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Selective enrichment of monospecific polyclonal antibodies for antibody-based proteomics efforts
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0001-8993-048X
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-0605-8417
2004 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1043, 33-40 p.Article in journal (Refereed) Published
Abstract [en]

A high stringency protocol, suitable for systematic purification of polyclonal antibodies, is described. The procedure is designed to allow the generation of target protein-specific antibodies suitable for functional annotation of proteins. Antibodies were generated by immunization with recombinantly produced affinity-tagged target proteins. To obtain stringent recovery of the antibodies, a two-step affinity chromatography principle was devised to first deplete the affinity tag-specific antibodies followed by a second step for affinity capture of the target protein-specific antibodies. An analytical dot-blot array system was developed to analyze the cross-reactivity of the affinity-purified antibodies. The results suggest that the protocol can be used in a highly parallel and automated manner to generate mono-specific polyclonal antibodies for large-scale, antibody-based proteomics efforts, i.e. affinity proteomics.

Place, publisher, year, edition, pages
2004. Vol. 1043, 33-40 p.
Keyword [en]
Affinity chromatography; Antibodies; Immobilized metal-ion affinity chromatography; Protein epitope signature tags; Proteomics
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-5915DOI: 10.1016/j.chroma.2004.06.008ISI: 000222728800006PubMedID: 15317410Scopus ID: 2-s2.0-3242702469OAI: oai:DiVA.org:kth-5915DiVA: diva2:10449
Note
QC 20100824. 23rd International Symposium on the Separation of Proteins Peptides and Polynucleotides. Delray Beach, FL. NOV 09-12, 2003Available from: 2006-06-02 Created: 2006-06-02 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Systems enabling antibody-mediated proteomics research
Open this publication in new window or tab >>Systems enabling antibody-mediated proteomics research
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

As many genome sequencing efforts today are completed, we are now provided with the genetic maps for several organisms, including man. With these maps at hand, the scientific focus is now shifting towards investigations of the functionality of proteins. This task is even more challenging than the genomic field since proteins, in contrast to DNA, do not allow themselves to be specifically probed or amplified by easy and generic methods. However, to achieve knowledge regarding protein function, useful information includes where, when and how much certain proteins are expressed in an organism. Such information can be obtained if protein-specific binding molecules are available as tools. One such class of target specific binders are the antibody molecules, traditionally employed in a broad variety of biotechnical applications, including protein localization studies on both cellular and sub cellular levels.

In a first serie of studies, new methodology for recombinant production and purification of antigens for generation of antibodies via immunization routes were investigated. Parallel affinity gene fusion-based expression systems were used for evaluation of different concepts for production of antigen and post-immunization antibody purification. Carefully designed protein antigens from different organisms were produced and used to raise antisera which were affinity purified on their respective antigens to obtain highly specific polyclonal antibodies (monospecific antibodies). One of the constructed expression systems includes an affinity handle, ZSPA-1, previously selected from a combinatorial protein library for its capacity to selectively bind protein A. This allows for convenient, non IgG-dependent, affinity purification of proteins on conventional protein A resins.

A strategy where highly target specific antibody preparations could be affinity purified in a more streamlined setup is also presented. By this strategy it was possible to fractionate antibodies showing reactivity to different parts of the antigen into separate fractions. This resulted in affinity purified antibodies showing monospecific but still multi-epitope reactivity. Purified monospecific antibodies were used in different studies including Western blot immunofluorescence and recovery applications. For affinity purification of endogenous target from its native surrounding a selective elution strategy where the recombinant antigen was used to competitively elute the captured target was developed.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006
Keyword
Antibody generation, dual expression, affinity purification, E. coli expression, Affibody molecules, polyclonal antibody, monospecific antibody
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-4025 (URN)91-7178-370-9 (ISBN)
Public defence
2006-06-14, Sal FD5, AlvaNova univ centrum, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Note
QC 20100824Available from: 2006-06-02 Created: 2006-06-02 Last updated: 2011-11-23Bibliographically approved

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Publisher's full textPubMedScopushttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TG8-4CP6BHG-6&_user=4478132&_handle=V-WA-A-W-AZ-MsSAYWW-UUA-U-AACADDDEAW-AAVEBCYDAW-EUYCCUVVA-AZ-U&_fmt=summary&_coverDate=07%2F16%2F2004&_rdoc=6&_orig=browse&_srch=%23toc%235248%232004%23989569998%23509619!&_cdi=5248&view=c&_acct=C000034958&_version=1&_urlVersion=0&_userid=4478132&md5=dc9ccda3f285d9f9139230ccaccea79a

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Uhlén, MathiasHober, Sophia

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