Detection of Individual Microbial Pathogens by Proximity Ligation

Gústafsdóttir, Sigrún M.; Nordengrahn, Ann; Simon, Fabrice; Wallgren, Per; Rivera, E.; Schallmeiner, Edith; Mérza, M.; Landegren, Ulf

doi:10.1373/clinchem.2005.065847

Cited by 77 publications

(52 citation statements)

References 19 publications

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“…This was shown by PLA, which is a modern technology to study protein-protein interactions. 32,33 PLA has been used in the past to detect parvovirus and an intracellular bacterium 34 and our data indicate that it may become an important tool to investigate and verify binding of native and modified viruses and other pathogen to cell surface molecules. The PLA assay also revealed that insertion of FWKT motif in the HI loop allowed for binding to SSTR 2 but did not completely abolish CAR binding.…”

Section: Oncolytic Adenovirus With Somatostatin Motifs J Leja Et Almentioning

confidence: 89%

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

Leja

Nilsson

et al. 2011

Gene Ther

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We have previously described the oncolytic adenovirus, Ad(CgA-E1A-miR122), herein denoted Ad5(CgA-E1A-miR122) that selectively replicates in and kills neuroendocrine cells, including freshly isolated midgut carcinoid cells from liver metastases. Ad5(CgA-E1A-miR122) is based on human adenovirus serotype 5 (Ad5) and infects target cells by binding to the coxsackieadenovirus receptor (CAR) and integrins on the cell surface. Some neuroendocrine tumor (NET) and neuroblastoma cells express low levels of CAR and are therefore poorly transduced by Ad5. However, they often express high levels of somatostatin receptors (SSTRs). Therefore, we introduced cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site for SSTRs in the virus fiber knob. We show that FWKT-modified Ad5 binds to SSTR 2 on NET cells and transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to greater extent than Ad5, indicating that fiber knob modification may prolong the systemic circulation time. We conclude that modification of adenovirus with the FWKT motif may be beneficial for NET therapy.

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Section: Oncolytic Adenovirus With Somatostatin Motifs J Leja Et Almentioning

confidence: 89%

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

Leja

Nilsson

et al. 2011

Gene Ther

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“…The most robust method aptamer-mediated amplifications have been obtained using the proximity ligation assay (PLA), in which adjacent binding of two aptamers to a protein or cellular target results in ligation and formation of a unique amplicon which can then be sensitively detected by real-time PCR [12][13][14][15][16][17]. Fredriksson and co-workers originally showed that aptamer-mediated PLA could be used for the detection of zeptomole amounts of plateletderived growth factor (PDGF) [12].…”

Section: Introductionmentioning

confidence: 99%

Real-time PCR detection of protein analytes with conformation-switching aptamers

Yang

Ellington

2008

Analytical Biochemistry

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We have developed a novel method that utilizes conformation-switching aptamers for real-time PCR analysis of protein analytes. The aptamers have been designed so that they assume one secondary structure in the absence of a protein analyte, and a different secondary structure in the presence of a protein such as thrombin or PDGF. The protein-bound structure in turn assembles a ligation junction for the addition of a real-time PCR primer. Protein concentrations could be specifically detected into the picomolar range, even in the presence of cell lysates. The method has advantages relative to both immunoPCR (since no signal is produced by background binding) and to the proximity ligation assay (PLA; since only one epitope on a protein surface must be bound, rather than two).

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“…Proximity ligation assay is an ultrasensitive and specific method for detection of proteins and other analytes (9,17,23 ). We used 3 monoclonal antibodies; PSA-API was captured to the solid phase by a mAb against tPSA and detected by 2 mAbs labeled with DNA probes, 1 mAb against API and 1 mAb against tPSA.…”

Section: Discussionmentioning

confidence: 99%

“…Sequences of the oligonucleotide probes, primers, and connector used in this assay have been described (17 ). We obtained the DNA connector and primers from TAG Copenhagen and TaqMan probe (5Ј FAM, 3Ј MGB) from Applied Biosystems UK.…”

Section: Oligonucleotides Used For Proximity Ligationmentioning

confidence: 99%

“…Then proximity probes (100 pmol/L) in 50 L of PBS-BSA buffer were added and incubated for 2 h at room temperature. After removal of unbound probes by washing 4 times with 200 L Milli-Q water, we added the ligation buffer and TaqMan PCR mix (50 L) (17 ). After ligation for 5 min at room temperature, we transferred the tubes to a real-time PCR instrument (Applied Biosystems PRISM 7000) for temperature cycling at 95°C for 2 min, followed by 95°C for 15 s and 60°C for 60 s, repeated 40 times.…”

Section: Proximity Ligation Assay For Psa-apimentioning

confidence: 99%

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Proximity Ligation Measurement of the Complex between Prostate Specific Antigen and α1-Protease Inhibitor

et al. 2009

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Background: Prostate specific antigen (PSA)–α1-protease inhibitor complex (PSA-API) is a minor form of PSA in serum. It may be useful for prostate cancer (PCa) diagnosis, but its specific detection is hampered by nonspecific background. To avoid this, we developed an immunoassay for PSA-API based on proximity ligation. Methods: We used a monoclonal antibody (mAb) to total PSA (tPSA) to capture PSA, while using another anti-tPSA mAb together with an anti-API mAb as probes. We measured PSA-API by quantification of amplified DNA strands conjugated to the probes. We measured serum PSA-API in 84 controls and 55 men with PCa who had PSA concentrations of 4.0–10 μg/L. Results: The detection limit of the assay was 6.6 ng/L. The proportion of PSA-API to tPSA (%PSA-API) tended to be lower in men with PCa (2.8%) than without cancer (3.3%) but was not statistically significant (P = 0.363). When used alone, %PSA-API [area under the curve (AUC) 0.546] did not improve detection of PCa, whereas %fPSA (AUC 0.710) and the sum of %fPSA and %PSA-API (AUC 0.723) did. At 90% diagnostic sensitivity, the diagnostic specificity for cancer was not significantly better for %fPSA + %PSA-API than for %fPSA alone (36% vs 30%). Conclusions: Proximity ligation eliminated nonspecific background, enabling accurate measurement of PSA-API in serum specimens with moderately increased tPSA. The combined use of %PSA-API and %fPSA provided a modest improvement for PCa detection, but based on the current study cohort, it is uncertain whether the improvement has clinical utility. .

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Detection of Individual Microbial Pathogens by Proximity Ligation

Cited by 77 publications

References 19 publications

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells

Real-time PCR detection of protein analytes with conformation-switching aptamers

Proximity Ligation Measurement of the Complex between Prostate Specific Antigen and α1-Protease Inhibitor

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