Change search
ReferencesLink to record
Permanent link

Direct link
Immunizations of inbred rabbits using the same antigen yield antibodies with similar, but not identical, epitopes
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).ORCID iD: 0000-0002-5248-8568
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).ORCID iD: 0000-0001-9423-0541
KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
Show others and affiliations
(English)Manuscript (preprint) (Other academic)
Abstract [en]

A problem for the generation of polyclonal antibodies is the potential difficulties to obtain 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 in-bred 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 gene 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 in-bred 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.

National Category
Medical Biotechnology Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-59559OAI: diva2:475955
QS 2012Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2014-11-11Bibliographically 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.
Trita-BIO-Report, ISSN 1654-2312 ; 2012:1
antibody, antibody validation, biomarker, epitope mapping, peptide array, proteomics, RBM3, staphylococcal surface display
National Category
Medical Biotechnology Biochemistry and Molecular Biology
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)
QC 20120111Available from: 2012-01-11 Created: 2012-01-11 Last updated: 2012-01-11Bibliographically approved

Open Access in DiVA

No full text

Search in DiVA

By author/editor
Hjelm, BarbaraForsström, BjörnLöfblom, JohnRockberg, JohanUhlén, Mathias
By organisation
Proteomics (closed 20130101)Science for Life Laboratory, SciLifeLab
Medical BiotechnologyBiochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 56 hits
ReferencesLink to record
Permanent link

Direct link