Proximity ligation assays with peptide conjugate 'burrs' for the sensitive detection of spores

The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide-oligonucleotide-phycoerythrin conjugates ('burrs') that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins.     

Proximity ligation assays with peptide conjugate ‘burrs’ for the sensitive detection of spores

The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide–oligonucleotide–phycoerythrin conjugates (‘burrs’) that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins.     

Peptide arrays for highly sensitive and specific antibody-binding fluorescence assays

We report a novel generation of peptide arrays fabricated by site-specific ligation of glyoxylyl peptides onto glass slides covered by a semicarbazide sol-gel layer. These arrays allowed the highly sensitive and specific detection of antibodies in very small blood samples from infected individuals using three model peptidic epitopes (HCV Core and NS4, EBV Capsid) in an immunofluorescence assay. Comparison with standard enzyme-linked immunosorbent assays (ELISAs) demonstrated a large gain in sensitivity and specificity. These unique properties, combined with the possibility to immobilize glycoproteins such as antibodies, offer the possibility to perform sandwich immunofluorescent assays in a highly parallel format.     

Proximity probing assays for simultaneous visualization of protein complexes in situ

Evaluation of: Leuchowius KJ, Clausson CM, Grannas K et al. Parallel visualization of multiple protein complexes in individual cells in tumor tissue. Mol. Cell Proteomics doi:10.1074/mcp.O112.023374 (2013) (Epub ahead of print).

Techniques for in situ detection and quantification of proteins in fixed tissue remain an important element of both basic biological analyses and clinical biomarker research. The practical importance of such techniques can be exemplified by the everyday clinical use of immunohistochemical detection of the estrogen receptor and HER2 in tissues from breast cancer patients. Several techniques are currently available for detection of single proteins and post-translational modifications, but very few are suitable for detection of protein complexes. Methods that enable simultaneous detection and quantification of protein complexes provide novel possibilities for understanding the biological role(s) of protein complexes and may open new opportunities to improve clinical biomarker research. Leuchowius et al. describe an improved proximity ligation assay for in situ detection of protein complexes, which is able to detect and quantify several protein complexes simultaneously in the same tissue specimen.     

Sensitive protein detection via triple-binder proximity ligation assays

The detection of weakly expressed proteins and protein complexes in biological samples represents a fundamental challenge. We have developed a new proximity-ligation strategy named 3PLA that uses three recognition events for the highly specific and sensitive detection of as little as a hundred molecules of the vascular endothelial growth factor (VEGF), the biomarkers troponin I, and prostate-specific antigen (PSA) alone or in complex with an inhibitor--demonstrating the versatility of 3PLA.     

Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 μl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.     

Two sensitive, convenient, and widely applicable assays for marker enzyme activities specific to endoplasmic reticulum

Two assay protocols are described for enzyme activities known to reside in the endoplasmic reticulum of a wide variety of species and tissue types, with the intent that they be used as marker enzyme assays in subcellular fractionations. The enzyme activities assayed are choline phosphotransferase and dolichol-P-mannosyl synthase, both of which result in synthesis of lipid products. The assays are constructed to make them easy to perform and sensitive enough to detect enzyme activity even using microgram quantities of cell protein. The assay methodologies are effective not only in vertebrate cells, but in insect cells and yeast cells as well. This implies that these assays should be useful as marker enzyme assays for a wide variety of eukaryotic cells.     

New antibody immobilization method via functional liposome layer for specific protein assays

A specific protein assay system based on functional liposome-modified gold electrodes has been demonstrated. To fabricate such assay system, a liposome layer was initially grown on top of a gold layer. The liposome layer contained two kinds of functional molecules: biotin molecules for the binding sites of streptavidin and N-(10,12-pentacosadiynoic)-acetylferrocene molecules for the facile redox probe in electrochemical detections. Then, streptavidin was attached on the functional liposme-modified layer using the interaction of streptavidin-sbiotin complex. On the streptavidin-attached surface, antibody molecules, anti-human serum albumin antibodies could be immobilized without any secondary antibodies. AFM imaging of the streptavidin-attached liposome surface revealed a uniform distribution of closely packed streptavidin molecules. In situ quartz-crystal microbalance and electrochemical measurements demonstrated that the wanted antibody-antigen reactions should occur with high specificity and selectivity. Our specific antibody assay system, based on a functional liposome modified electrode, can be developed further to yield sophisticated structures for numerous protein chips and immunoassay sensors.     

A sensitive proximity ligation assay for active PSA

Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. The utility of PSA tests is limited by their inability to differentiate prostate cancer from non-malignant conditions such as benign prostatic hyperplasia and prostatitis. In circulation, PSA occurs in various complexed and free forms, and specific determination of some of these can be used to improve the diagnostic accuracy of PSA tests. We have previously identified peptides that specifically bind to enzymatically active PSA and using such a peptide we have developed an immunopeptidometric assay for this form of PSA. However, the sensitivity of that assay is too low to measure active PSA at clinically important levels. Recently a novel sensitive immunoassay for analysis of proteins, termed the proximity ligation assay, has been established. Here we describe a sensitive implementation of the proximity ligation assay, which utilizes a PSA-binding peptide and antibody as probes to detect active PSA. The assay has a sensitivity of 0.07 microg/l, which is approximately ten-fold lower than that of our previous assay. It does not cross-react with inactive proPSA or the highly similar kallikrein hK2. Our results show that a highly sensitive immunopeptidometric assay can be developed using proximity ligation. This principle should facilitate establishment of specific assays for active forms of other proteases.     

Self-assembly of proximity probes for flexible and modular proximity ligation assays

Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.     

PCR assays for specific and sensitive detection of Pseudomonas tolaasii,the cause of brown blotch disease of mushrooms

AIMS: The present study describes PCR assays to detect specifically Pseudomonas tolaasii from various samples. METHODS AND RESULTS: Two sets of PCR primers were developed to amplify genes required for tolaasin production. Only a PCR product of 449 bp or 249 bp was produced in PCR reactions with the Pt-1A/Pt-1D1 or Pt-PM/Pt-QM primer sets, respectively, and DNA and cells of Ps. tolaasii. Nested and immunocapture-nested PCR could detect to 3 cells of Ps. tolaasii and amplify the Ps. tolaasii-specific DNA from a sample containing 10 000 times more other bacterial cells than Ps. tolaasii, respectively. CONCLUSIONS: The PCR assays are simple, rapid and reliable methods for detection and identification of Ps. tolaasii. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocols can effectively distinguish Ps. tolaasii from other bacteria and detect Ps. tolaasii from various samples for studying ecology of the bacterium and preventing the use of contaminated water or spawn or medium in mushroom cultivation.     

C-terminal glyoxylyl peptides for sensitive enzyme-linked immunosorbent assays

Summary A model peptide antigen derived from HCV Core protein was modified by a hydrophilic glyoxylamide arm using a tartaramide-based linker and used in an enzyme-linked immunosorbent assay (ELISA) on hydrazide plates. A comparative study with the standard non-covalent adsorption procedures demonstrated a large gain in sensitivity for the detection of antibodies in HCV-positive sera.     

Nine surface plasmon resonance assays for specific protein quantitation during cell culture and process development

Quantitation of protein is essential during pharmaceutical development, and a variety of methods and technologies for determination of total and specific protein concentration are available. Here we describe the development of a streamlined assay platform for specific quantitation assays using surface plasmon resonance (SPR) technology. A total of nine different assays were developed using similar conditions, of which eight assays were for quantitation of different human blood plasma proteins (IgG, IgG1–4 subclasses, IgA, transferrin, and albumin) from a chromatography-based IgG plasma process. Lastly, an assay for monitoring the concentration of a recombinant monoclonal antibody during 13 days of CHO cell culturing was developed. Assay performances were compared with enzyme-linked immunosorbent assay (ELISA), nephelometry, ARCHITECT, and Cobas c501. SPR assays were shown to have higher sensitivity than analysis using nephelometry, ARCHITECT, and Cobas and to have significantly lower analysis and hands-on time compared with ELISA. Furthermore, the SPR assays were robust enough to be used for up to 12 days, allowing specific protein concentration measurement of a sample to be completed at line within 10 min. Using the same platform with only few varied parameters between different assays has saved time in the lab as well as for evaluation and presentation of results.     

C-terminal glyoxylyl peptides for sensitive enzyme-linked immunosorbent assays

A model peptide antigen derived from HCV Coreprotein was modified by a hydrophilic glyoxylamide arm using atartaramide-based linker and used in an enzyme-linkedimmunosorbent assay (ELISA) on hydrazide plates. A comparativestudy with the standard non-covalent adsorption proceduresdemonstrated a large gain in sensitivity for the detection ofantibodies in HCV-positive sera.     

Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

Convenient and well-performing protein detection methods for a wide range of targets are in great demand for biomedical research and future diagnostics. Assays without the need for washing steps while still unaffected when analyzing complex biological samples are difficult to develop. Herein, we report a well-characterized nucleic acid proximity-based assay using antibodies, called Proximity Extension Assay (PEA), showing good performance in plasma samples. Target-specific antibody pairs are linked to DNA strands that upon simultaneous binding to the target analyte create a real-time PCR amplicon in a proximity-dependent manner enabled by the action of a DNA polymerase. 3'Exonuclease-capable polymerases were found to be clearly superior in sensitivity over non-3'exonuclease ones. A PEA was set up for IL-8 and GDNF in a user-friendly, homogenous assay displaying femtomolar detection sensitivity, good recovery in human plasma, high specificity and up to 5-log dynamic range in 1 μL samples. Furthermore, we have illustrated the use of a macro-molecular crowding matrix in combination with this homogeneous assay to drive target binding for low-affinity antibodies, thereby improving the sensitivity and increasing affinity reagent availability by lowering assay development dependency on high-affinity antibodies. Assay performance was also confirmed for a multiplex version of PEA.     

Protein detection using proximity-dependent DNA ligation assays

The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 x 10(-21) mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.     

Protein biomarker validation via proximity ligation assays

The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PLA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Fluorescent single-stranded DNA binding protein as a probe for sensitive, real-time assays of helicase activity

The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases.