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Extraction,isolation, and concentration of calprotectin antigen (S100A8/S100A9) fromgranulocytes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry. Gentian Technology AS, Moss, Norway.ORCID iD: 0000-0002-6097-6318
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.ORCID iD: 0000-0003-3161-0402
2018 (English)In: Health Science Reports, Vol. 1, no 5, article id e35Article in journal (Refereed) Published
Abstract [en]

Background

Calprotectin is a promising biomarker for granulocyte activation. It is mainly measured in faeces as a marker for inflammatory bowel disease. A limitation is that there is no widely accepted calibrator.

Aim

To establish a method for purification of calprotectin from human granulocytes that is easily reproducible, reliable, and could contribute to a better agreement between different calprotectin methods.

Methods and results

Calprotectin was purified from granulocyte extracts using ion‐exchange chromatography. The granulocytes were separated from blood bags. The purity was analysed by analysing pixel density of a picture of the sodium dodecyl sulfate polyacrylamide gel electrophoresis and by size exclusion chromatography. The calprotectin concentration of the pure antigen solution was determined using Biuret method. The purity was >95% for 3 preparations, and their concentrations were 1079, 1080, and 1813 mg/L.

Conclusion

It is possible to reproducibly prepare highly purified calprotectin antigen from human granulocytes. The preparations can be used for preparing calibrators, controls for immunological calprotectin assays, and immunisation for raising antibodies against human calprotectin in hens.

Place, publisher, year, edition, pages
Wiley-Blackwell Publishing Inc., 2018. Vol. 1, no 5, article id e35
National Category
Clinical Laboratory Medicine
Identifiers
URN: urn:nbn:se:uu:diva-355220DOI: 10.1002/hsr2.35OAI: oai:DiVA.org:uu-355220DiVA, id: diva2:1227929
Available from: 2018-06-27 Created: 2018-06-27 Last updated: 2018-10-15Bibliographically approved
In thesis
1. Avian antibodies applied in particle enhanced turbidimetric immunoassay: Developement of serum/plasma calprotectin immunoassay and its clinical performance as a marker for bacterial infections
Open this publication in new window or tab >>Avian antibodies applied in particle enhanced turbidimetric immunoassay: Developement of serum/plasma calprotectin immunoassay and its clinical performance as a marker for bacterial infections
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Calprotectin is a cytosolic protein in the granulocytes, consisting of S100A8 and S100A9. On the site of inflammation, the neutrophils release the cytosol as an inflammatory response. The circulating calprotectin concentration increases and can therefore be used as marker for neutrophil activation and inflammation.

To raise specific antibodies, it is crucial to immunize with pure calprotectin antigen. We purified calprotectin from human granulocytes by ion-exchange chromatography, dialysed it towards saline and concentrated it to required levels, suited for immunisation of the hens. The purified antigen solutions were assigned concentration values by the Biuret method and the purity was checked by SDS PAGE and size exclusion chromatography. The yield was approximately 2 mg purified antigens per unit of 450 ml blood.

A prototype calprotectin particle enhanced turbidimetric immunoassay was developed from the purified antigen and the affinity purified antibodies. The antigen was spiked into PBS to prepare calibrators and controls. The antibodies were coated to latex particles to prepare immunoparticles. The performance of the immunoassay was technically tested on a clinical chemistry analyser. LoQ, antigen excess, linearity, precision and calibration stability met the pre-set criteria.

In the production process of immunoparticles there are several factors affecting the performance of the assay. Investigating eight factors applying a Taguchi L12 screening, we experienced that conductivity and pH of conjugate buffer, coating grade and conductivity of dialysis buffer II affected the sensitivity and antigen excess the most.

The assay was used to measure clinical samples. Serum samples from elderly people aged 70+ were collected. Only patients with no infections were included to establish a reference interval for this patient group. The reference interval in serum was 0.3 mg/L to 2.5 mg/L for both genders. Furthermore, the plasma calprotectin immunoassay was tested clinically on critically ill patients to assess the ability of plasma calprotectin as an early marker for detection of bacterial infections. It showed promising results. Calprotectin was a better predictive marker for sepsis than procalcitonin and white blood cell count. Because some patients with an inflammation have low numbers of granulocytes, we examined the correlation between white blood cell count and the calprotectin levels in a group of patients with an inflammation. There was a weak correlation between the number of white blood cells and calprotectin concentration.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 54
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1507
Keywords
Sepsis, avian antibodies, calprotectin antigen, particle enhanced turbidimetric immunoassay.
National Category
Clinical Laboratory Medicine
Research subject
Clinical Chemistry
Identifiers
urn:nbn:se:uu:diva-363261 (URN)978-91-513-0480-9 (ISBN)
Public defence
2018-12-06, Robergssalen, ingång 40, Akademiska Sjukhuset,, Uppsala, 10:00 (Norwegian)
Opponent
Supervisors
Funder
The Research Council of Norway, 223022/O30
Available from: 2018-11-15 Created: 2018-10-15 Last updated: 2018-11-30

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Nilsen, TomLarsson, Anders

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