uu.seUppsala University Publications
Change search
ReferencesLink to record
Permanent link

Direct link
Microfluidic analysis of antibody specificity in a compact disk format
KTH, Proteomik.
KTH, Skolan för bioteknologi (BIO).
KTH, Skolan för bioteknologi (BIO).
Show others and affiliations
2006 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 5, no 7, 1568-1574 p.Article in journal (Refereed) Published
Abstract [en]

A new and flexible technology for high throughput analysis of antibody specificity and affinity is presented. The method is based on microfluidics and takes advantage of compact disks (CDs) in which the centrifugal force moves fluids through microstructures containing immobilized metal affinity chromatography columns. Analyses are performed as a sandwich assay, where antigen is captured to the column via a genetically attached His(6)-tag. The antibodies to be analyzed are applied onto the columns. Thereafter, fluorescently labeled secondary antibodies recognize the bound primary antibodies, and detection is carried out by laser-induced fluorescence. The CDs contain 104 microstructures enabling analysis of antibodies against more than 100 different proteins using a single CD. Importantly, through the three- dimensional visualization of the binding patterns in a column it is possible to separate high affinity from low affinity binding. The method presented here is shown to be very sensitive, flexible and reproducible.

Place, publisher, year, edition, pages
2006. Vol. 5, no 7, 1568-1574 p.
Keyword [en]
microfluidics, miniaturization, compact disk, biochip, gyrolab, antibody specificity, IMMUNOSORBENT-ASSAY ELISA, PROTEOMICS, MICROARRAYS, GENOME, CHIP
National Category
Industrial Biotechnology
URN: urn:nbn:se:uu:diva-208954DOI: 10.1021/pr050447cISI: 000238838500007OAI: oai:DiVA.org:uu-208954DiVA: diva2:655451

QC 20100712

Available from: 2010-07-12 Created: 2013-10-11 Last updated: 2015-10-01
In thesis
1. Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
Open this publication in new window or tab >>Mass Spectrometry and Affinity Based Methods for Analysis of Proteins and Proteomes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteomics is a fast growing field and there has been a tremendous increase of knowledge the last two decades. Mass spectrometry is the most used method for analysis of complex protein samples. It can be used both in large scale discovery studies as well as in targeted quantitative studies. In parallel with the fast improvements of mass spectrometry-based proteomics there has been a fast growth of affinity-based methods. A common challenge is the large dynamic range of protein concentrations in biological samples. No method can today cover the whole dynamic range. If affinity and mass spectrometry-based proteomics could be used in better combination, this would be partly solved. The challenge for affinity-based proteomics is the poor specificity that has been seen for many of the commercially available antibodies. In mass spectrometry, the challenges are sensitivity and sample throughput. In this thesis, large scale approaches for validation of antibodies and other binders are presented. Protein microarrays were used in four validation studies and one was based on mass spectrometry. It is shown that protein microarrays can be valuable tools to check the specificity of antibodies produced in a large scale production. Mass spectrometry was shown to give similar results as Western blot and Immunohistochemistry regarding specificity, but did also provide useful information about which other proteins that were bound to the antibody.

Mass spectrometry has many applications and in this thesis two methods contributing with new knowledge in animal proteomics are presented. A combination of high affinity depletion, SDS PAGE and mass spectrometry revealed 983 proteins in dog cerebrospinal fluid, of which 801 were marked as uncharacterized in UniProt. A targeted quantitative study of cat serum based on parallel reaction monitoring showed that mass spectrometry can be an applicable method instead of ELISA in animal proteomic studies. Mass spectrometry is a generic method and has the advantage of shorter and less expensive development costs for specific assays that are not hampered by cross-reactivity.

Mass spectrometry supported by affinity based applications will be an attractive tool for further improvements in the proteomic field.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 82 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1272
Mass spectrometry, proteomics, microarray, protein, antibody, antigen, affinity, validation
National Category
Analytical Chemistry
Research subject
Chemistry with specialization in Analytical Chemistry
urn:nbn:se:uu:diva-259623 (URN)978-91-554-9300-4 (ISBN)
Public defence
2015-09-25, C4:305, BMC, Husargatan 3, Uppsala, 10:15 (Swedish)
Available from: 2015-09-03 Created: 2015-08-10 Last updated: 2015-10-01

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Eriksson, CeciliaAgaton, CharlottaKånge, RikardSundberg, MartenNilsson, PeterUhlen, MathiasHober, Sophia
In the same journal
Journal of Proteome Research
Industrial Biotechnology

Search outside of DiVA

GoogleGoogle Scholar

Altmetric score

Total: 166 hits
ReferencesLink to record
Permanent link

Direct link