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Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Fred Nyberg)
Norwegian Medicines Agency.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
2010 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 31, no 16, 2722-2729 p.Article in journal (Refereed) Published
Abstract [en]

Although protein biomarkers have a great potential as biomarkers for diagnosis of diseases, they are seldom used in hospitals. There are many reasons for this, for instance, the difficulties to (i) find a biomarker for which the concentration in body fluids clearly differs between patients and healthy subjects, (ii) attain purification of the biomarker close to 100%, which is required for production of conventional protein antibodies as well as artificial gel antibodies for selective capture of a biomarker, (iii) design a standard curve for rapid and accurate determination of the concentration of the biomarker in the body fluid because of adsorption of the biomarker onto vials, pipettes, etc., (iv) determine accurately the sample volume delivered by a pipette, (v) avoid polymerization of the biomarker upon storage and to decide whether it is in the form not only of monomers, but also of dimers, trimers, etc., in the native state, (vi) determine the degree of possible glycosylation and amidation of the biomarker and (vii) decide whether glycosylation and amidation positively or negatively affects the possibility to use the protein as a biomarker. In this article, we discuss in quantitative terms the difficulties (iii-vii) and how to overcome them, which also may help to overcome the difficulty (ti), which in turn minimizes difficulty (i).

Place, publisher, year, edition, pages
2010. Vol. 31, no 16, 2722-2729 p.
Keyword [en]
Artificial gel antibodies, Biomarker, Free zone electrophoresis, Growth hormone, Polyacrylamide gels
National Category
Chemical Sciences
Research subject
URN: urn:nbn:se:uu:diva-122455DOI: 10.1002/elps.201000243ISI: 000281732900004OAI: oai:DiVA.org:uu-122455DiVA: diva2:310171
Uppdaterad från Manuskript till Artikel; 20101202Available from: 2010-04-16 Created: 2010-04-13 Last updated: 2011-05-05Bibliographically approved
In thesis
1. Application of Artificial Gel Antibodies for the Detection and Quantification of Proteins in Biological Fluids
Open this publication in new window or tab >>Application of Artificial Gel Antibodies for the Detection and Quantification of Proteins in Biological Fluids
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The molecular-imprinting method has previously been used for the synthesis of artificial gel antibodies, highly selective for various proteins. In present study, we have synthesized artificial gel antibodies against haemoglobin, albumin and different forms of growth hormone with the aim to develop a simple and rapid procedure to measure the concentration of these protein biomarkers in samples of clinical interest.  A spectrophotometric method was developed to design a standard curve in the form of a straight line, whereby the true absorption (not the recorded “apparent” absorption) was plotted against a known protein concentration. The procedure, applied to quantitative analysis of albumin in human plasma and cerebrospinal fluid (CSF) from patients with ALS, indicated that  the concentration of this protein was significantly enhanced in CSF from patients with amyotrophic lateral sclerosis (ALS), compared to control samples. A low level of albumin was observed in plasma from ALS patients compared to controls. Additionally, free zone electrophoresis was employed to detect human growth hormone (GH) activity in hormone preparations purified from human pituitaries. We have successfully synthesized antibodies capable of discriminating between dimeric and monomeric GH in samples of clinical origin. To quantify these proteins a calibration curve has been designed, i.e. a plot of the electrophoretic mobility of the complex GH/gel antibody against the protein concentration in the sample, for instance serum or CSF.

This method was also employed for qualitative and quantitative determinations of Somatropin, a non-glycosylated GH and glycosylated-GH in a body liquid.

Our results indicate that by this technique one can “fish out” with high accuracy various proteins from both body fluids containing a great number of other proteins. It might well apply also to biomarker proteins for other diseases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 64 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 741
National Category
Medicinal Chemistry Pharmaceutical Sciences
Research subject
Pharmaceutical Science
urn:nbn:se:uu:diva-122457 (URN)978-91-554-7802-5 (ISBN)
Public defence
2010-05-18, C4:305, Uppsala biomedicinska centrum BMC, Husarg. 3, Uppsala, 13:00 (English)
Available from: 2010-04-27 Created: 2010-04-13 Last updated: 2010-05-17Bibliographically approved

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