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Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.ORCID iD: 0000-0002-5248-8568
KTH, Centres, Science for Life Laboratory, SciLifeLab.
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2011 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, no 11, 1824-1835 p.Article in journal (Refereed) Published
Abstract [en]

A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.

Place, publisher, year, edition, pages
2011. Vol. 20, no 11, 1824-1835 p.
Keyword [en]
epitope mapping, monospecific antibody, affinity chromatography
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-47969DOI: 10.1002/pro.716ISI: 000296273700009Scopus ID: 2-s2.0-80054717087OAI: oai:DiVA.org:kth-47969DiVA: diva2:457225
Funder
Knut and Alice Wallenberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20111117Available from: 2011-11-17 Created: 2011-11-15 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Epitope mapping of antibodies towards human protein targets
Open this publication in new window or tab >>Epitope mapping of antibodies towards human protein targets
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis, based on five research papers, presents results from development and evaluation ofmethods for identifying the interaction site of antibodies on their antigens and the functional investigation of these in different assays. As antibodies have proven to be invaluable tools in diagnostics, therapy and basic research, the demand of characterizing these binding molecules has increased. Techniques for epitope mapping in a streamlined manner are therefore needed, particularly in high throughput projects as the Human Protein Atlas that aims to systematically generate two antibodies with separate epitopes towards all human proteins. 

In paper I we describe an approach to map the epitopes of polyclonal and monoclonal antibodies for the first time using staphylococcal display. This method was combined with peptide scanning and alanine scanning using suspension bead arrays, to create a streamlined approach of highresolution characterization of epitopes recognized by antibodies as demonstrated in paper II. Single epitopes were identified for the monoclonal antibodies and several (one to five) separate epitopes scattered throughout the antigen sequence were determined for each polyclonal antibody. Further, antibodies of different species origin showed overlapping binding epitopes.

In paper III we studied the epitope patterns of polyclonal antibodies generated with the same antigen in different animals. Although common epitope regions could be identified the exact epitope pattern was not repeated, as some epitopes did not reoccur in the repeated immunizations. In paper IV, a potential biomarker for colon cancer, RBM3, was investigated using validated antibodies by epitope mapping and siRNA analysis.

Finally, in paper V, a method for generating epitope-specific antibodies based on affinity purification of a polyclonal antibody is described. The generated antibodies were used in several immunoassays and showed a great difference in functionality. Paired antibodies with separate epitopes were successfully generated and could be used in a sandwich assay or to validate each other in immunohistochemistry.

Taken together, in these studies we have demonstrated valuable concepts for the characterization of antibody epitopes.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. xi, 46 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2012:1
Keyword
antibody, antibody validation, biomarker, epitope mapping, peptide array, proteomics, RBM3, staphylococcal surface display
National Category
Medical Biotechnology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-59529 (URN)978-91-7501-222-3 (ISBN)
Public defence
2012-01-27, FD5, Albanova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20120111Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2012-01-11Bibliographically approved
2. Antibody based plasma protein profiling
Open this publication in new window or tab >>Antibody based plasma protein profiling
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided.

Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V).

As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. viii, 68 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2013:12
Keyword
protein profiling, plasma, antibody, affinity proteomics, biomark- ers, multiplex, assay development
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-126270 (URN)978-91-7501-829-4 (ISBN)
Public defence
2013-09-06, Gamma house lecture hall, SciLifeLab, Tomtebodavägen 23a, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20130821

Available from: 2013-08-21 Created: 2013-08-20 Last updated: 2013-08-21Bibliographically approved
3. Characterization of antibody specificity using peptide array technologies
Open this publication in new window or tab >>Characterization of antibody specificity using peptide array technologies
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antibodies play an important role in the natural immune response to invading pathogens. The strong and specific binding to their antigens also make them indispensable tools for research, diagnostics and therapy.

This thesis describes the development of methods for characterization of an- tibody specificity and the use of these methods to investigate the polyclonal antibody response after immunization. Paper I describes the development of an epitope-specific serum fractionation technique based on epitope map- ping using overlapping peptides followed by chromatographic separation of polyclonal serum. This technique together with another epitope mapping technique based on bacterial display of protein fragments were then used to generate antibody sandwich pairs (Paper I), investigate epitope variations of repeated immunizations (Paper II) and to determine the ratio of antibodies targeting linear and conformational epitopes of polyclonal antibodies (Paper III). Paper IV describes the optimization of in situ-synthesized high-density peptide arrays for epitope mapping and how different peptide lengths influ- ence epitope detection and resolution. In Paper V we show the development of planar peptide arrays covering the entire human proteome and how these arrays can be used for epitope mapping and off-target binding analysis. In Paper VI we show how polyclonal antibodies targeting linear epitopes can be used for peptide enrichment in a rapid, absolute protein quantification protocol based on mass spectrometry.

Altogether these investigations demonstrate the usefulness of peptide arrays for fast and straightforward characterization of antibody specificity. The work also contributes to a deeper understanding of the polyclonal anti- body response obtained after immunization with recombinant protein frag- ments.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. xi, 49 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:16
Keyword
Antibody, Epitope mapping, Peptide array, Suspension bead array, Antigen, Specificity, Cross-reactivity, Immunization, Immunogenicity
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-155723 (URN)978-91-7595-316-8 (ISBN)
Public defence
2014-11-28, Gardaulan, Nobels väg 18, Solna, 10:15 (English)
Opponent
Supervisors
Note

QC 20141111

Available from: 2014-11-11 Created: 2014-11-11 Last updated: 2015-02-18Bibliographically approved

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Forsström, BjörnLundberg, EmmaSchwenk, Jochen M.Uhlen, Mathias

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