Clinical Chemistry
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Clinical Chemistry 54: 1218-1225, 2008. First published May 22, 2008; 10.1373/clinchem.2007.099424
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Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 2008;54:1218-1225.)
© 2008 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Use of Proximity Ligation to Screen for Inhibitors of Interactions between Vascular Endothelial Growth Factor A and Its Receptors

Sigrun M. Gustafsdottir1, Stefan Wennström1, Simon Fredriksson2, Edith Schallmeiner1, Andrew D. Hamilton3, Said M. Sebti4 and Ulf Landegren1,a

1 Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, SE-75185 Uppsala, Sweden; 2 Olink Bioscience, Dag Hammarskjöldsväg 54A, 75183 Uppsala, Sweden; 3 Yale University, New Haven, CT; 4 H. Lee Moffitt Research Institute, University of South Florida, Tampa, FL.

aAddress correspondence to this author at: Rudbeck Laboratory, Department of Genetics and Pathology, Uppsala University, Dag Hammarskjöldsväg 20, SE-751 85 Uppsala, Sweden. Fax 46-18-4714808; e-mail ulf.landegren{at}genpat.uu.se.

Background: Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs.

Methods: We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule.

Results: The PLAs were successful for monitoring the formation and inhibition of VEGF-A–receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC50)] from a dose–response curve.

Conclusions: The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique provides dose–response curves, allowing IC50 values to be calculated.






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