Rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays.
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.
Kijanka
Gregor
G
Centre for Human Proteomics, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland.
Ipcho
Simon
S
Baars
Sabine
S
Chen
Hong
H
Hadley
Katie
K
Beveridge
Allan
A
Gould
Edith
E
Murphy
Derek
D
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
D000906:Antibodies; D000939:Epitopes; D011994:Recombinant Proteins