Recombinant Human CD22β/Siglec-2

Recombinant Human CD22β/Siglec-2 is a valuable tool for a wide range of research applications due to its well-characterized biological activity, structural features, and relevance in both physiological and pathological contexts. Here are several compelling reasons to use Recombinant Human CD22β/Siglec-2 in your research:

1. Well-Defined Biological Function

CD22 (Siglec-2) is a key inhibitory receptor on B cells, regulating B cell activation and serving as a co-receptor for the B cell receptor (BCR). It binds preferentially to α2,6-linked sialic acid ligands, making it essential for studying B cell signaling, immune regulation, and cell-cell interactions.

2. High Purity and Bioactivity

Recombinant CD22β/Siglec-2 proteins are typically produced with high purity (>95%) and guaranteed bioactivity, as confirmed by functional assays such as ELISA and SDS-PAGE. This ensures reliable and reproducible results in your experiments.

3. Consistent Quality Across Lots

Stringent quality control (QC) testing is performed on each production lot, ensuring lot-to-lot consistency in both activity and purity. This is critical for long-term studies and comparative experiments.

4. Versatile Applications

Recombinant CD22β/Siglec-2 can be used in a variety of research settings, including:

• Binding and interaction studies: Investigate ligand-receptor interactions, especially with sialylated glycans.
• Functional assays: Study B cell signaling, inhibition, and modulation.
• In vitro and in vivo studies: Suitable for cell-based assays, apoptosis studies, and animal models.
• Drug and therapeutic development: Useful for screening inhibitors, designing targeted therapies, and evaluating immunotoxins or CAR-T cell constructs.

5. Structural Insights

The recombinant protein contains the full extracellular domain, including the N-terminal Ig-like V-type domain and multiple Ig-like C2-type domains, allowing for detailed structural and functional studies. This is particularly useful for understanding ligand binding, domain interactions, and the effects of post-translational modifications.

6. Relevance to Disease Models

CD22 is a validated target in B cell malignancies (e.g., B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma) and autoimmune disorders. Using recombinant CD22β/Siglec-2 enables the development and testing of novel therapeutics, such as CAR-T cells, immunotoxins, and small molecule inhibitors.

7. Facilitates High-Throughput Screening

The availability of recombinant CD22β/Siglec-2 in bulk quantities makes it suitable for high-throughput screening of potential ligands, inhibitors, and therapeutic candidates.

8. Supports Personalized Medicine Approaches

The use of recombinant CD22β/Siglec-2 in cell-based assays and NMR studies allows for the direct assessment of ligand and inhibitor affinity using patient-derived cells, facilitating the design of tailored therapeutics and personalized medicine strategies.

9. Compatibility with Advanced Techniques

Recombinant CD22β/Siglec-2 is compatible with advanced techniques such as NMR spectroscopy, surface plasmon resonance (SPR), and flow cytometry, enabling detailed molecular and cellular analyses.

10. Broad Research Community Support

CD22/Siglec-2 is well-studied and widely used in the scientific community, with extensive literature and established protocols available to support your research.

In summary, Recombinant Human CD22β/Siglec-2 is a robust, reliable, and versatile reagent that can significantly enhance the quality and scope of your research in immunology, oncology, and drug development.

Yes, recombinant human CD22β/Siglec-2 can be used as a standard for quantification and calibration in ELISA assays, provided certain conditions are met.

Suitability as an ELISA Standard

Recombinant CD22β/Siglec-2 proteins are appropriate for ELISA calibration applications. The predominant CD22β isoform encodes an 847 amino acid polypeptide with an N-terminal Ig-like V-type domain, six Ig-like C2-type domains, a transmembrane region, and a cytoplasmic tail containing four immunoreceptor tyrosine-based inhibition motifs (ITIMs). This structural composition makes it suitable for recognition by antibody pairs used in sandwich ELISA formats.

Key Considerations for Use

Protein Formulation: When selecting recombinant CD22β/Siglec-2 for use as an ELISA standard, choose the formulation with carrier protein (such as BSA) rather than carrier-free preparations. Carrier proteins stabilize the recombinant protein and prevent non-specific adsorption to assay surfaces, which is critical for maintaining accurate standard curves.

Protein Purity and Characterization: Ensure the recombinant protein has high purity (ideally >90%) and has been characterized for molecular weight and biological activity. The protein should be verified through appropriate methods such as SDS-PAGE or mass spectrometry to confirm its identity and integrity.

Concentration Determination: Before preparing your standard curve, accurately determine the protein concentration using appropriate analytical methods such as HPLC or spectrophotometric analysis. This ensures that your calibration points are precise and traceable.

Standard Curve Preparation: Prepare serial dilutions from a stock solution of known concentration, typically spanning a range that encompasses your expected sample values. Most commercial ELISA kits using CD22β/Siglec-2 standards operate within detection ranges of approximately 3.13-200 ng/mL or 94-6000 pg/mL, depending on the assay format.

Quality Control Recommendations

Validate your standard curve by confirming linearity and reproducibility across multiple assay runs. Intra-assay precision should be <8% coefficient of variation, while inter-assay precision should be <10%. Recovery rates in biological matrices (serum, plasma) should fall within 85-115%.

Recombinant Human CD22β/Siglec-2 has been validated for several applications in published research, primarily focusing on its role as a B cell-specific receptor and its utility in both basic research and therapeutic development. Key applications include:

1. Bioassays and Functional Studies:

   • Used in bioassays to study B cell activation, inhibition, and signaling pathways. For example, CD22 ligands conjugated to antibodies have been shown to suppress B cell activation, demonstrating its role in immune modulation (e.g., suppression of B cell activation by anti-IgD-CD22L conjugates) .
   • Employed in functional ELISA and other binding assays to assess ligand-receptor interactions and to characterize high-affinity ligands for CD22/Siglec-2 [3, 9].

2. Therapeutic Target Validation:

   • CD22/Siglec-2 is a validated target for chimeric antigen receptor (CAR) T cell therapies in B cell malignancies, including B-cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin lymphoma (NHL) [2, 4].
   • Used in the development and testing of antibody-drug conjugates (ADCs) and immunotoxins, such as inotuzumab ozogamicin and moxetumomab pasudotox, for the treatment of CD22-expressing B cell malignancies [4, 12].

3. Structural and Ligand Binding Studies:

   • Applied in structural characterization studies using techniques like NMR to investigate the molecular interactions between CD22/Siglec-2 and its ligands, including high-affinity sialosides and synthetic ligands [3, 6, 9].
   • Used to assess the affinity and specificity of novel ligands and inhibitors for CD22/Siglec-2, supporting the design of targeted therapeutics [3, 6, 9].

4. In Vivo Studies:

   • Utilized in in vivo assays to evaluate the efficacy and safety of CD22-targeted therapies, including CAR T cells and immunotoxins, in animal models of B cell malignancies [1, 2].

5. Apoptosis and Cell Death Assays:

   • Employed in apoptosis assays to study the effects of CD22-targeted therapies on B cell survival and death, particularly in the context of antibody-dependent cellular cytotoxicity (ADCC) and immunotoxin-induced cell killing [2, 4].

These applications highlight the versatility of Recombinant Human CD22β/Siglec-2 in both research and clinical settings, particularly in the fields of immunology, oncology, and drug development.

To reconstitute and prepare Recombinant Human CD22β/Siglec-2 protein for cell culture experiments, follow these steps to ensure protein stability and biological activity:

1. Centrifuge the vial before opening to collect all lyophilized powder at the bottom.
2. Warm the vial to room temperature before opening to minimize condensation.
3. Add sterile distilled water or PBS:
   • For most applications, reconstitute to a final concentration of 0.1–0.5 mg/mL in sterile distilled water.
   • Some protocols recommend sterile PBS at 100 μg/mL for certain Fc-chimera forms. Always check the specific datasheet for your protein variant.
   • Do not exceed 1 mg/mL to avoid solubility issues.
4. Gently mix by swirling or inverting the vial. Do not vortex or pipette vigorously to prevent protein denaturation.
5. Incubate at 4°C for several hours or overnight if solubility is incomplete.
6. Aliquot the reconstituted protein to minimize freeze-thaw cycles. For long-term storage, add a carrier protein (e.g., 0.1% BSA, 5% HSA, 10% FBS, or 5% trehalose) to enhance stability.
7. Storage:
   • Short-term (≤1 month): 2–8°C under sterile conditions.
   • Long-term (≤3 months): –20°C to –70°C.
   • Avoid repeated freeze-thaw cycles as this can denature the protein.
8. Confirm protein recovery by running a small aliquot on SDS-PAGE if needed.

Additional notes for cell culture use:

• If using in cell-based assays, ensure the buffer is compatible with your cells (e.g., avoid high salt or non-physiological pH).
• If the protein is carrier-free and you require higher stability or lower adsorption to plastic, supplement with a carrier protein as above.
• Always filter-sterilize the final solution if sterility is required and not already ensured by the reconstitution process.

Summary of best practices:

• Centrifuge, reconstitute gently, aliquot, and store appropriately.
• Avoid vigorous mixing and repeated freeze-thaw cycles.
• Use carrier proteins for long-term stability if compatible with your assay.

These steps will help maintain the integrity and activity of recombinant CD22β/Siglec-2 for reliable cell culture experiments.