Recombinant Human Apo-Serum Amyloid A (Apo-SAA)

Recombinant Human Apo-Serum Amyloid A (Apo-SAA) is widely used in research applications because it is a critical acute-phase apolipoprotein involved in inflammation, immune cell recruitment, and amyloidosis, making it highly relevant for studies on inflammatory diseases, immune responses, and biomarker development.

Key scientific reasons to use recombinant Apo-SAA in research include:

• Modeling Acute and Chronic Inflammation: Apo-SAA is a major acute-phase protein whose levels rise dramatically during inflammation, infection, trauma, and malignancy. Recombinant Apo-SAA allows controlled in vitro and in vivo studies of its role in both protective acute responses and detrimental chronic inflammation.
• Immune Cell Chemotaxis and Activation: Recombinant Apo-SAA induces directional migration (chemotaxis) of monocytes, neutrophils, and T lymphocytes, modulates adhesion molecule expression, and enhances immune cell recruitment to sites of injury or infection. These properties are essential for dissecting mechanisms of immune cell trafficking and inflammatory signaling.
• Cytokine Induction and Immune Modulation: Apo-SAA stimulates the production of both pro-inflammatory (e.g., TNF-α, IL-6, IL-8) and anti-inflammatory (e.g., IL-10) cytokines in immune cells, and can skew macrophages toward regulatory (M2) phenotypes. Recombinant protein enables precise mechanistic studies of these effects.
• Biomarker and Therapeutic Target Research: SAA is a sensitive biomarker for disease activity in rheumatic and autoinflammatory diseases, often outperforming CRP or ESR in clinical scenarios. Recombinant Apo-SAA is used to develop and validate assays, and to investigate its potential as a therapeutic target in conditions such as rheumatoid arthritis, COVID-19, and metabolic diseases.
• Amyloidosis and Lipoprotein Interaction Studies: Apo-SAA is central to amyloid formation and interacts with HDL and other lipoproteins, affecting their function and structure. Recombinant Apo-SAA is essential for studying amyloidogenesis and lipoprotein biology.
• Innate Immunity and Antimicrobial Activity: SAA participates in pathogen recognition and has demonstrated antibacterial and antifungal activities, making recombinant Apo-SAA valuable for exploring innate immune mechanisms.

Best practices for using recombinant Apo-SAA include:

• Employing it in dose-response and time-course experiments to model physiological and pathological concentrations.
• Using it in cell-based assays to study chemotaxis, cytokine induction, and immune cell differentiation.
• Applying it in animal models to investigate its systemic effects on inflammation, immune regulation, and amyloidosis.

In summary, recombinant human Apo-SAA is a versatile tool for investigating the molecular and cellular mechanisms of inflammation, immune regulation, and amyloid diseases, and for developing sensitive biomarkers and therapeutic strategies.

Recombinant Human Apo-Serum Amyloid A (Apo-SAA) can be used as a standard for quantification or calibration in ELISA assays, provided it is well-characterized, pure, and matches the isoform(s) detected by your assay. Several commercial ELISA kits for human SAA use recombinant SAA as their calibration standard, demonstrating its suitability for this purpose.

Key considerations for use as a standard:

• Isoform specificity: Ensure your recombinant Apo-SAA matches the isoform(s) recognized by the antibodies in your ELISA. Most kits target SAA1, SAA2, or both, and some distinguish between full-length and truncated forms.
• Purity and quantification: The recombinant protein should be highly pure (typically >95%) and accurately quantified, as impurities or inaccurate concentration will affect your standard curve and quantification.
• Matrix effects: If your samples are in serum, plasma, or other complex matrices, consider preparing your standard curve in the same matrix or using a suitable diluent to minimize matrix effects.
• Validation: It is best practice to validate the performance of your recombinant standard in your specific ELISA system. This includes confirming parallelism between the standard curve and serially diluted native samples, and ensuring recovery and linearity are acceptable.

Supporting details:

• Many commercial SAA ELISA kits use recombinant human SAA as their standard, and these kits are validated for quantification in serum, plasma, and other fluids.
• Published studies and kit protocols confirm that calibration with recombinant SAA is standard practice, provided the recombinant protein is well-characterized and matches the assay’s target.
• Some kits and protocols recommend using clinical samples as standards for certain applications, but this is less common and primarily for animal SAA assays.

Summary:
You can use recombinant human Apo-SAA as a standard for ELISA quantification if it is pure, accurately quantified, and matches the isoform(s) detected by your assay. Always validate its performance in your specific ELISA system to ensure accurate quantification.

Recombinant Human Apo-Serum Amyloid A (Apo-SAA) has been validated for several key applications in published research, primarily in studies of inflammation, immune response, and disease biomarker development.

Validated Applications:

• Functional Assays: Recombinant Apo-SAA is widely used in in vitro and in vivo functional assays to study its biological activities, such as chemoattraction of monocytes and neutrophils, induction of cytokine production, and modulation of immune cell responses.
• Cell Recruitment Studies: Apo-SAA has been validated for its ability to induce recruitment of neutrophils and mononuclear cells, both directly and in cooperation with chemokines (e.g., CXCL8), often via FPR2-dependent mechanisms.
• Antimicrobial Activity Assays: Recombinant Apo-SAA is used to assess its role as a pattern recognition molecule, binding to bacterial outer membrane proteins (e.g., OmpA) and promoting phagocytosis and bacterial killing, including disruption of bacterial membranes.
• Inflammatory Disease Models: Apo-SAA is employed in models of inflammatory diseases such as inflammatory bowel disease (IBD), rheumatoid arthritis (RA), and atherosclerosis to study its effects on disease activity, immune cell infiltration, and cytokine induction.
• Biomarker Validation: Recombinant Apo-SAA is used in quantitative assays (e.g., ELISA, mass spectrometric immunoassay) to validate its utility as a biomarker for disease activity, prognosis, and response to therapy in conditions like IBD, RA, and other rheumatic diseases.
• Lipid Metabolism and HDL Remodeling: Apo-SAA is validated in studies investigating its role in HDL remodeling, cholesterol recycling, and removal of toxic lipids during inflammation.
• Endothelial Dysfunction and Atherosclerosis: Apo-SAA is used to study its impact on endothelial cells, nitric oxide synthase activity, and the development of atherosclerotic plaques.

Supporting Details:

• Recombinant Apo-SAA is typically expressed in E. coli and purified to high homogeneity for use in these assays.
• Its chemoattractant activity is validated across a range of concentrations, and its effects on immune cell migration and cytokine induction are well-documented.
• Functional validation includes both direct effects (e.g., cell recruitment, antimicrobial activity) and indirect effects (e.g., enhancement of chemokine-mediated responses, modulation of T-cell differentiation).

Summary Table of Key Applications

These applications are supported by multiple peer-reviewed studies and reviews, confirming the utility of recombinant human Apo-SAA in both basic and translational research contexts.

To reconstitute and prepare Recombinant Human Apo-Serum Amyloid A (Apo-SAA) protein for cell culture experiments, dissolve the lyophilized protein in sterile, high-purity water at a minimum concentration of 100 µg/mL. Gently mix to ensure complete dissolution, as the protein may appear as a film at the bottom of the vial. Avoid vigorous vortexing to prevent protein denaturation.

Detailed protocol and best practices:

• Reconstitution:

  • Briefly centrifuge the vial before opening to collect all material at the bottom.
  • Add sterile distilled water (or 10 mM PBS, pH 7.4, if specified for your application) to achieve at least 100 µg/mL final concentration.
  • Gently mix by pipetting or slow inversion. Do not vortex.
  • Allow several minutes for complete reconstitution.

• Carrier protein (optional):

  • For extended storage or to prevent adsorption to plastic, add 0.1% BSA (bovine serum albumin) after reconstitution.

• Aliquoting and storage:

  • Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
  • Store aliquots at –20°C or –80°C for long-term stability.
  • For short-term use, reconstituted protein is stable for up to 1 week at 2–8°C.

• Preparation for cell culture:

  • Dilute the stock solution into your cell culture medium (e.g., RPMI-1640 or DMEM) immediately before use.
  • If using serum-free medium, consider adding a carrier protein (e.g., HSA or BSA) to minimize loss due to adsorption.
  • Filter sterilize if necessary, using a low-protein binding filter.

• Quality control:

  • Confirm protein concentration by absorbance at 280 nm or a suitable protein assay.
  • Check for endotoxin contamination if sensitive cell types are used; further purification may be required for ultra-low endotoxin applications.

Summary of key steps:

• Dissolve in sterile water at ≥100 µg/mL.
• Gently mix; do not vortex.
• Add 0.1% BSA for storage.
• Aliquot and freeze at –20°C or –80°C.
• Dilute into cell culture medium just before use.

These steps ensure optimal solubility, stability, and biological activity of recombinant Apo-SAA for cell-based assays.