Rapid characterization of binding specificity and cross-reactivit.pdf (279.81 kB)

Rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays.

Download (279.81 kB)
journal contribution
posted on 22.11.2019, 15:18 by Gregor Kijanka, Simon IpCho, Sabine Baars, Hong Chen, Katie Hadley, Allan Beveridge, Edith Gould, Derek Murphy

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.

History

Comments

The definitive version is available at http://www.sciencedirect.com

Published Citation

Kijanka G. IpCho S. Barrs S. Chen H. Hadley K. Beveridge A. Gould E. Murphy D. Rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays. Journal of Immunological Methods. 2009; 340(2):132-7.

Publication Date

30/01/2009

Publisher

North-Holland Pub. Co.

PubMed ID

18996391

Exports