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Next generation of labeling reagents for quantitative and multiplexing immunoassays by use of LA-ICP-MS
KTH, School of Biotechnology (BIO), Protein Technology.ORCID iD: 0000-0002-4751-2519
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Abstract. Immuno imaging by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a growing research field in the life sciences as biology and biomedicine. Various element labeling strategies for antibodies have been developed for the application of multiplex immunoassays analyzed by use of LA-ICP-MS. High multiplexing capabilities, a wide linear dynamic range and the possibility of absolute quantification are the main advantages of ICP-MS. But in the context of immuno imaging by use of LA-ICP-MS quantification of analytes is limited due to a non-controllable antibody labeling chemistry. in the presented proof-of-principle A novel antibody labeling technique has been investigated which results in a controlled labeling degree. A small affinity protein based on the C2 domain of protein G was modified with conventional metal coded tags (MeCAT) after introducing a cysteine in the C-terminus of the protein. The modified C2 domain photo crosslinks to the Fc or Fab region of the IgG and allows specific and covalent labeling of antibodies for multiplex immunoassays analyzed by use of LA-ICP-MS. In combination with a house-made calibration membrane the amount of labeled antibody-antigen complexes in a multiplex Western Blot immunoassay was determined by LA-ICP-MS. 

National Category
Engineering and Technology
Identifiers
URN: urn:nbn:se:kth:diva-191160OAI: oai:DiVA.org:kth-191160DiVA: diva2:955165
Available from: 2016-08-24 Created: 2016-08-24 Last updated: 2016-08-29Bibliographically approved
In thesis
1. Engineering of small IgG binding domains for antibody labelling and purification
Open this publication in new window or tab >>Engineering of small IgG binding domains for antibody labelling and purification
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In protein engineering, rational design and selection from combinatorial libraries are methods used to develop proteins with new or improved features. A very important protein for the biological sciences is the antibody that is used as a detecting agent in numerous laboratory assays. Antibodies used for these purposes are often ”man-made”, by immunising animals with the desired target, or by selections from combinatorial libraries. Naturally, antibodies are part of the immune defence protecting us from foreign attacks from e.g. bacteria or viruses. Some bacteria have evolved surface proteins that can bind to proteins abundant in the blood, like antibodies and serum albumin. By doing so, the bacteria can cover themselves in the host’s own proteins and through that evade being detected by the immune system. Two such proteins are Protein A from Staphylococcus aureus and Protein G from group C and G Streptococci. Both these proteins contain domains that bind to antibodies, one of which is denoted C2 (from Protein G) and another B (from Protein A). The B domain have been further engineered to the Z domain.

In this thesis protein engineering has been used to develop variants of the C2 and Z domains for site-specific labelling of antibodies and for antibody purification with mild elution. By taking advantage of the domains’ inherent affinity for antibodies, engineering and design of certain amino acids or protein motifs of the domains have resulted in proteins with new properties. A photo crosslinking amino acid, p-benzoylphenylalanine, have been introduced at different positions to the C2 domain, rendering three new protein domains that can be used for site-specific labelling of antibodies at the Fc or Fab fragment. These domains were used for labelling antibodies with lanthanides and used for detection in a multiplex immunoassay. Moreover, a library of calcium-binding loops was grafted onto the Z domain and used for selection of a domain that binds antibodies in a calcium dependent manner. This engineered protein domain can be used for the purification of antibodies using milder elution conditions, by calcium removal, as compared to traditional antibody purification. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. 82 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:15
Keyword
Antibody, labelling, purification, Protein G, Protein A, protein engineering, protein design, combinatorial selection
National Category
Engineering and Technology Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-191303 (URN)978-91-7729-093-3 (ISBN)
External cooperation:
Public defence
2016-09-30, M2, Brinellvägen 64, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2016-09-09 Created: 2016-08-26 Last updated: 2016-09-09Bibliographically approved

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Kanje, Sara
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