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- Gullberg, Mats, et al.
(författare)
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Cytokine detection by antibody-based proximity ligation
- 2004
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 101:22, s. 8420-4
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- 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 microg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-microl samples, and we exemplify its utility in situations when only minute sample amounts are available.
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| 2. |
- Gustafsdottir, Sigrun M., et al.
(författare)
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Detection of individual microbial pathogens by proximity ligation
- 2006
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 52:6, s. 1152-1160
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Nucleic acid amplification allows the detection of single infectious agents. Protein-based assays, although they provide information on ongoing infections, have substantially less detection sensitivity.METHODS: We used proximity ligation reactions to detect proteins on bacteria and virus particles via nucleic acid amplification. Antibodies recognizing viral or bacterial surface proteins were equipped with DNA strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents.RESULTS: Detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium.CONCLUSIONS: This method enables detection of ligated DNA strands with good sensitivity by real-time PCR and could be of value for early diagnosis of infectious disease and in biodefense.
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| 3. |
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| 5. |
- Gustafsdottir, Sigrun M, et al.
(författare)
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Use of Proximity Ligation to Screen for Inhibitors of Interactions between Vascular Endothelial Growth Factor A and Its Receptors
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:7, s. 1218-1225
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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