title: Antibody array analysis of labelled proteomes: how should we control specificity?
abstract: Researchers who use protein binders in multiplexed assays can be divided into two camps. One believes that arrays with proteome-wide coverage will become a reality once we have developed binders for all proteins. The sceptics claim that detection with immobilized protein binders and sample labelling will not provide the required specificity. In this article, we review the evidence showing that antibody array analysis of labelled samples can provide meaningful data and discuss the issues raised by the sceptics. We argue that direct the evidence for monospecificity has yet to be published. This will require assays designed to resolve the proteins captured by each binder. One option is to combine array measurement with protein separation. We have developed an assay where labelled sample proteins are separated by size exclusion chromatography (SEC) before contact with microsphere-based arrays (Size-MAP; size exclusion chromatography-resolved microsphere-based affinity proteomics). The effect is an 'antibody array Western blot' where reactivity of immobilized binders is resolved against the size of the proteins in the sample. We show that Size-MAP is useful to discriminate monospecific- and polyreactive antibodies and for automatic detection of reacting with the same target. The possibility to test specificity directly in array-based measurement should be useful to select the best binders and to determine whether the DNA microarray for the proteome is a realistic goal or not.
authors.affiliation: Department of Immunology, Clinic of Specialized Medicine and Surgery Oslo University Hospital, Rikshospitalet, N-0027 Oslo, Norway.
authors.forename: Anders
authors.initials: A
authors.lastname: Holm
authors.forename: Weiwei
authors.initials: W
authors.lastname: Wu
authors.forename: Fridtjof
authors.initials: F
authors.lastname: Lund-Johansen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D002850:Chromatography, Gel; D016000:Cluster Analysis; D006801:Humans; D008863:Microspheres; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D015203:Reproducibility of Results; D013194:Staining and Labeling
chemical_list: D000906:Antibodies; D020543:Proteome