Use of proximity ligation to screen for inhibitors of interactions between vascular endothelial growth factor A and its receptors.

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 (IC(50))] 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 IC(50) values to be calculated

Cytokine detection by antibody-based proximity ligation

Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 μg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-μl samples, and we exemplify its utility in situations when only minute sample amounts are available

In vitro analysis of DNA–protein interactions by proximity ligation

Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1–10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites

Recent progress in developing proximity ligation assays for pathogen detection

The effective management of infectious diseases depends on the early detection of the microbes responsible, since pathogens are most effectively eliminated in the initial stages of infection. Current immunodiagnostic methods lack the sensitivity for earliest possible diagnosis. Nucleic acid-based tests (NATs) are more sensitive, but the detection of microbial DNA does not definitively prove the presence of a viable microorganism capable of causing a given infection. Proximity assays combine the specificity of antibody-based detection of proteins with the sensitivity and dynamic range of NATs, and their use may allow earlier as well as more clinically relevant detection than is possible with current NATs or immunoassays. However, the full potential of proximity assays for pathogen detection remains to be fulfilled, mainly due to the challenges associated with identifying suitable antibodies and antibody combinations, sensitivity issues arising from non-specific interactions of proximity probes and the longer incubation times required to carry out the assays