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Characterization of antibody specificity using peptide array technologies
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-5248-8568
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 [en]
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: urn:nbn:se:kth:diva-155723ISBN: 978-91-7595-316-8 (print)OAI: oai:DiVA.org:kth-155723DiVA: diva2:762237
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
List of papers
1. Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
Open this publication in new window or tab >>Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
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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.

Keyword
epitope mapping, monospecific antibody, affinity chromatography
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-47969 (URN)10.1002/pro.716 (DOI)000296273700009 ()2-s2.0-80054717087 (Scopus ID)
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
2. Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes
Open this publication in new window or tab >>Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 12, e45817- p.Article in journal (Refereed) Published
Abstract [en]

A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

Keyword
animal experiment, antigen binding, article, breast cancer, cancer tissue, controlled study, epitope mapping, human, human tissue, immunization, immunohistochemistry, nonhuman, prediction, protein structure, rabbit, Staphylococcus carnosus, Western blotting
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-111858 (URN)10.1371/journal.pone.0045817 (DOI)000312694300002 ()23284606 (PubMedID)2-s2.0-84871314636 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceKnut and Alice Wallenberg FoundationSwedish Research CouncilVinnova
Note

QC 20130115

Available from: 2013-01-14 Created: 2013-01-14 Last updated: 2017-12-06Bibliographically approved
3. Dissecting antibodies withregards to linear and conformational epitopes
Open this publication in new window or tab >>Dissecting antibodies withregards to linear and conformational epitopes
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-155725 (URN)
Note

QS 2014

Available from: 2014-11-11 Created: 2014-11-11 Last updated: 2014-11-11Bibliographically approved
4. High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays
Open this publication in new window or tab >>High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays
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2012 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, no 12, 1790-1800 p.Article in journal (Refereed) Published
Abstract [en]

Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against linear epitopes of the human proteome and obtained very detailed descriptions of the involved specificities. The epitopes identified ranged from 4 to 12 amino acids in size. In general, the antibodies were of exquisite specificity, frequently disallowing even single conservative substitutions. In several cases, multiple distinct epitopes could be identified for the same target protein, suggesting an efficient approach to the generation of paired antibodies. Two alternative epitope mapping approaches identified similar, although not necessarily identical, epitopes. These results show that ultrahigh-density peptide microarrays can be used for linear epitope mapping. With an upper theoretical limit of 2,000,000 individual peptides per array, these peptide microarrays may even be used for a systematic validation of antibodies at the proteomic level.

Keyword
antibody affinity, antibody specificity, article, controlled study, DNA sequence, epitope mapping, high throughput screening, human, linear system, molecular size, peptide synthesis, priority journal, protein expression, protein microarray, protein targeting, signal transduction, substitution reaction
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-110177 (URN)10.1074/mcp.M112.020800 (DOI)000313557000023 ()2-s2.0-84870659704 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceEU, FP7, Seventh Framework Programme, 222773
Note

QC 20130111

Available from: 2013-01-11 Created: 2013-01-10 Last updated: 2017-12-06Bibliographically approved
5. Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays
Open this publication in new window or tab >>Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays
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2014 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 6, 1585-1597 p.Article in journal (Refereed) Published
Abstract [en]

Antibodies are of importance for the field of proteomics, both as reagents for imaging cells, tissues, and organs and as capturing agents for affinity enrichment in mass-spectrometry-based techniques. It is important to gain basic insights regarding the binding sites (epitopes) of antibodies and potential cross-reactivity to nontarget proteins. Knowledge about an antibody's linear epitopes is also useful in, for instance, developing assays involving the capture of peptides obtained from trypsin cleavage of samples prior to mass spectrometry analysis. Here, we describe, for the first time, the design and use of peptide arrays covering all human proteins for the analysis of antibody specificity, based on parallel in situ photolithic synthesis of a total of 2.1 million overlapping peptides. This has allowed analysis of on-and off-target binding of both monoclonal and polyclonal antibodies, complemented with precise mapping of epitopes based on full amino acid substitution scans. The analysis suggests that linear epitopes are relatively short, confined to five to seven residues, resulting in apparent off-target binding to peptides corresponding to a large number of unrelated human proteins. However, subsequent analysis using recombinant proteins suggests that these linear epitopes have a strict conformational component, thus giving us new insights regarding how antibodies bind to their antigens.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-147955 (URN)10.1074/mcp.M113.033308 (DOI)000337239500016 ()2-s2.0-84901938911 (Scopus ID)
Note

QC 20140711

Available from: 2014-07-11 Created: 2014-07-10 Last updated: 2017-12-05Bibliographically approved
6. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins
Open this publication in new window or tab >>Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins
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2014 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 13, no 6, 1611-1624 p.Article in journal (Refereed) Published
Abstract [en]

AThe combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-147956 (URN)10.1074/mcp.M113.034140 (DOI)000337239500018 ()2-s2.0-84901952830 (Scopus ID)
Note

QC 20150217

Available from: 2014-07-11 Created: 2014-07-10 Last updated: 2017-12-05Bibliographically approved

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