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Avian antibodies applied in particle enhanced turbidimetric immunoassay: Developement of serum/plasma calprotectin immunoassay and its clinical performance as a marker for bacterial infections
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry. Gentian AS.ORCID iD: 0000-0002-6097-6318
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 [en]
Sepsis, avian antibodies, calprotectin antigen, particle enhanced turbidimetric immunoassay.
National Category
Clinical Laboratory Medicine
Research subject
Clinical Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-363261ISBN: 978-91-513-0480-9 (print)OAI: oai:DiVA.org:uu-363261DiVA, id: diva2:1256049
Public defence
2018-12-06, Robergssalen, ingång 40, Akademiska Sjukhuset,, Uppsala, 10:00 (Norwegian)
Opponent
Supervisors
Funder
The Research Council of Norway, 223022/O30Available from: 2018-11-15 Created: 2018-10-15 Last updated: 2018-11-30
List of papers
1. Extraction,isolation, and concentration of calprotectin antigen (S100A8/S100A9) fromgranulocytes
Open this publication in new window or tab >>Extraction,isolation, and concentration of calprotectin antigen (S100A8/S100A9) fromgranulocytes
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
National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-355220 (URN)10.1002/hsr2.35 (DOI)
Available from: 2018-06-27 Created: 2018-06-27 Last updated: 2018-10-15Bibliographically approved
2. A new turbidimetric immunoassay for serum calprotectin for fully automatized clinical analysers
Open this publication in new window or tab >>A new turbidimetric immunoassay for serum calprotectin for fully automatized clinical analysers
2015 (English)In: Journal of Inflammation, ISSN 1476-9255, E-ISSN 1476-9255, Vol. 12, article id 45Article in journal (Refereed) Published
Abstract [en]

Serum and plasma calprotectin concentration is shown to be elevated when neutrophils are activated, and may therefore be used as a marker for inflammatory diseases. A serum calprotectin immunoassay was developed based on calprotectin values observed in samples from the intensive care unit. The polyclonal avian antibodies were raised and affinity purified with calprotectin antigens. The performance was tested and it was observed that the assay was linear in the range 0.3-24.7 mg/L, the limit of quantitation was observed to be lower than 0.3 mg/L, no antigen excess was observed up to 54 mg/L, all CVs were lower than 1.8 % in the precision study, the calibration curve stability was longer than 6 weeks, and there was no significant interference detected for haemoglobin, intralipid or bilirubin. The serum calprotectin immunoassay presented in this paper performs well within the criteria carefully set from the limited clinical experience obtained in both serum and plasma. In addition it is commutable with Bühlmann MRP8/14 ELISA.

National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-259346 (URN)10.1186/s12950-015-0090-3 (DOI)000358486900002 ()26213499 (PubMedID)
Note

Economical support was given from the Norwegian Research Council.

Available from: 2015-07-31 Created: 2015-07-31 Last updated: 2018-10-15Bibliographically approved
3. Influence of 8 parameters when coating avian calprotectin antibodies to latex particles
Open this publication in new window or tab >>Influence of 8 parameters when coating avian calprotectin antibodies to latex particles
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Aim: To find the parameters affecting sensitivity and security zone in the preparation process of immunoparticles with avian antibodies for use in particle enhanced turbidimetric immunoassays. Method: 12 combinations of 8 parameters were tested in the preparation process of the immunoparticles. The study was designed according to Taguchi L12 screening method and analysed using DOE KISS PRO XL. Results/Conclusion: The parameters affecting the sensitivity and security zone the most were conductivity of the conjugate buffer, coating grade and pH of the conjugate buffer.

National Category
Clinical Laboratory Medicine
Research subject
Clinical Chemistry
Identifiers
urn:nbn:se:uu:diva-363262 (URN)
Funder
The Research Council of Norway
Available from: 2018-10-15 Created: 2018-10-15 Last updated: 2018-10-17
4. Serum calprotectin levels in elderly males and females without bacterial or viral infections
Open this publication in new window or tab >>Serum calprotectin levels in elderly males and females without bacterial or viral infections
2014 (English)In: Clinical Biochemistry, ISSN 0009-9120, E-ISSN 1873-2933, Vol. 47, no 12, p. 1065-1068Article in journal (Refereed) Published
Abstract [en]

OBJECTIVES: Calprotectin is released from activated leukocytes and calprotectin can thus be used as a marker for leukocyte activation. Faeces calprotectin is not only used as a marker for inflammatory bowel disease but can also be used to detect leukocyte activation in other body fluids. The aim of the present study was to study serum calprotectin levels in non-infected elderly individuals to establish reference intervals for the marker.

METHODS: Serum calprotectin was analyzed by immunoturbidimetry in 75year old females and males without known infections. Individuals with CRP>20mg/L were excluded as this could indicate a subclinical infection. The calprotectin levels in the remaining 713 individuals were used to calculate reference values for this population. The Spearman rank correlations between calprotectin and 27 other laboratory biomarkers were also investigated.

RESULTS: There was a strong positive Spearman rank correlation between calprotectin and CRP (p<0.000001) and alkaline phosphatase (p<0.000001). There were also significant negative correlations between calprotectin and ApoA1 and direct HDL-cholesterol.

CONCLUSIONS: The reference interval for serum-calprotectin for all study subjects was 0.3-2.6mg/L. Leukocyte alkaline phosphatase contributes to serum alkaline phosphatase levels.

National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:uu:diva-220516 (URN)10.1016/j.clinbiochem.2014.01.003 (DOI)000340995900016 ()24440500 (PubMedID)
Available from: 2014-03-17 Created: 2014-03-17 Last updated: 2018-10-15Bibliographically approved
5. Calprotectin as an early biomarker of bacterial infections in critically ill patients: an exploratory cohort assessment
Open this publication in new window or tab >>Calprotectin as an early biomarker of bacterial infections in critically ill patients: an exploratory cohort assessment
Show others...
2017 (English)In: Criminology & Public Policy, ISSN 1441-2772, E-ISSN 1941-1006, Vol. 19, no 3, p. 205-213Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Calprotectin is the most abundant protein in the cytosolic fraction of neutrophils, and neutrophil degranulation is a major response to bacterial infections.

OBJECTIVES: To assess the value of plasma calprotectin as an early marker of bacterial infections in critically ill patients and compare it with the corresponding values for procalcitonin (PCT), C-reactive protein (CRP) and white blood cell count (WBC).

METHODS: We measured daily plasma calprotectin levels in 110 intensive care unit patients using a newly developed turbidimetric assay run on clinical chemistry analysers. The likelihood of infection was determined according to the International Sepsis Forum criteria.

RESULTS: Overall, 58 patients (52.7%) developed a suspected or confirmed bacterial infection. Plasma calprotectin predicted such infections within 24 hours with an area under the receiver operating characteristics curve (ROC area) of 0.78 (95% CI, 0.68-0.89). The ROC area for calprotectin was significantly greater than the corresponding ROC areas for WBC (P < 0.001) and PCT (P = 0.02) but only marginally better than the ROC area for CRP (0.71; 95% CI, 0.68-0.89).

CONCLUSION: Plasma calprotectin appears to be a useful early marker of bacterial infections in critically ill patients, with better predictive characteristics than WBC and PCT.

National Category
Anesthesiology and Intensive Care
Identifiers
urn:nbn:se:uu:diva-329205 (URN)000408400800003 ()28866970 (PubMedID)
Available from: 2017-09-14 Created: 2017-09-14 Last updated: 2018-10-15Bibliographically approved

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