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Application of Artificial Gel Antibodies for the Detection and Quantification of Proteins in Biological Fluids
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Fred Nyberg)
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. , p. 64
Series
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
Identifiers
URN: urn:nbn:se:uu:diva-122457ISBN: 978-91-554-7802-5 (print)OAI: oai:DiVA.org:uu-122457DiVA, id: diva2:310187
Public defence
2010-05-18, C4:305, Uppsala biomedicinska centrum BMC, Husarg. 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2010-04-27 Created: 2010-04-13 Last updated: 2018-05-29Bibliographically approved
List of papers
1. Highly selective artificial gel antibodies for detection and quantification of biomarkers in clinical samples: I. Spectrophotometric approach to design the calibration curve for the quantification
Open this publication in new window or tab >>Highly selective artificial gel antibodies for detection and quantification of biomarkers in clinical samples: I. Spectrophotometric approach to design the calibration curve for the quantification
2008 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 31, no 22, p. 3945-3953Article in journal (Refereed) Published
Abstract [en]

High selectivity of a biomarker is a basic requirement when it is used for diagnosis, prognosis and treatment of a disease. The artificial gel antibodies, which we synthesise by a molecular imprinting method, have this property not only for proteins, but also for bioparticles, such as viruses and bacteria. However, diagnosis of a disease requires not only that the biomarker can be "fished out" from a body fluid with high selectivity, but also that its concentration in the sample can rapidly be determined and preferably by a simple technique. This paper deals primarily with the development of a spectrophotometric method, which is so simple and fast that it can be used with advantage in a Doctor's Office. The development of this method was not straight-forward. However, by modifications of the performance of these measurements we can now design standard curves in the form of a straight line, when we plot the true (not the recorded "apparent" absorption) against known protein concentrations. In an additional publication (see the following paper in this issue of JSS) we show an application of such a plot: determination of the concentration of albumin in serum and cerebrospinal fluid from patients with neurological disorders to investigate whether albumin is a biomarker for these diseases.

Keywords
Apparent absorption, artificial antibodies, CBB, molecular imprinting, true absorption
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-104856 (URN)10.1002/jssc.200800385 (DOI)000262167500017 ()19065619 (PubMedID)
Available from: 2009-05-29 Created: 2009-05-29 Last updated: 2018-01-13Bibliographically approved
2. Highly selective artificial gel antibodies for detection and quantification of biomarkers in clinical samples: II. Albumin in body fluids of patients with neurological disorders
Open this publication in new window or tab >>Highly selective artificial gel antibodies for detection and quantification of biomarkers in clinical samples: II. Albumin in body fluids of patients with neurological disorders
2008 (English)In: Journal of Separation Science, ISSN 1615-9306, E-ISSN 1615-9314, Vol. 31, no 22, p. 3954-3958Article in journal (Refereed) Published
Abstract [en]

We have previously used the molecular-imprinting method for the synthesis of artificial gel antibodies, highly selective for various proteins. In the present work, we have synthesized artificial gel antibodies against human albumin with the aim to develop a simple and rapid procedure to measure the concentration of this protein in samples of clinical interest. The procedure, based on the design of a standard curve (see the preceding paper), was applied on a quantitative analysis of albumin in human plasma and cerebrospinal fluid (CSF). We found that our technique permitted detection of albumin in these body fluids with high precision and that the concentration of this protein was significantly enhanced in CSF from patients with amyotrophic lateral sclerosis (ALS), compared to control samples. This finding is in agreement with results from earlier studies, which confirms the validity of our analysis technique and suggests that the barrier permeability may be affected in ALS, perhaps also for other proteins. No enhancement in plasma levels of albumin was seen in patients with ALS, but rather a decrease. The results further indicate that our approach might also apply well to other biomarkers for the actual neurological disease and other disorders.

Keywords
Amyotrophic lateral sclerosis, artificial gel antibodies, cerebrospinal fluid, human albumin, plasma
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-104857 (URN)10.1002/jssc.200800386 (DOI)000262167500018 ()19065610 (PubMedID)
Available from: 2009-05-29 Created: 2009-05-29 Last updated: 2018-01-13Bibliographically approved
3. Application of artificial gel antibodies for investigating molecular polymorphisms of human pituitary growth hormone
Open this publication in new window or tab >>Application of artificial gel antibodies for investigating molecular polymorphisms of human pituitary growth hormone
2011 (English)In: Amino Acids, ISSN 0939-4451, E-ISSN 1438-2199, Vol. 40, no 4, p. 1249-1255Article in journal (Refereed) Published
Abstract [en]

Artificial gel-antibodies were used to probe human growth hormone (GH) activity in hormone preparations purified from human pituitaries. A partially purified fraction containing differently sized forms of the hormone was further processed to yield monomeric and dimeric forms of GH activity suitable for synthesizing artificial polyacrylamide gel antibodies. These two types of GH antibodies were used for probing GH activity in experiments analyzing the two forms of the hormone by HPLC gel-permeation and ion-exchange chromatography. In the size-exclusion experiments both hormone fractions eluted as homogenous peaks, whereas the ion exchanger resolved the hormones in several active components. The antibodies towards the GH monomer were more potent to recognize monomeric GH compared to antibodies against dimeric GH. The opposite was found for the dimeric GH antibodies. It was concluded that these two sets of antibodies it might be useful for discriminating between dimeric and monomeric GH in samples of clinical origin.

Keywords
Assay, Electrophoresis, Growth hormone (GH), HPLC, Human, Pituitary, Polyacrylamide gel antibodies, Purification, Quantification
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Biochemistry
Identifiers
urn:nbn:se:uu:diva-122448 (URN)10.1007/s00726-011-0840-3 (DOI)000288546700021 ()21312046 (PubMedID)
Available from: 2010-04-16 Created: 2010-04-13 Last updated: 2018-01-12Bibliographically approved
4. Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics
Open this publication in new window or tab >>Precautions to improve the accuracy of quantitative determinations of biomarkers in clinical diagnostics
2010 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 31, no 16, p. 2722-2729Article 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).

Keywords
Artificial gel antibodies, Biomarker, Free zone electrophoresis, Growth hormone, Polyacrylamide gels
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-122455 (URN)10.1002/elps.201000243 (DOI)000281732900004 ()
Note
Uppdaterad från Manuskript till Artikel; 20101202Available from: 2010-04-16 Created: 2010-04-13 Last updated: 2017-12-12Bibliographically approved

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