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Microfluidic analysis of antibody specificity in a compact disk format
KTH, School of Biotechnology (BIO), Proteomics.
KTH, School of Biotechnology (BIO).
KTH, School of Biotechnology (BIO).
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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
Identifiers
URN: urn:nbn:se:kth:diva-14074DOI: 10.1021/pr050447cISI: 000238838500007Scopus ID: 2-s2.0-33745862053OAI: oai:DiVA.org:kth-14074DiVA: diva2:329577
Note
QC 20100712Available from: 2010-07-12 Created: 2010-07-12 Last updated: 2011-09-20Bibliographically approved
In thesis
1. Affinity based proteomics research tools
Open this publication in new window or tab >>Affinity based proteomics research tools
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Listen to the mantra; the mapping of the genome was finished in 2001, and the sequel research challenge is the thorough survey of the corresponding human proteome. This was stated almost a decade ago, it has been repeated over and over, and is still most certainly a hard nut to crack. The workload is daunting, much because there is no protein amplification method and no binary system for the detection of proteins, and because the complexity of the proteome is larger than that of the genome as it seems. Hence, there is a need for high throughput technologies that, at sufficiently low limits of detection and with satisfying sensitivities, may investigate protein content in human samples. With this aim, the Human Proteome Resource (HPR) project was initiated in 2003.

All work presented in this thesis relate to protein interactions; binders are either utilized such as for the depletion of high abundant proteins from serum, or analyzed such as in the validation of monospecific antibody specificity, or the epitope mapping of the same. In Paper I, the Gyrolab system is utilized in a setup for the specificity analysis of monospecific antibodies towards their antigen, and the setup is compared to planar protein arrays. Gyrolab technology is used again, in Paper II, where epitope mapping of monospecific antibodies is performed in order to analyze antibody specificity. Also, mapping serves to compare the immune-responses from parallel immunizations using the same antigen, thereby assessing reproducibility in the regeneration of antibodies. Paper III describes a high throughput approach for the depletion of high abundant proteins, in serum and plasma samples, taking advantage of Affibody molecules as binders. The last two papers, IV and V, utilize monospecific antibodies for protein analysis; in Paper IV pull out experiments show that competitive elution using the PrEST antigen can be a fruitful approach to increase specificity. And finally, in Paper V, a setup for the semi-quantitative protein content analysis in fluid samples is suggested. Again, the Gyrolab technology is used, and the setup is tested on a simplified model system.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. 86 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:15
National Category
Immunology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-11184 (URN)978-91-7415-422-1 (ISBN)
Public defence
2009-10-09, FR4 Oskar Klein, AlbaNova, Roslagstullsbacken 33, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100712Available from: 2009-10-01 Created: 2009-10-01 Last updated: 2010-07-16Bibliographically approved

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Uhlen, MathiasHober, Sophia

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