Western blotting via proximity ligation for high performance protein analysis.

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 ß 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.

Reference values for the Quality of Life Index in the general Swedish population 18-80 years of age.

PURPOSE: The aim in the present study was to establish underlying dimensions of quality of life in Sweden, measured by QLI, and to obtain reference values among a representative sample between 18 and 80 years of age from the general Swedish population. METHOD: A total of 1,680 randomly selected persons completed the questionnaire (57% response rate). All data were coded and entered into the statistical software. Factor analysis, maximum-likelihood method with oblique rotation, was employed to explore and reveal underlying dimensions of the QLI. To describe QLI total and subscale reference values for different age groups and men and women, respectively, means and 95% CI as well as medians and quartiles were used. For comparisons related to demographic and background variables, parametric and non-parametric analyses were used (alpha=0.01). All data were analysed using SPSS 14.0 statistical software. RESULTS: Four underlying dimensions emerged: Family and friends, Health and functioning, Social and economic and Psychological/spiritual. Mean values for the total QLI and the four subscales ranged between 17.2 and 23.7 (possible range=0.0-30.0). CONCLUSIONS: The overall QLI and subscale scores correspond with those presented by other researchers. Population-based measures of generic quality of life and underlying dimensions are important considering the gain when results from specific patient groups are viewed.

Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay.

The activity of proteins is typically regulated by secondary modifications and by interactions with other partners, resulting in the formation of protein complexes whose functions depend on the participating proteins. Accordingly, it is of central importance to monitor the presence of interaction complexes as well as their localization, thus providing information about the types of cells where the proteins are located and in what sub-cellular compartment these interactions occur. Several methods for visualizing protein interactions in situ have been developed during the last decade. These methods in most cases involve genetic constructs, and they have been successfully used in assays of living cell maintained in tissue culture, but they cannot easily be implemented in studies of clinical specimens. For such samples, affinity reagents like antibodies can be used to target the interacting proteins. In this review we will describe the in situ proximity ligation assays (in situ PLA), a method that is suitable for visualizing protein interactions in both tissue sections and in vitro cell lines, and we discuss research tasks when this or other method may be selected.

Construct validity of the Assessment of Work Performance (AWP).

The instrument Assessment of Work Performance (AWP 1.1) can be used to assess an individual's skills during work performance - how efficient and appropriate a client performs a work task. The instrument is currently used by over 300 assessors working in a variety of work rehabilitation settings in Sweden, and it has been used with over 10,000 clients. In this study, the construct validity of the AWP 1.1 was tested with 364 assessments of clients with a variety of various work-related problems assessed by six occupational therapists in a Social Insurance Office in Sweden between 2004 and 2005. Principal Component Analysis shows construct validity of the AWP 1.1. Further, the findings indicate that the instrument is sensitive and discriminates between clients, and no gender related patterns were identified.

Content validity and utility of the Assessment of Work Performance (AWP).

In the area of work rehabilitation, many decisions about future interventions for the client are based on the results of various kinds of assessments. Therefore, it is important that the assessment instruments used are adequate, useful, and reliable. The purpose of this study was to investigate the content validity and utility of the instrument Assessment of Work Performance (AWP) which is used to assess an individual's observable (working) skills during work performance, i.e. how efficient and appropriate a client performs a work activity. A questionnaire was answered by 67 respondents who used the AWP in various work rehabilitation settings in Sweden. The result indicates content validity and utility for the AWP that supports further testing of the instrument.

Proximity ligation: a specific and versatile tool for the proteomic era.

Knowledge about the total human genome sequence now provides opportunities to study its myriad gene products. However, the presence of alternative splicing, post-translational modifications, and innumerable protein-protein interactions among proteins occurring at widely different concentrations, all combine to place extreme demands on the specificity and sensitivity of assays. The choice of method also depends on matters such as whether proteins will be analyzed in body fluids and lysates, or localized inside single cells. In this review we discuss commonly used detection methods and compare these to the recently-developed proximity ligation technique.

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.

Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments.

We present a method to specifically select large sets of DNA sequences for parallel amplification by PCR using target-specific oligonucleotide constructs, so-called selectors. The selectors are oligonucleotide duplexes with single-stranded target-complementary end-sequences that are linked by a general sequence motif. In the selection process, a pool of selectors is combined with denatured restriction digested DNA. Each selector hybridizes to its respective target, forming individual circular complexes that are covalently closed by enzymatic ligation. Non-circularized fragments are removed by exonucleolysis, enriching for the selected fragments. The general sequence that is introduced into the circularized fragments allows them to be amplified in parallel using a universal primer pair. The procedure avoids amplification artifacts associated with conventional multiplex PCR where two primers are used for each target, thereby reducing the number of amplification reactions needed for investigating large sets of DNA sequences. We demonstrate the specificity, reproducibility and flexibility of this process by performing a 96-plex amplification of an arbitrary set of specific DNA sequences, followed by hybridization to a cDNA microarray. Eighty-nine percent of the selectors generated PCR products that hybridized to the expected positions on the array, while little or no amplification artifacts were observed.

Highly sensitive and specific protein detection via combined capillary isoelectric focusing and proximity ligation.

Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.

Analyzing genes using closing and replicating circles.

During the past two years, significant breakthroughs have been achieved in genetic analyses through the application of technologies based on analytical DNA-circularization reactions. Padlock probes and molecular inversion probes have enabled parallel, high-throughput single nucleotide polymorphism (SNP) genotyping at increased scales, whereas, at the other end of the analysis spectrum, DNA molecules in individual cells have been genotyped, in situ, using padlock probes and rolling-circle amplification (RCA). This review describes the recent developments in the technologies that use specific DNA circularization, coupled to DNA amplification through PCR or rolling-circle amplification, and addresses the great potential of these tools.

Real-time monitoring of rolling-circle amplification using a modified molecular beacon design.

We describe a method to monitor rolling-circle replication of circular oligonucleotides in dual-color and in real-time using molecular beacons. The method can be used to study the kinetics of the polymerization reaction and to amplify and quantify circularized oligonucleotide probes in a rolling-circle amplification (RCA) reaction. Modified molecular beacons were made of 2'-O-Me-RNA to prevent 3' exonucleolytic degradation by the polymerase used. Moreover, the complement of one of the stem sequences of the molecular beacon was included in the RCA products to avoid fluorescence quenching due to inter-molecular hybridization of neighboring molecular beacons hybridizing to the concatemeric polymerization product. The method allows highly accurate quantification of circularized DNA over a broad concentration range by relating the signal from the test DNA circle to an internal reference DNA circle reporting in a distinct fluorescence color.

New technologies for DNA analysis--a review of the READNA Project.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

A sense of closeness: protein detection by proximity ligation.

Highly specific and sensitive procedures will be required to evaluate proteomes. Proximity ligation is a recently introduced mechanism for protein analysis. In this technique, the convergence of sets of protein-binding reagents on individual target molecules juxtaposes attached nucleic acid sequences. Through a ligation reaction a DNA reporter sequence is created, which can be amplified. The procedure thus encodes detected proteins as specific nucleic acid sequences in what may be viewed as a reverse translation reaction.

Making ends meet in genetic analysis using padlock probes.

Padlock probes are molecular tools that combine highly specific target sequence recognition with the potential for multiplexed analysis of large sets of target DNA or RNA sequences. In this brief review, we exemplify the ability of these probes to distinguish single-nucleotide target sequence variants. We further discuss means to detect the location of target sequences in situ, and to amplify reacted padlock probes via rolling-circle replication, as well as to sort reaction products on tag-arrays. We argue that the probes have the potential to render high-throughput genetic analyses precise and affordable.

An efficient method to perform milliliter-scale PCR utilizing highly controlled microwave thermocycling.

This communication describes the development of a controlled microwave methodology for rapid milliliter-scale PCR.

Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability.

Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.

Self-efficacy beliefs and fear of childbirth in nulliparous women.

OBJECTIVE: To explore how childbirth self-efficacy, i.e. outcome expectancy and efficacy expectancy, was associated with fear of childbirth (FOC) and how efficacy expectancy and FOC, respectively were related to socio-demographic characteristics, mental problems and preference for a caesarean section. METHODS: In this cross-sectional study, a consecutive sample of 1000 pregnant nulliparous women was sent the Wijma Delivery Expectancy Questionnaire and Childbirth Self-Efficacy Inventory. Statistical analyses were performed on data from 423 women. RESULTS: Outcome expectancy and efficacy expectancy correlated significantly and positively, FOC correlated significantly and negatively with both outcome expectancy and efficacy expectancy. Women with severe FOC (20.8%) had a significantly lower level of education (p = 0.001), and had more often sought help because of mental problems (p = 0.004). They were more likely to have low-efficacy expectancy (p < 0.001) and to prefer a caesarean section instead of a vaginal birth (p < 0.001). CONCLUSIONS: Lower efficacy expectancy was associated with higher FOC while preference for a caesarean section was not. Improvement of self-efficacy could be a part of care for women with FOC during pregnancy; however, it would not be enough for fearful women who wish to have a caesarean section.

Molecular tools for companion diagnostics.

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.

Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum.

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.

In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method.

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor beta (PDGFRbeta) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRbeta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRbeta in porcine aortic endothelial cells transfected with the beta-receptor, but not in cells transfected with the alpha-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRbeta. We furthermore visualized tyrosine phosphorylated PDGFRbeta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.