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Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity
Attana AB.
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Solid State Electronics. (Thin Films Group)
IFM, Linköping University.
Attana AB.
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2010 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 398, no 2, 161-168 p.Article in journal (Refereed) Published
Abstract [en]

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We have shown that successful immobilization is highly dependent on surface pKa, antibody pI and pH of immobilization buffer. By use of EDC/sulfo-NHS activation reagents, the effect of the intrinsic surface pKa is avoided and immobilization also at very low pH has been made possible which is of importance for immobilization of acidic proteins. Generic immobilization conditions were demonstrated on a panel of antibodies which resulted in an average coefficient of variation of 4% for the immobilization of these antibodies.

Antigen binding capacity as a function of immobilization pH was studied. In most cases the antigen binding capacity followed the immobilization response. However, the antigen to antibody binding ratio differed between the antibodies investigated, and for one of the antibodies, the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab antibodies on different antibody surfaces showed that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.

Place, publisher, year, edition, pages
2010. Vol. 398, no 2, 161-168 p.
Keyword [en]
antibody, orientation, biosensor, immobilization, sensor surface, QCM
National Category
Other Industrial Biotechnology Analytical Chemistry
Research subject
Surface Biotechnology
URN: urn:nbn:se:uu:diva-107233DOI: 10.1016/j.ab.2009.11.038ISI: 000274615100003PubMedID: 19962366OAI: oai:DiVA.org:uu-107233DiVA: diva2:228373
Available from: 2009-07-30 Created: 2009-07-30 Last updated: 2010-12-15Bibliographically approved
In thesis
1. Development of Electroacoustic Sensors for Biomolecular Interaction Analysis
Open this publication in new window or tab >>Development of Electroacoustic Sensors for Biomolecular Interaction Analysis
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biomolecular interaction analysis to determine the kinetics and affinity between interacting partners is important for the fundamental understanding of biology, as well as for the development of new pharmaceutical substances. A quartz crystal microbalance instrument suitable for kinetics and affinity analyses of interaction events was developed. The functionality of the sensor system was demonstrated by development of an assay for relative affinity determination of lectin-carbohydrate interactions.

Sensor surfaces allowing for effective immobilization of one interacting partner is a key functionality of a biosensor. Here, three different surfaces and immobilization methods were studied. First, optimized preparation conditions for sensor surfaces based on carboxyl-terminated self assembled monolayers were developed and were demonstrated to provide highly functional biosensor surfaces with low non-specific binding. Second, a method allowing for immobilization of very acidic biomolecules based on the use of an electric field was developed and evaluated. The electric field made it possible to immobilize the highly acidic C-peptide on a carboxylated surface. Third, a method for antibody immobilization on a carboxyl surface was optimized and the influence of immobilization pH on the immobilization level and antigen binding capacity was thoroughly assessed. The method showed high reproducibility for a set of antibodies and allowed for antibody immobilization also at low pH.

Three broadly different strategies to increase the sensitivity of electroacoustic sensors were explored. A QCM sensor with small resonator electrodes and reduced flow cell dimensions was demonstrated to improve the mass transport rate to the sensor surface. The use of polymers on QCM sensor surfaces to enhance the sensor response was shown to increase the response of an antibody-antigen model system more than ten-fold. Moreover, the application of high frequency thin film bulk acoustic resonators for biosensing was evaluated with respect to sensing range from the surface. The linear detection range of the thin film resonator was determined to be more than sufficient for biosensor applications involving, for instance, antibody-antigen interactions. Finally, a setup for combined frequency and resistance measurements was developed and was found to provide time resolved data suitable for kinetics determination.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 68 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 658
biosensor, protein interactions, kinetics, affinity, QCM, quartz crystal microbalance, piezoelectric resonators, dissipation, motional resistance
National Category
Other Industrial Biotechnology Analytical Chemistry
Research subject
Engineering Science with specialization in Microsystems Technology
urn:nbn:se:uu:diva-107211 (URN)978-91-554-7572-7 (ISBN)
Public defence
2009-09-11, Å80101, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:30 (English)
Available from: 2009-08-20 Created: 2009-07-29 Last updated: 2014-11-25Bibliographically approved

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