Recombinant Human ICAM-1 Fc Chimera

Using Recombinant Human ICAM-1 in research applications is essential for studying cell adhesion, immune cell trafficking, inflammation, pathogen interactions, and for developing targeted drug delivery systems. Recombinant ICAM-1 provides a consistent, well-characterized, and species-specific tool for in vitro and in vivo experiments, enabling precise mechanistic studies and translational research.

Key reasons to use recombinant human ICAM-1 include:

• Modeling Human-Specific Interactions: Recombinant human ICAM-1 allows you to study interactions with human leukocyte integrins (such as LFA-1 and Mac-1), which are critical for immune cell adhesion, transmigration, and immune synapse formation. This is particularly important because ICAM-1 is upregulated during inflammation and is central to leukocyte recruitment and tissue infiltration.

• Pathogen Binding Studies: Human ICAM-1 serves as a receptor for major group human rhinoviruses and other pathogens, making recombinant protein essential for virology and infectious disease research.

• Drug Discovery and Targeted Delivery: ICAM-1 is a validated target for drug delivery systems (DDS), especially in inflammatory and immune-mediated diseases. Recombinant ICAM-1 enables the screening and optimization of antibodies, nanocarriers, aptamers, and other ligands for specific binding and internalization.

• Bioassays and Functional Studies: Recombinant ICAM-1 is used in cell adhesion assays, migration studies, and to assess the functional consequences of ligand binding, such as T cell activation or endothelial barrier modulation.

• Structural and Biophysical Analysis: The recombinant protein is critical for structural studies (e.g., crystallography, cryo-EM) to elucidate binding domains and mechanisms of interaction with integrins or pathogens.

• Reproducibility and Standardization: Recombinant proteins offer batch-to-batch consistency, defined post-translational modifications (when expressed in mammalian systems), and eliminate variability inherent to primary cell or tissue-derived ICAM-1.

• Versatility in Conjugation and Engineering: Recombinant ICAM-1 can be engineered with tags, Fc domains, or conjugated to other molecules (e.g., aptamers, fluorophores) for advanced applications in imaging, biosensing, or therapeutic development.

In summary, recombinant human ICAM-1 is a versatile and indispensable reagent for dissecting the molecular and cellular mechanisms of adhesion, inflammation, infection, and for advancing translational research in immunology, oncology, and drug delivery.

Yes, recombinant human ICAM-1 can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to the assay system. Recombinant ICAM-1 is commonly used as a standard in commercial ELISA kits for quantitative detection of soluble ICAM-1 in biological samples.

Key considerations and best practices:

• Standard Curve Preparation: Recombinant human ICAM-1 is typically reconstituted and serially diluted to generate a standard curve, which is essential for quantifying unknown samples in ELISA. The concentration range should match the assay’s sensitivity and expected sample levels.

• Assay Compatibility: Ensure the recombinant ICAM-1 standard is compatible with the antibodies used in your ELISA. Most sandwich ELISA kits for human ICAM-1 are designed to recognize both natural and recombinant forms, and validation data often show parallelism between curves generated from recombinant and natural ICAM-1.

• Protein Characteristics: The recombinant standard should have the same epitope availability and post-translational modifications (if relevant) as the native protein in your samples. Most commercial standards are produced in mammalian or insect cells to ensure proper folding and glycosylation, but this should be confirmed for your specific application.

• Calibration and Quantification: Use the recombinant standard to calibrate your assay and quantify ICAM-1 concentrations in unknown samples by comparing their absorbance values to the standard curve. Always run the standard curve in parallel with your samples for each assay.

• Validation: It is recommended to validate the use of your recombinant ICAM-1 standard by checking for parallelism between the standard curve and serial dilutions of a representative sample. This ensures accurate quantification and confirms that the assay detects both recombinant and endogenous ICAM-1 equivalently.

• Documentation: Record the source, lot number, and concentration of the recombinant standard used, as minor differences in protein preparation can affect quantification.

Summary Table: Use of Recombinant Human ICAM-1 as ELISA Standard

In conclusion, recombinant human ICAM-1 is widely accepted and scientifically appropriate as a standard for ELISA quantification, provided it is validated for your specific assay system and sample type.

Recombinant Human ICAM-1 has been validated for several key applications in published research, primarily in studies involving cell adhesion, immune cell trafficking, and molecular interaction assays.

Validated Applications:

• Bioassay:
  The most common application is as a substrate in cell adhesion assays, where recombinant ICAM-1 is immobilized to measure the adhesion of various cell types, such as T cells, neutrophils, and leukemic cells. These assays are used to study integrin-mediated adhesion, immune cell recruitment, and the effects of signaling molecules on cell adhesion.

• Flow Cytometry:
  Recombinant ICAM-1 has been used as a ligand in flow cytometry to analyze the binding and activation of leukocyte integrins (e.g., LFA-1) and to characterize cell surface interactions.

• Surface Plasmon Resonance (SPR):
  It is employed in SPR assays to quantify the binding kinetics and affinity between ICAM-1 and its ligands, such as antibodies, aptamers, or integrins.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  Recombinant ICAM-1 serves as a positive control or standard in ELISA to detect soluble ICAM-1 or to measure antibody binding in immunological studies.

• Western Blotting:
  Used as a control protein to validate antibody specificity and to quantify ICAM-1 expression in various samples.

• Mass Cytometry (CyTOF):
  Applied in high-dimensional immune profiling to study ICAM-1 expression and its role in cell signaling and immune responses.

• Spatial Biology and Vaccine Development:
  Utilized in advanced imaging and vaccine research to investigate ICAM-1’s distribution and function in tissues.

• Drug Delivery and Targeting Studies:
  Recombinant ICAM-1 is used to validate targeting strategies for drug delivery systems, such as nanoparticles or aptamers, especially in the context of inflammatory diseases and enzyme replacement therapies.

Representative Published Research Applications:

• Cell Adhesion and Migration:
  Studies have used recombinant ICAM-1 to investigate neutrophil and T cell adhesion, migration, and signaling pathways relevant to inflammation, autoimmune disease, and cancer.

• Aptamer and Antibody Binding:
  Recombinant ICAM-1 is a target for the selection and validation of DNA aptamers and monoclonal antibodies, which are then used for therapeutic or diagnostic purposes.

• Targeted Drug Delivery:
  ICAM-1-targeted nanocarriers have been validated for intracellular trafficking and delivery of therapeutic enzymes in lysosomal storage diseases and other conditions.

Summary Table of Validated Applications

Additional Notes:

• Recombinant ICAM-1 is frequently used in both in vitro and in vivo models to study immune cell interactions, inflammatory responses, and as a target for therapeutic development.
• It is also a key reagent in the development and validation of diagnostic tools and targeted therapies, including aptamer-based and antibody-based approaches.

If you require protocol details or specific experimental setups for any of these applications, please specify the intended use.

To reconstitute and prepare Recombinant Human ICAM-1 protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS to a concentration of at least 100–400 μg/mL, depending on the specific product and downstream application. Use sterile technique throughout to maintain protein integrity and prevent contamination.

Step-by-step protocol:

• Equilibrate the vial to room temperature before opening to minimize condensation.
• Add sterile PBS (or sterile deionized water if specified by the manufacturer) directly to the lyophilized protein. For most recombinant human ICAM-1 proteins, a concentration of 400 μg/mL in sterile PBS is recommended. Some protocols suggest reconstitution at 100–1000 μg/mL; check your product datasheet for the optimal range.
• Gently mix by pipetting up and down or by gentle inversion. Avoid vigorous vortexing, which can denature the protein.
• Allow the protein to dissolve at room temperature for 10–30 minutes. If necessary, briefly centrifuge to collect the solution at the bottom of the vial.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which can degrade protein activity.
• Store aliquots at –20 °C or –80 °C for long-term storage. Avoid repeated freeze-thaw cycles.

Preparation for cell culture experiments:

• Dilute the reconstituted stock to the desired working concentration using sterile PBS or cell culture medium immediately before use. Typical working concentrations for coating plates or functional assays range from 1–20 μg/mL, but optimal conditions should be determined empirically for your specific assay.
• If using for plate coating (e.g., adhesion assays), incubate plates with the diluted protein (e.g., 12.5 μg/mL, 100 μL/well) at 4 °C overnight or at room temperature for 1–2 hours, then wash with PBS before adding cells.
• If adding directly to cell cultures, ensure the protein is endotoxin-free and compatible with your cell type and assay conditions.

Additional notes:

• Always consult the specific product datasheet for any unique instructions or buffer requirements, as formulations may vary (e.g., presence of stabilizers like trehalose or sorbitol).
• If the protein is provided in a buffer containing glycerol or other additives, ensure compatibility with your downstream application.
• For highly sensitive applications, consider filtering the reconstituted protein through a 0.2 μm sterile filter.

Summary of key points:

• Reconstitute in sterile PBS (typically 400 μg/mL).
• Mix gently and allow to dissolve fully.
• Aliquot and store at –20 °C or –80 °C.
• Dilute to working concentration immediately before use.
• Avoid repeated freeze-thaw cycles.

If your application requires a different buffer or concentration, adjust accordingly and always verify with your product’s technical datasheet.