title: Dissecting antibodies with regards to linear and conformational epitopes.
abstract: An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets.
authors: Science for Life Laboratory, KTH-Royal Institute of Technology, SE-171 21 Stockholm, Sweden.
authors: Björn
authors: B
authors: Forsström
authors: Department of Proteomics, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
authors: Barbara Bisławska
authors: BB
authors: Axnäs
authors: Department of Proteomics, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
authors: Johan
authors: J
authors: Rockberg
authors: Department of Proteomics, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
authors: Hanna
authors: H
authors: Danielsson
authors: Department of Proteomics, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
authors: Anna
authors: A
authors: Bohlin
authors: Science for Life Laboratory, KTH-Royal Institute of Technology, SE-171 21 Stockholm, Sweden; Department of Proteomics, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), Stockholm, Sweden.
authors: Mathias
authors: M
authors: Uhlen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002846:Chromatography, Affinity / Q000379:methods*; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007114:Immunization; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D000906:Antibodies; D000939:Epitopes; D010455:Peptides; D011994:Recombinant Proteins