Biological resources (cells, tissues, bodily fluids or biomolecules) are considered essential raw material for the advancement of health-related biotechnology, for research and development in life sciences, and for ultimately improving human health. Stored in local biobanks, access to the human biological samples and related medical data for transnational research is often limited, in particular for the international life science industry. The recently established pan-European Biobanking and BioMolecular resources Research Infrastructure-European Research Infrastructure Consortium (BBMRI-ERIC) aims to improve accessibility and interoperability between academic and industrial parties to benefit personalized medicine, disease prevention to promote development of new diagnostics, devices and medicines. BBMRI-ERIC is developing the concept of Expert Centre as public-private partnerships in the precompetitive, not-for-profit field to provide a new structure to perform research projects that would face difficulties under currently established models of academic-industry collaboration. By definition, Expert Centres are key intermediaries between public and private sectors performing the analysis of biological samples under internationally standardized conditions. This paper presents the rationale behind the Expert Centres and illustrates the novel concept with model examples.European Journal of Human Genetics advance online publication, 19 November 2014; doi:10.1038/ejhg.2014.235.
We report the development of a new database of technology services and products for analysis of biobank samples in biomedical research. BARCdb, the Biobanking Analysis Resource Catalogue (http://www.barcdb.org), is a freely available web resource, listing expertise and molecular resource capabilities of research centres and biotechnology companies. The database is designed for researchers who require information on how to make best use of valuable biospecimens from biobanks and other sample collections, focusing on the choice of analytical techniques and the demands they make on the type of samples, pre-analytical sample preparation and amounts needed. BARCdb has been developed as part of the Swedish biobanking infrastructure (BBMRI.se), but now welcomes submissions from service providers throughout Europe. BARCdb can help match resource providers with potential users, stimulating transnational collaborations and ensuring compatibility of results from different labs. It can promote a more optimal use of European resources in general, both with respect to standard and more experimental technologies, as well as for valuable biobank samples. This article describes how information on service and reagent providers of relevant technologies is made available on BARCdb, and how this resource may contribute to strengthening biomedical research in academia and in the biotechnology and pharmaceutical industries.
The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of three or more copies of the specific protein. We have developed a SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 10 (7)-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at hundred-fold higher concentration. Using either of the two monoclonal anti-PrP antibodies 3F4 and 6H4 we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases.
The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.
The in situ proximity ligation assay (isPLA) is an increasingly used technology for in situ detection of protein interactions, post-translational modifications, and spatial relationships of antigens in cells and tissues, in general. In order to test its performance we compared isPLA with immunofluorescence microscopy by analyzing protein interactions in cytoplasmic protein aggregates, so-called Mallory Denk bodies (MDBs). These structures represent protein inclusions in hepatocytes typically found in human steatohepatitis and they can be generated in mice by feeding of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). We investigated the colocalization of all three key MDB components, namely keratin 8 (K8), keratin 18 (K18), and p62 (sequestosome 1) by isPLA and immunofluorescence microscopy. Sensitivity and specificity of isPLA was assessed by using Krt8-/- and Krt18-/- mice as biological controls, along with a series of technical controls. isPLA signal visualization is a robust technology with excellent sensitivity and specificity. The biological relevance of signals generated critically depends on the performance of antibodies used, which requires careful testing of antibodies like in immunofluorescence microscopy. There is a clear advantage of isPLA in visualizing protein co-localization, particularly when antigens are present at markedly different concentrations. Furthermore, isPLA is superior to confocal microscopy with respect to spatial resolution of colocalizing antigens. Disadvantages compared to immunofluorescence are increased costs and longer duration of the laboratory protocol.
Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ~5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.
The microtubule-affinity regulating kinase (MARK) family consists of four highly conserved members that have been implicated in phosphorylation of tau protein, causing formation of neurofibrillary tangles in Alzheimers disease (AD). Understanding of roles by individual MARK isoform in phosphorylating tau has been limited due to lack of antibodies selective for each MARK isoform. In this study, we first applied the proximity ligation assay on cells to select antibodies specific for each MARK isoform. In cells, a CagA peptide specifically and significantly inhibited tau phosphorylation at Ser²?² mediated by MARK4 but not other MARK isoforms. We then used these antibodies to study expression levels of MARK isoforms and interactions between tau and individual MARK isoforms in postmortem human brains. We found a strong and significant elevation of MARK4 expression and MARK4-tau interactions in AD brains, correlating with the Braak stages of the disease. These results suggest the MARK4-tau interactions are of functional importance in the progression of AD and the results also identify MARK4 as a promising target for AD therapy.
Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.
Neuroendocrine tumors (NET) are malignant solid tumors that arise in hormone-secreting tissue of the diffuse neuroendocrine system or endocrine glands. Although traditionally understood to be a rare disease, the incidence and prevalence of NET have increased greatly in the past 3 decades. However, during this time, progress in diagnosis and outcome of NET has generally been modest. In order to achieve improved outcome in NET, a better understanding of NET biology combined with more reliable serum markers and better techniques to identify tumor localization and small lesions are needed. Although some NET biomarkers exist, sensitive and specific markers that predict tumor growth and behavior are generally lacking. In addition, the integration of new molecular imaging technologies in patient diagnosis and follow-up has the potential to enhance care. To discuss developments and issues required to improve diagnostics and management of NET patients, with specific focus on the latest advances in molecular imaging and biomarker science, 17 global leaders in the fields of NET, molecular imaging and biomarker technology gathered to participate in a 2-day meeting hosted by Prof. Kjell Öberg at the University of Uppsala in Sweden. During this time, findings were presented regarding methods with potential prognostic and treatment applications in NET or other types of cancers. This paper describes the symposium presentations and resulting discussions.
After the conclusion of the second five-year period of the European Science Foundation (ESF) programme on functional genomics, it is time to take stock and evaluate its accomplishments. The programme networked leading scientists from a large number of European countries for strategy discussions about the promotion of functional genomics research, and to arrange scientific meetings and exchange programmes. In brief, I believe this programme has punched above its weight, and that it has successfully contributed to the overall organisation of molecular biosciences in Europe. With a modest annual budget the programme has created several interesting new opportunities, some of which may have yet to show their full impact. However, these mini-reviews are intended to provide a personal perspective on this functional genomics effort, and accordingly I focus on my personal experiences from the ESF programme.
Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.
Western blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting. This combination allowed the use of isothermal rolling circle amplification of DNA molecules formed in target-specific ligation reaction, for 16-fold or greater increase in detection sensitivity. The increased specificity because of dual antibody recognition ensured highly selective assays, detecting the specific band when combinations of two cross-reactive antitubulin antibodies were used (i.e. both producing distinct nonspecific bands in traditional Western blotting). We also demonstrated detection of phosphorylated platelet-derived growth factor receptor ? by proximity ligation with one antibody directed against the receptor and another directed against the phosphorylated tyrosine residue. This avoided the need for stripping and re-probing the membrane or aligning two separate traditional blots. We demonstrate that the high-performance in situ proximity ligation-based Western blotting described herein is compatible with detection via enhanced chemiluminescence and fluorescence detection systems, and can thus be readily employed in any laboratory.
Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use.
Gene expression - a key feature for modulating cell fate-is regulated in part by histone modifications, which modulate accessibility of the chromatin to transcription factors. Until now, protein-DNA interactions (PDIs) have mostly been studied in bulk without retrieving spatial information from the sample or with poor sequence resolution. New tools are needed to reveal proteins interacting with specific DNA sequences in situ for further understanding of the orchestration of transcriptional control within the nucleus. We present herein an approach to visualise individual PDIs within cells, based on the in situ proximity ligation assay (PLA). This assay, previously used for the detection of protein-protein interactions in situ, was adapted for analysis of target PDIs, using padlock probes to identify unique DNA sequences in complex genomes. As a proof-of-principle we detected histone H3 interacting with a 26 bp consensus sequence of the Alu-repeat abundantly expressed in the human genome, but absent in mice. However, the mouse genome contains a highly similar sequence, providing a model system to analyse the selectivity of the developed methods. Although efficiency of detection currently is limiting, we conclude that in situ PLA can be used to achieve a highly selective analysis of PDIs in single cells.
Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (? 6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.
While minimum information about a microarray experiment (MIAME) standards have helped to increase the value of the microarray data deposited into public databases like ArrayExpress and Gene Expression Omnibus (GEO), limited means have been available to assess the quality of this data or to identify the procedures used to normalize and transform raw data. The EMERALD FP6 Coordination Action was designed to deliver approaches to assess and enhance the overall quality of microarray data and to disseminate these approaches to the microarray community through an extensive series of workshops, tutorials, and symposia. Tools were developed for assessing data quality and used to demonstrate how the removal of poor-quality data could improve the power of statistical analyses and facilitate analysis of multiple joint microarray data sets. These quality metrics tools have been disseminated through publications and through the software package arrayQualityMetrics. Within the framework provided by the Ontology of Biomedical Investigations, ontology was developed to describe data transformations, and software ontology was developed for gene expression analysis software. In addition, the consortium has advocated for the development and use of external reference standards in microarray hybridizations and created the Molecular Methods (MolMeth) database, which provides a central source for methods and protocols focusing on microarray-based technologies.
Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A? peptides are cornerstones in the pathology of Alzheimers disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of A? protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.
Alternative splicing creates diverse mRNA isoforms from single genes and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns along transcripts into a library of binary strings of nucleic acid tags that encode the exon composition of individual mRNA molecules. The exon inclusion pattern of each analyzed transcript is thus represented as binary data, and the abundance of different splice variants is registered by counts of individual molecules. The technique is illustrated in a model experiment by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits many genes to be analyzed in parallel and it will be valuable for elucidating the complex effects of combinatorial splicing.
The current arsenal of molecular tools for site-directed cleavage of single-stranded DNA (ssDNA) is limited. Here, we describe a method for targeted DNA cleavage that requires only the presence of an A nucleotide at the target position. The procedure involves hybridization of a complementary oligonucleotide probe to the target sequence. The probe is designed to create a deliberate G:A mismatch at the desired position of cleavage. The DNA repair enzyme MutY glycosylase recognizes the mismatch structure and selectively removes the mispaired A from the duplex to create an abasic site in the target strand. Addition of an AP-endonuclease, such as Endonuclease IV, subsequently cleaves the backbone dividing the DNA strand into two fragments. With an appropriate choice of an AP-cleaving enzyme, the 3- and 5-ends of the cleaved DNA are suitable to take part in subsequent enzymatic reactions such as priming for polymerization or joining by DNA ligation. We define suitable standard reaction conditions for glycosylase/AP-cleaving enzyme (G/AP) cleavage, and demonstrate the use of the method in an improved scheme for in situ detection using target-primed rolling-circle amplification of padlock probes.
Detection of proteins released in the bloodstream from tissues damaged by disease can promote early detection of pathological conditions, differential diagnostics, and follow-up of therapy. Despite these prospects and a plethora of candidate biomarkers, efforts in recent years to establish new protein diagnostic assays have met with limited success. One important limiting factor has been the challenge of detecting proteins present at trace levels in complex bodily fluids. To achieve robust, sensitive, and specific detection, we have developed a microparticle-based solid-phase proximity ligation assay, dependent on simultaneous recognition of target proteins by three antibody molecules for added specificity. After capture on a microparticle, solid-phase pairs of proximity probes are added followed by washes, enabling detection and identification of rare protein molecules in blood while consuming small amounts of sample. We demonstrate that single polyclonal antibody preparations raised against target proteins of interest can be readily used to establish assays where detection depends on target recognition by three individual antibody molecules, recognizing separate epitopes. The assay was compared with state-of-the-art sandwich ELISAs for detection of vascular endothelial growth factor, interleukin-8 and interleukin-6, and it was found to be superior both with regard to dynamic range and minimal numbers of molecules detected. Furthermore, the assays exhibited excellent performance in undiluted plasma and serum as well as in whole blood, producing comparable results for nine different antigens. We thus show that solid-phase proximity ligation assay is suitable for validation of a variety of protein biomarkers over broad dynamic ranges in clinical samples.
The in situ proximity ligation assay (PLA) allows a protein or protein complex to be represented as an amplifiable DNA molecule. Recognition is mediated by proximity probes consisting of antibodies coupled with oligonucleotides. Upon dual binding of the proximity probes, the oligonucleotides direct the formation of a circular DNA molecule, which is then amplified by rolling-circle replication. The localized concatemeric product is then detected with fluorescent probes. The in situ PLA enables localized detection of individual native proteins or interacting protein pairs in fixed cells or tissue sections, thus providing an important tool for basic and clinical research.
The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990 s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor beta signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery.
Changes in mucin protein expression and in glycosylation are common features in pre-neoplastic lesions and cancer and are therefore used as cancer-associated markers. De novo expression of intestinal mucin MUC2 and cancer-associated sialyl-Tn antigen are frequently observed in intestinal metaplasia (IM) and gastric cancer. However, despite that these antigens often co-localize, MUC2 has not been demonstrated to be a carrier of sialyl-Tn. By using the in situ proximity ligation assay (in situ PLA), we herein could show that MUC2 is a major carrier of the sialyl-Tn antigen in all IM cases and in most gastric carcinoma cases. The requirement by in situ PLA for the presence of both antigens in close proximity increases the selectivity compared to measurement of co-localization, as determined by immunohistochemistry. Identification of the mucin which is the carrier of a carbohydrate structure offers unique advantages for future development of more accurate diagnostic and prognostic markers.
Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site.
Interactions between members of the epidermal growth factor receptor (EGFR) family mediates cellular responses to ligand stimulation. Measurement of these interactions could provide important information and may prove useful as prognostic markers in malignancy. Therefore, to develop methods to study these interactions in genetically unmodified cells, such as clinical samples, in a sensitive and selective way, with good statistical accuracy, is important. The in situ proximity ligation assay (in situ PLA) was used to quantify homo- and heteromeric interactions between EGFR and HER2 in cultured cells, using flow cytometry as the readout method. Cells were monitored for changes in dimerization patterns and phosphorylation status upon stimulation. The different cell lines displayed varying amounts of interactions between EGFR and HER2, but the amount of dimerization was not found to be affected significantly upon stimulation by EGF. Activation of EGFR could be visualized by in situ PLA, but not by immunofluorescence staining. In situ PLA was successfully used to study receptor dimerization and activation of the EGF-receptor family with high selectivity and sensitivity. The combination of in situ PLA and flow cytometry provided a statistically powerful way of analyzing protein-protein interactions and post-translational modifications on a single-cell basis.
Prostate specific antigen (PSA)-alpha1-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.
Recent outbreaks of avian influenza in different parts of the world have caused major economic losses for the poultry industry, affected wildlife seriously and present a significant threat even to human public health, due to the risk for zoonotic transmission. The ability to recognize avian influenza viruses (AIVs) early is of paramount importance to ensure that appropriate measures can be taken quickly to contain the outbreak. In this study, the performance of a proximity ligation assay (PLA) for the detection of AIV antigens in biological specimens was evaluated. It is shown that PLA: (i) as a novel principle of highly sensitive antigen detection is extending the arsenal of tools for the diagnosis of AIV; (ii) is very specific, nearly as sensitive as a commonly used reference real-time PCR assay, and four orders of magnitude more sensitive than a sandwich ELISA, utilizing the same antibody; (iii) avoids the necessity of nucleic acids extraction, which greatly facilitates high-throughput implementations; (iv) allows the use of inactivated samples, which safely can be transported from the field to diagnostic laboratories for further analysis. In summary, the results demonstrate that PLA is suited for rapid, accurate and early detection of AIV.
Analytical reactions in which short DNA strands are used in combination with DNA ligases have proven useful for measuring, decoding, and locating most classes of macromolecules. Given the need to accumulate large amounts of precise molecular information from biological systems in research and in diagnostics, ligation reactions will continue to offer valuable strategies for advanced analytical reactions. Here, we provide a basis for further development of methods by reviewing the history of analytical ligation reactions, discussing the properties of ligation reactions that render them suitable for engineering novel assays, describing a wide range of successful ligase-based assays, and briefly considering future directions.
A new concept for multiplex detection and quantification of microbes is here demonstrated on a range of infectious fungal species. Padlock probe methodology in conjunction with qPCR and Luminex technology was used for simultaneous detection of ten fungal species in one single experiment. By combining the multiplexing properties of padlock probes and Luminex detection with the well established quantitative characteristics of qPCR, quantitative microbe detection was done in 10-plex mode. A padlock probe is an oligonucleotide that via a ligation reaction forms circular DNA when hybridizing to specific target DNA. The region of the padlock probe that does not participate in target DNA hybridization contains generic primer sequences for amplification and a tag sequence for Luminex detection. This was the fundament for well performing multiplexing. Circularized padlock probes were initially amplified by rolling circle amplification (RCA), followed by a SybrGreen real time PCR which allowed an additive quantitative assessment of target DNA in the sample. Detection and quantification of amplified padlock probes were then done on color coded Luminex microspheres carrying anti-tag sequences. A novel technique, using labeled oligonucleotides to prevent reannealing of amplimers by covering the flanks of the address sequence, improved the signal to noise ratio in the detection step considerably. The method correctly detected fungi in a variety of clinical samples and offered quantitative information on fungal nucleic acid.
Dyslexia, or specific reading disability, is the unexpected failure in learning to read and write when intelligence and senses are normal. One of the susceptibility genes, DYX1C1, has been implicated in neuronal migration, but little is known about its interactions and functions. As DYX1C1 was suggested to interact with the U-box protein CHIP (carboxy terminus of Hsc70-interacting protein), which also participates in the degradation of estrogen receptors alpha (ERalpha) and beta (ERbeta), we hypothesized that the effects of DYX1C1 might be at least in part mediated through the regulation of ERs. ERs have shown to be important in brain development and cognitive functions. Indeed, we show that DYX1C1 interacts with both ERs in the presence of 17beta-estradiol, as determined by co-localization, co-immunoprecipitation and proximity ligation assays. Protein levels of endogenous ERalpha or exogenous ERbeta were reduced upon over-expression of DYX1C1, resulting in decreased transcriptional responses to 17beta-estradiol. Furthermore, we detected in vivo complexes of DYX1C1 with ERalpha or ERbeta at endogenous levels along neurites of primary rat hippocampal neurons. Taken together, our data suggest that DYX1C1 is involved in the regulation of ERalpha and ERbeta, and may thus affect the brain development and regulate cognitive functions. These findings provide novel insights into the function of DYX1C1 and link neuronal migration and developmental dyslexia to the estrogen-signaling effects in the brain.
Multiplex protein quantification has been constrained by issues of assay specificity, sensitivity and throughput. This research presents a novel approach that overcomes these limitations using antibody-oligonucleotide conjugates for immuno-polymerase chain reaction (immuno-PCR) or proximity ligation, coupled with competitive PCR and MALDI-TOF mass spectrometry. Employing these combinations of technologies, we demonstrate multiplex detection and quantification of up to eight proteins, spanning wide dynamic ranges from femtomolar concentrations, using only microliter sample volumes.
Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.
Management of data to produce scientific knowledge is a key challenge for biological research in the 21st century. Emerging high-throughput technologies allow life science researchers to produce big data at speeds and in amounts that were unthinkable just a few years ago. This places high demands on all aspects of the workflow: from data capture (including the experimental constraints of the experiment), analysis and preservation, to peer-reviewed publication of results. Failure to recognise the issues at each level can lead to serious conflicts and mistakes; research may then be compromised as a result of the publication of non-coherent protocols, or the misinterpretation of published data. In this report, we present the results from a workshop that was organised to create an ontological data-modelling framework for Laboratory Protocol Standards for the Molecular Methods Database (MolMeth). The workshop provided a set of short- and long-term goals for the MolMeth database, the most important being the decision to use the established EXACT description of biomedical ontologies as a starting point.
While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-mediated affinity-based assays, such as immuno-PCR and proximity ligation assays (PLA), use oligonucleotides attached to affinity reagents as reporter molecules. Conjugation of oligonucleotides to affinity reagents generally employs chemistries that target primary amines or cysteines. Because of the random nature of these processes neither the number of oligonucleotides conjugated per molecule nor their sites of attachment can be accurately controlled for affinity reagents with several available amines and cysteines. Here, we present a straightforward and convenient approach to functionalize recombinant affinity reagents for PLA by expressing the reagents as fusion partners with SNAP protein tags. This allowed us to conjugate oligonucleotides in a site-specific fashion, yielding precisely one oligonucleotide per affinity reagent. We demonstrate this method using designed ankyrin repeat proteins (DARPins) recognizing the tumor antigen HER2 and we apply the conjugates in different assay formats. We also show that SNAP or CLIP tags, expressed as fusion partners of transfected genes, allow oligonucleotide conjugations to be performed in fixed cells, with no need for specific affinity reagents. The approach is used to demonstrate induced interactions between the fusion proteins FKBP and FRB by allowing the in situ conjugated oligonucleotides to direct the production of templates for localized rolling circle amplification reactions.
The heterogeneous nature of cancer results in highly variable therapeutic responses even among patients with identical stages and grades of a malignancy. The move towards personalised medicine in cancer therapy has therefore been motivated by a need to customise therapy according to molecular features of individual tumours. Companion diagnostics serves to support early drug development, it can provide surrogate markers in clinical trials, and also guide selection of individual therapies and monitoring of responses in routine clinical care. The era of companion diagnostics can be said to have begun with the introduction of the HercepTest - a first-of-a-kind diagnostic tool developed by DakoCytomation in 1998 to select patients for therapy with the anticancer drug Herceptin (trastuzumab). Herceptin and the paired test proved that companion diagnostics can help guide patient-tailored therapies. We will discuss herein technologies to analyse companion diagnostics markers at the level of DNA, RNA or protein, focusing on a series of methods developed in our laboratory that can facilitate drug development and help stratify patients for therapy.
Fundamental open questions in signal transduction remain concerning the sequence and distribution of molecular signaling events among individual cells. In this work, we have characterized the intercellular variability of transforming growth factor ?-induced Smad interactions, providing essential information about TGF-? signaling and its dependence on the density of cell populations and the cell cycle phase. By employing the recently developed in situ proximity ligation assay, we investigated the dynamics of interactions and modifications of Smad proteins and their partners under native and physiological conditions. We analyzed the kinetics of assembly of Smad complexes and the influence of cellular environment and relation to mitosis. We report rapid kinetics of formation of Smad complexes, including native Smad2-Smad3-Smad4 trimeric complexes, in a manner influenced by the rate of proteasomal degradation of these proteins, and we found a striking cell to cell variation of signaling complexes. The single-cell analysis of TGF-? signaling in genetically unmodified cells revealed previously unknown aspects of regulation of this pathway, and it provided a basis for analysis of these signaling events to diagnose pathological perturbations in patient samples and to evaluate their susceptibility to drug treatment.
Improved protein assays promise to offer new insights into biological processes as well as the identification of new, clinically important biomarkers. In recent years, a number of approaches have been developed where protein-binding reagents, typically antibodies, are equipped with DNA strands to enable protein analyses via powerful nucleic acid detection reactions for improved performance. In this review, we provide a background to this emerging field, and we describe several different ways in which these reagents can improve protein analyses by lowering detection thresholds, improving multiplexing and extending the range of biomolecules available for analysis, both in research settings and in clinical routine.
Despite great interest, investments, and efforts, the ongoing search for plasma protein biomarkers for disease so far has come up surprisingly empty-handed. Although discovery programs have revealed large numbers of biomarker candidates, the clinical utility has been validated for only a very small number of these. While this disappointing state of affairs may suggest that plasma protein biomarkers have little more to offer for diagnostics, we take the perspective that experimental conditions might not have been optimal and that analyses will be required that offer far greater sensitivity than currently available, in terms of numbers of molecules needed for unambiguous detection. Accordingly, techniques are needed to search deep and wide for protein biomarker candidates. The requirements and feasibility of such assays will be discussed.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.