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BMC research notes2013; 6; 152; doi: 10.1186/1756-0500-6-152

Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum–a pilot study.

Abstract: Serum amyloid A (SAA) is useful as a diagnostic marker of systemic inflammation in horses, but only heterologous assays based on non-equine calibration and standardization are available for measurements of equine SAA. More accurate measurements could be obtained using purified species-specific SAA in native conformation for assay calibration and standardization. Further knowledge about the biochemical properties of SAA would facilitate a future production of native species-specific calibration material Therefore, the aim of the study was an investigation of the solubility and potentials for purification of equine SAA based on biochemical properties.Freeze dried equine acute phase serum was dissolved in 70% 2-propanol, 8 M urea, and milli-Q water, respectively. Supercritical fluid extraction (SFE), size-exclusive chromatography (FPLC-SEC), and preparative isoelectric focusing (IEF) were performed in the attempt to purify. Immunostaining of IEF blots were used for isoform-specific detection of SAA in the preparations and purity was assessed by silverstained SDS-PAGE. Results: SAA was soluble in 70% 2-propanol, 8 M urea and Milli-Q water. SAA was not separated in the lipophilic or ampipathic fractions following SFE. SAA was included in a FPLC-SEC-fraction of 237 kDa, despite the molecular weight known to be much smaller, suggesting binding to other serum constituents. SAA precipitated following separation of other serum proteins by preparative IEF. Conclusions: No effective purification of SAA was achieved in the present study, but findings important for future investigations were made. The study suggested that SAA is not exclusively hydrophobic, but appears less hydrophobic when interacting with other serum components. These results suggest more complex aspects of solubility than previously believed, and indicate potentials for purification of native SAA.
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Publication Date: 2013-04-16 PubMed ID: 23590853PubMed Central: PMC3637563DOI: 10.1186/1756-0500-6-152Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study aimed to investigate the solubility (ability to dissolve) and potential purification methods of Serum Amyloid A (SAA), a marker of systemic inflammation in horses, from equine acute phase serum (a substance produced during an early stage of disease).

Purpose

  • This research was conducted to improve the accuracy of measurements detecting systemic inflammation in horses, particularly by using a purified version of SAA, a protein that responds during the early phase of inflammation or infection.
  • Currently, heterologous assays not specific to horses are used making measurements of equine SAA less accurate.
  • Understanding the biochemical properties of SAA may allow for the development of native species-specific calibration material, improving assay performance.

Methodology

  • The team used freeze-dried equine acute phase serum that was dissolved using 70% 2-propanol, 8 M urea, and milli-Q water.
  • They tried to purify SAA using supercritical fluid extraction (SFE), size-exclusion chromatography (also known as FPLC-SEC), and preparative isoelectric focusing (IEF).
  • They then used immunostaining of IEF blots for isoform-specific detection of SAA in the preparations and assessed purity using silver-stained SDS-PAGE, a common technique for protein analysis.

Results

  • The researchers found that SAA could dissolve in 70% 2-propanol, 8 M urea, and milli-Q water.
  • However, they also found that SAA didn’t separate out in lipophilic or amphipathic fractions after SFE.
  • During size-exclusion chromatography, SAA was part of a 237 kDa fraction, despite its molecular weight being significantly lower, suggesting it binds to other serum constituents.
  • During preparative IEF, other serum proteins separated but SAA precipitated.

Conclusions

  • Purification of SAA was not achieved in this study, but the research uncovered important findings for future investigations.
  • The study indicated that the properties of SAA are not only hydrophobic as previously believed, but also appear less hydrophobic (water repelling) when interacting with other serum components.
  • The newfound complexities of SAA’s solubility could offer potential pathways for its purification in future studies which would enhance its use as a diagnostic tool in equine medicine.

Cite This Article

APA
Christensen MB, Sørensen JC, Jacobsen S, Kjelgaard-Hansen M. (2013). Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum–a pilot study. BMC Res Notes, 6, 152. https://doi.org/10.1186/1756-0500-6-152

Publication

BMC research notes
ISSN: 1756-0500
NlmUniqueID: 101462768
Country: England
Language: English
Volume: 6
Pages: 152

Researcher Affiliations

Christensen, Michelle B
  • Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Groennegaardsvej 3, ground floor, Frederiksberg C 1870, Denmark. mic@sund.ku.dk
Sørensen, Jens Christian
    Jacobsen, Stine
      Kjelgaard-Hansen, Mads

        MeSH Terms

        • Acute-Phase Reaction / blood
        • Acute-Phase Reaction / veterinary
        • Animals
        • Calibration
        • Chromatography, Gel
        • Horses
        • Hydrophobic and Hydrophilic Interactions
        • Inflammation / blood
        • Inflammation / veterinary
        • Isoelectric Focusing
        • Pilot Projects
        • Serum Amyloid A Protein / isolation & purification
        • Silver Staining
        • Solubility

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        Citations

        This article has been cited 2 times.
        1. Jacobsen S, Vinther AM, Kjelgaard-Hansen M, Nielsen LN. Validation of an equine serum amyloid A assay with an unusually broad working range. BMC Vet Res 2019 Dec 19;15(1):462.
          doi: 10.1186/s12917-019-2211-3pubmed: 31856804google scholar: lookup
        2. Souto PC, Santos MR, Orozco AMO, Bento LD, Ramirez-Lopez CJ, Girardi FM, Machado JCA, de Oliveira LL, da Fonseca LA. Enzyme-Linked Immunosorbent Assay (ELISA) Development for Equine Serum Amyloid A (SAA) Determination Using Recombinant Proteins. Methods Protoc 2025 Apr 7;8(2).
          doi: 10.3390/mps8020037pubmed: 40278511google scholar: lookup
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