Validation of antibodies for protein profiling A study using immunohistochemistry on tissue microarrays

Abstract: The field of proteomics has rapidly expanded due to the completion of the human genome sequence. This thesis validates affinity-purified monospecific antibodies of polyclonal origin, for protein profiling in a broad spectrum of normal tissues and cells. Validation of antibodies is crucial for development of reliable binders for target proteins and this thesis evaluates the generation and application of large sets of msAbs in different settings. MsAbs were generated towards recombinant Protein Epitope Signature Tag (PrEST) antigens using a stringent affinity-purification strategy, presented in the first study. The specificity of msAbs was studied using reverse phase protein arrays and immunohistochemistry (IHC), and results presented over 90% success rate in the protein array analysis. In IHC, 81% of the msAbs displayed apparent specific staining in normal tissues. MsAbs were also compared with commercial analogs (cAbs) using IHC and Western blot. Results presented similar outcome between msAbs and cAbs in both applications, although interpretation suggested more extensive IHC staining patterns with msAbs than with monoclonal analogs. For antibody validation, an approach called paired antibodies was presented and involved the generation of two msAbs towards non-overlapping epitopes on the same protein. Similarities in protein detection between paired antibodies were studied using three different antibody-based methods. Similar results were observed in several applications, indicating that this strategy can be a useful tool for studying known and unknown proteins. Given the reliability of msAbs, they were also applied in a study investigating the impact of tissue fixatives on protein detection. The study showed that different fixation mechanisms appeared to affect protein recognition by indicating that aldehyde-based fixation, e.g. induced by neutral buffered formalin, was preferred for tissues used in IHC and non-aldehyde based fixation was applicable for tissues used in protein extraction analysis and Western blotting.Conclusively, validation results suggest that msAbs are reliable affinity binders that can be used as valuable tools for proteome-wide protein profiling in tissues and cells.