply remove the required amount from the refrigerator and add it directly to your culture medium.

Long-term Storage

Store the lyophilized protein at −20°C to −80°C for up to one year from the date of receipt. After reconstitution, the protein is stable at −20°C for approximately 3 months. Minimize repeated freeze-thaw cycles, as these degrade protein activity.

Optimization for Cell Culture Applications

Carrier Protein Addition

For diluted working solutions intended for cell culture, add a carrier protein to prevent the protein from adhering to tube walls and losing activity. Recommended carrier proteins include:

• 0.1% bovine serum albumin (BSA)
• 10% fetal bovine serum (FBS)
• 5% human serum albumin (HSA)

For serum-free culture or in vivo experiments, use trehalose as the carrier protein instead, as it avoids introducing animal or human proteins into your system.

Aliquoting Strategy

Divide the reconstituted protein into smaller aliquots and store at −20°C to −80°C. This approach minimizes exposure to freeze-thaw cycles and maintains protein stability throughout your experimental period.

Quality Considerations

Ensure the protein meets purity standards of ≥95% as determined by SDS-PAGE and has endotoxin levels <0.1 EU/μg by LAL method. The recombinant protein is typically expressed in HEK293 cells with a 6×His tag at the N-terminus, containing amino acids Asp48–Ser321 of human CD23.