RESEARCH ARTICLE Dissecting Antibodies with Regards to Linear

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 epi-topes. 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 rela-tive 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 as-says. 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 re-sults 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 whol-ly or partially denatured protein targets