title: Rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays.
abstract: Antibodies are routinely used as research tools, in diagnostic assays and increasingly as therapeutics. Ideally, these applications require antibodies with high sensitivity and specificity; however, many commercially available antibodies are limited in their use as they cross-react with non-related proteins. Here we describe a novel method to characterize antibody specificity. Six commercially available monoclonal and polyclonal antibodies were screened on high-density protein arrays comprising of ~10,000 recombinant human proteins (Imagenes). Two of the six antibodies examined; anti-pICln and anti-GAPDH, bound exclusively to their target antigen and showed no cross-reactivity with non-related proteins. However, four of the antibodies, anti-HSP90, anti-HSA, anti-bFGF and anti-Ro52, showed strong cross-reactivity with other proteins on the array. Antibody-antigen interactions were readily confirmed using Western immunoblotting. In addition, the redundant nature of the protein array used, enabled us to define the epitopic region within HSP90 of the anti-HSP90 antibody, and identify possible shared epitopes in cross-reacting proteins. In conclusion, high-density protein array technology is a fast and effective means for determining the specificity of antibodies and can be used to further improve the accuracy of antibody applications.
authors.affiliation: Centre for Human Proteomics, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland.
authors.forename: Gregor
authors.initials: G
authors.lastname: Kijanka
authors.forename: Simon
authors.initials: S
authors.lastname: Ipcho
authors.forename: Sabine
authors.initials: S
authors.lastname: Baars
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.forename: Katie
authors.initials: K
authors.lastname: Hadley
authors.forename: Allan
authors.initials: A
authors.lastname: Beveridge
authors.forename: Edith
authors.initials: E
authors.lastname: Gould
authors.forename: Derek
authors.initials: D
authors.lastname: Murphy
mesh_terms: D000906:Antibodies / Q000032:analysis* / Q000276:immunology*; D000918:Antibody Specificity; D003429:Cross Reactions / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D006801:Humans; D040081:Protein Array Analysis / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology
chemical_list: D000906:Antibodies; D000939:Epitopes; D011994:Recombinant Proteins