Antiviral interferon response at single-cell resolution.

Experimental studies of the innate immune response of mammalian cells to viruses reveal pervasive heterogeneity at the level of single cells. Interferons are induced only in a fraction of virus-infected cells; subsequently a fraction of cells exposed to interferons upregulate interferon-stimulated genes. Nevertheless, quantitative experiments and linked mathematical models show that the interferon response can be effective in curbing viral spread through two distinct mechanisms. First, paracrine interferon signals from scattered source cells can protect many uninfected cells, and the self-amplification of interferon production might serve to calibrate response amplitude to strength of viral infection. Second, models of the tug-of-war between viral replication and the innate interferon response imply a pivotal role of interferon action on already infected cells in curbing viral spread, through effectively lowering virus replication rate. This finding is in line with the observation that several pathogenic viruses selectively abrogate interferon action on infected cells. Thus, interferons may delay viral spread in acute infections by acting as sentinels, warning uninfected cells of imminent danger, or as negative feedback regulators of virus replication in infected cells. The timing of the interferon response relative to the onset of viral replication is critical for its effectiveness in curbing viral spread.

The genesis and evolution of bead-based multiplexing.

Multiplexed analysis has the advantage of allowing for simultaneous detection of multiple analytes in a single reaction vessel which reduces time, labor, and cost as compared to single-reaction-based detection methods. Microsphere-based suspension array technologies, such as the Luminex® xMAP® system, offer high-throughput detection of both protein and nucleic acid targets in multiple assay chemistries. After Luminex's founding in 1995, it quickly became the leader in bead-based multiplexing solutions. Today, xMAP Technology is the most widely adopted bead-based multiplexing platform with over 35,000 peer-reviewed publications, an installed base of approximately 15,500 instruments, and over 70 Luminex Partners offering more than 1300 research use kits as well as custom assay solutions. Because of the open architecture of the xMAP platform it has been implemented in a variety of applications that range from transplant medicine, biomarker discovery and validation, pathogen detection, drug discovery, vaccine development, personalized medicine, neurodegeneration, and cancer research.

Review: Diagnosis of Helicobacter pylori infection.

Endoscopic imaging of the stomach is improving. In addition to narrow band imaging, other methods, for example, blue light imaging and linked color imaging, are now available and can be combined with artificial intelligence systems to obtain information on the gastric mucosa and detect early gastric cancer. Immunohistochemistry is only recommended as an ancillary stain in case of chronic active gastritis without Helicobacter pylori detection by standard staining, and recommendations to exclude false negative H. pylori results have been made. Molecular methods using real-time PCR, droplet digital PCR, or amplification refractory mutation system PCR have shown a high accuracy, both for detecting H. pylori and for clarithromycin susceptibility testing, and can now be used in clinical practice for targeted therapy. The most reliable non-invasive test remains the 13 C-urea breath test. Large data sets show that DOB values are higher in women and that the cut-off for positivity could be decreased to 2.74 DOB. Stool antigen tests using monoclonal antibodies are widely used and may be a good alternative to UBT, particularly in countries with a high prevalence of H. pylori infection. Attempts to improve serology by looking at specific immunodominant antigens to distinguish current and past infection have been made. The interest of Gastropanel® which also tests pepsinogen levels was confirmed.

United States and European Multicenter Prospective Study for the Analytical Performance and Clinical Validation of a Novel Sensitive Fully Automated Immunoassay for Calcitonin.

BACKGROUND: The objective of this study is the validation and proof of clinical relevance of a novel electrochemiluminescence immunoassay (ECLIA) for the determination of serum calcitonin (CT) in patients with medullary thyroid carcinoma (MTC) and in different diseases of the thyroid and of calcium homeostasis. METHODS: This was a multicenter prospective study on basal serum CT concentrations performed in 9 US and European referral institutions. In addition, stimulated CT concentrations were measured in 50 healthy volunteers after intravenous calcium administration (2.5 mg/kg bodyweight). RESULTS: In total, 1929 patients and healthy controls were included. Limits of blank, detection, and quantification for the ECLIA were 0.3, 0.5, and 1 ng/L, respectively. Highest intra- and interassay coefficients of variation were 7.4% (CT concentration, 0.8 ng/L) and 7.0% (1.1 ng/L), respectively. Medians (interval) of serum CT concentrations in 783 healthy controls were 0.8 ng/L (<0.5-12.7) and 3 ng/L (<0.5-18) for females and males, respectively (97.5th percentile, 6.8 and 11.6 ng/L, respectively). Diagnostic sensitivity and specificity were 100%/97.1% and 96.2%/96.4%, for female/males, respectively. Patients (male/female) with primary hyperparathyroidism, renal failure, and neuroendocrine tumors showed CT concentrations >97.5th percentile in 33%/4.7%, 18.5%/10%, and 8.3%/12%, females/males, respectively. Peak serum CT concentrations were reached 2 min after calcium administration (161.7 and 111.8 ng/L in males and females, respectively; P < 0.001). CONCLUSIONS: Excellent analytical performance, low interindividual variability, and low impact of confounders for increased CT concentrations in non-MTC patients indicate that the investigated assay has appropriate clinical utility. Calcium-stimulated CT results suggest good test applicability owing to low interindividual variability.

[Paradigm shift in ANCA diagnostics : New international consensus recommendations]. / Paradigmenwechsel in der ANCA-Diagnostik : Neue internationale Konsensusempfehlungen.

BACKGROUND: Up to now indirect immunofluorescence (IIF) followed by an antigen-specific assay specific for proteinase 3 (PR3) or myeloperoxidase (MPO) has been the standard method for the detection of antineutrophil cytoplasmic antibodies (ANCA). The development of more sensitive and highly specific PR3-ANCA and MPO-ANCA immunoassays for the diagnosis of ANCA-associated vasculitis (AAV) has raised doubts about the two-stage diagnostic strategy currently recommended for ANCA detection. OBJECTIVE: Presentation and discussion of the new international consensus recommendations on ANCA testing in AAV. METHODS: This article presents the new guidelines for ANCA testing that have been developed based on the results of a recent large multicenter study by the European Vasculitis Society (EUVAS). The draft of the author committee was revised by each contributor and subsequently distributed to 12 experts on 4 continents. After further revision the final document was returned for ratification and submitted for publication. RESULTS/CONCLUSION: The current study results confirm the superiority of the diagnostic value of antigen-specific immunoassays compared to IIF. The current consensus recommendations support the primary use of PR3-ANCA and MPO-ANCA immunoassays for diagnostic evaluation of patients with AAV without the categorical need for additional IIF.

Antineutrophil cytoplasmic antibodies (ANCA) testing: detection methods and clinical application.

Antineutrophil cytoplasmic antibodies (ANCA) are considered the diagnostic biomarker of some necrotising vasculitis such as granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and, to a lesser extent, eosinophilic granulomatosis with polyangiitis (EGPA). According to the current recommendations, combining indirect immunofluorescence and proteinase 3 (PR3) and myeloperoxidase (MPO) antigen specific immunometric assays, in the proper clinical setting, assures the best diagnostic specificity. When such conditions are satisfied, ANCA are detected in up to 90% of patients with active generalised GPA and MPA and in about 40% of patients with EGPA. Cytoplasmic ANCA (C-ANCA) with specificity for PR3 are usually found in patients with GPA whereas perinuclear ANCA (P-ANCA) in patients with MPA and EGPA. However, ANCA antigen specificity is more closely associated with disease phenotype and prognosis than clinical diagnosis. The clinical value of serial ANCA testing in monitoring disease activity is still debated. Recently, new promising developments in methodology and techniques (computer-based image analysis of immunofluorescence patterns, novel generation of PR3-/MPO-ANCA immunometric assays and multiplex technology) have been proposed but studies comparing the performances of the different assays are scarce.

Current and emerging technologies for rapid detection and characterization of Salmonella in poultry and poultry products.

Salmonella is the leading cause of foodborne illnesses in the United States, and one of the main contributors to salmonellosis is the consumption of contaminated poultry and poultry products. Since deleterious effects of Salmonella on public health and the economy continue to occur, there is an ongoing need to develop more advanced detection methods that can identify Salmonella accurately and rapidly in foods before they reach consumers. Rapid detection and identification methods for Salmonella are considered to be an important component of strategies designed to prevent poultry and poultry product-associated illnesses. In the past three decades, there have been increasing efforts towards developing and improving rapid pathogen detection and characterization methodologies for application to poultry and poultry products. In this review, we discuss molecular methods for detection, identification and genetic characterization of Salmonella associated with poultry and poultry products. In addition, the advantages and disadvantages of the established and emerging rapid detection and characterization methods are addressed for Salmonella in poultry and poultry products. The methods with potential application to the industry are highlighted in this review.

Clinical utility and analytical challenges in measurement of cerebrospinal fluid amyloid-ß(1-42) and τ proteins as Alzheimer disease biomarkers.

BACKGROUND: Over the past 2 decades, clinical studies have provided evidence that cerebrospinal fluid (CSF) amyloid ß(1-42) (Aß(1-42)), total τ (t-τ), and τ phosphorylated at Thr181 (p-τ(181)) are reliable biochemical markers of Alzheimer disease (AD) neuropathology. CONTENT: In this review, we summarize the clinical performance and describe the major challenges for the analytical performance of the most widely used immunoassay platforms [based on ELISA or microbead-based multianalyte profiling (xMAP) technology] for the measurement of CSF AD biomarkers (Aß(1-42), t-τ, and p-τ(181)). With foundational immunoassay data providing the diagnostic and prognostic values of CSF AD biomarkers, the newly revised criteria for the diagnosis of AD include CSF AD biomarkers for use in research settings. In addition, it has been suggested that the selection of AD patients at the predementia stage by use of CSF AD biomarkers can improve the statistical power of clinical trial design. Owing to the lack of a replenishable and commutable human CSF-based standardized reference material (SRM) and significant differences across different immunoassay platforms, the diagnostic-prognostic cutpoints of CSF AD biomarker concentrations are not universal at this time. These challenges can be effectively met in the future, however, through collaborative ongoing standardization efforts to minimize the sources of analytical variability and to develop reference methods and SRMs. SUMMARY: Measurements of CSF Aß(1-42), t-τ, and p-τ(181) with analytically qualified immunoassays reliably reflect the neuropathologic hallmarks of AD in patients at the early predementia stage of the disease and even in presymptomatic patients. Thus these CSF biomarker tests are useful for early diagnosis of AD, prediction of disease progression, and efficient design of drug intervention clinical trials.

Nanoparticle-based immunoassays in the biomedical field.

Recent research has looked to develop innovative, powerful and novel biofunctionalized nanoparticles, controlling and tailoring their properties in a very predictable manner to meet the needs of clinic immunoassays in the biomedical field. This minireview briefly summarizes recent advances covering the last 3 years, exploiting nanoparticle-based electrochemical, optical, mass-sensitive, colorimetric and immunodipstick assays. The enormous signal enhancement associated with the use of nanoparticles and formation of nanoparticle-antibody-antigen assemblies provide the basis for sensitive detection of disease-related proteins or biomarkers. Rather than being exhaustive, this minireview focuses on selected examples to illustrate novel concepts and promising applications. Finally, a small amount of speculation of possible future developments in nanoparticle-based immunoassays is provided.

[Autoantibody diagnostics in neurology using native and recombinant antigenic substrates]. / Autoantikörperdiagnostik in der Neurologie mittels nativer und rekombinanter Antigensubstrate.

Modern diagnostics for the determination of neurologically relevant autoantibodies are based on indirect immunofluorescence using tissue sections of the hippocampus, cerebellum and other tissues. For monospecific detection human embryonic kidney (HEK) cells transfected with different neurological antigens are used. Biochip mosaics are designed to give a quick overview and contain 20 or more substances positioned next to each other on a reaction field, which are incubated with the serum or cerebrospinal fluid (CSF) sample. Western blots based on cerebellum or hippocampus extracts or line blots containing defined recombinant antigens are used additionally. Initial investigations should always comprise the parallel analysis of all major antineural autoantibodies instead of performing only single parameter tests. Up until a few years ago autoantibodies against intracellular neuronal antigens were mainly investigated. Antibodies against structures of the neural cell surface, however, are much more frequently found, especially those against glutamate receptors (type NMDA).

Analytical chemiluminescence and bioluminescence: latest achievements and new horizons.

Chemiluminescence (CL) and bioluminescence (BL) are the detection techniques of choice for the development of highly sensitive analytical methods, from immunoassays and nucleic acid hybridization assays to whole-cell biosensors. Nevertheless, basic and applied research on CL and BL aimed at further improving their analytical performance is still very active. This research covers diverse and complementary fields, including (among others) enhancing the light emission efficiency of CL systems, the use of nanomaterials to catalyze or enhance CL/BL reactions, the study of BL proteins to elucidate the color modulation mechanism, the discovery of new BL systems, the production of thermostable BL protein mutants with altered emission spectra, the development of BL imaging techniques to expand our understanding of living systems, and the implementation of CL/BL detection in miniaturized analytical devices. In the near future, we expect even greater diffusion of CL/BL-based analytical methods, especially in portable analytical devices intended for applications ranging from environmental analysis to companion diagnostics for personalized medicine.

Immunology and immunodiagnosis of cystic echinococcosis: an update.

Cystic echinococcosis (CE) is a cosmopolitan zoonosis caused by the larval cystic stage of the dog tapeworm Echinococcus granulosus. This complex multicellular pathogen produces various antigens which modulate the host immune response and promote parasite survival and development. The recent application of modern molecular and immunological approaches has revealed novel insights on the nature of the immune responses generated during the course of a hydatid infection, although many aspects of the Echinococcus-host interplay remain unexplored. This paper summarizes recent developments in our understanding of the immunology and diagnosis of echinococcosis, indicates areas where information is lacking, and suggests possible new strategies to improve serodiagnosis for practical application.

Updates in immunoassays: bacteriology.

There are many immunoassays available that provide rapid, accurate and sensitive results. The intent of this article was to provide a brief overview of some of the products and methodologies available for clinical use and to discuss some of the principles behind the methodology and instrumentation. In the area of infectious disease, the use of immunoassays ensures rapid turnaround times that will result in the administration of prompt, accurate treatment for the patient. Ultimately, this will improve overall patient outcomes while possibly decreasing the costs associated with increased hospital stay. In conclusion, immunoassays are essentially easy to perform, cost-effective, produce highly sensitive and specific results, and allow the medical laboratory professional the ability to report accurate results in a timely manner.

Updates in immunoassays: virology.

Virus identification is a challenge to the clinical microbiologist since growing viruses in traditional cell culture is labor intensive, time consuming, and subject to contamination. The advent of rapid and automated immunoassays has eliminated this problem by generating positive results in minutes to hours. For example, testing for infectious mononucleosis can yield a positive result in 3-8 minutes as seen with the Beckman Coulter, Inc. ICON Mono test or in 5-15 minutes with the MONO Mononucleosis Rapid Test Device marketed by ACON Laboratories, Inc. Fully automated immunoassay analyzers provide fast, accurate, sensitive results that aid in a prompt and accurate diagnosis for the patient. Turnaround times are shortened, allowing for timely medical intervention and treatment. The priority in any hospital or medical facility is to treat the patient as quickly and appropriately as possible. By using immunoassays, clinical laboratory professionals are able to report out correct results in a timely manner, ensuring overall positive patient outcomes and improved quality of healthcare.

Updates in immunoassays: parasitology.

Although most clinical laboratories use microscopy and routine O&P procedures when identifying parasitic infections, there are several parasites that are better detected through serological means. Toxoplasma, Giardia, and Cryptosporidium were discussed along with immunoassays used for their detection. Immunoassays provide quick results and are less labor intensive than specimen concentration and slide preparation for microscopic examination. These assays are easy to use and provide sensitive and specific results. Some clinical laboratories no longer perform O&Ps in house and refer specimens to reference laboratories for evaluation. By using immunoassays, some of the more common parasites can be identified in a timely manner reducing turn-around times. Some controversy exists over the use of IIF and EIA tests used for ANA testing along with measuring CRPs and PCT as predictors of bacterial sepsis and septic shock. Regardless of the methodology discussed in this series of articles, there are pros and cons to the various immunoassays available. Determining the most appropriate assay based on patient population and volume is governed by the institution and its patients' needs. In conclusion, immunoassays, whether manual or automated, are easy to use, cost effective and allow the medical laboratory professional to provide quick and accurate results to the clinician so the most appropriate treatment can be administered to the patient. The ultimate goal of healthcare professionals is to provide the highest quality of medical care in a timely manner. The use of immunoassays in the clinical laboratory allows the healthcare team to successfully achieve this goal.

Systemic sclerosis - focus on dermatological aspects. Part 2: diagnostics, therapy.

Systemic sclerosis is a chronic inflammatory multiorgan disease which may involve the skin and internal organs to a varying extent. Pathogenetically the vasculature, connective tissue and the immune system are involved in a yet to be defined sequence and impact. Case history and results of physical as well as laboratory examinations will determine individually adapted further organ imaging or invasive procedures. Based on their results therapy is initiated which may include supportive measures such as physiotherapy as well as basic skin care and avoidance of any trauma. Many agents are available for the circulatory problems including Raynaud phenomenon and digital ulcers such as calcium channel blockers, ACE inhibitors and intravenous prostacyclin derivatives, as well as endothelin receptor blockers and phosphodiesterase inhibitors. Immunosuppressive and immunomodulatory agents are of varying efficacy depending on organ involvement. Though various therapeutic measures are available, beneficial effects are limited and associated with various unwanted effects. In any case, the therapy has to be individually adapted to the disease stage and course of the disease.

Simple Western: Bringing the Western Blot into the Twenty-First Century.

The Western blot is widely used in the study of protein biochemistry, but it is notoriously labor-intensive, and it is limited in its reproducibility and quantification, among many other challenges. By contrast, capillary-based protein separation and immunodetection, known as Simple Western™, overcomes many of the challenges associated with the traditional Western blot, and it is quickly gaining traction as a replacement for traditional Western blot analysis. The advantages that capillary-based immunoassay offers include ease of use, automation, reproducibility, quantification, and even built-in total protein normalization. In this chapter, we describe protocols for the two basic types of capillary-based immunodetection assays, one by molecular weight separation and the other by charge separation. In both methods, protein samples are separated in the capillary followed seamlessly by immunodetection with chemiluminescent or fluorescent antibodies for highly sensitive and specific detection of target proteins.

Autoantibody Discovery, Assay Development and Adoption: Death Valley, the Sea of Survival and Beyond.

Dating to the discovery of the Lupus Erythematosus (LE) cell in 1948, there has been a dramatic growth in the discovery of unique autoantibodies and their cognate targets, all of which has led to the availability and use of autoantibody testing for a broad spectrum of autoimmune diseases. Most studies of the sensitivity, specificity, commutability, and harmonization of autoantibody testing have focused on widely available, commercially developed and agency-certified autoantibody kits. However, this is only a small part of the spectrum of autoantibody tests that are provided through laboratories world-wide. This manuscript will review the wider spectrum of testing by exploring the innovation pathway that begins with autoantibody discovery followed by assessment of clinical relevance, accuracy, validation, and then consideration of regulatory requirements as an approved diagnostic test. Some tests are offered as "Research Use Only (RUO)", some as "Laboratory Developed Tests (LDT)", some enter Health Technology Assessment (HTA) pathways, while others are relegated to a "death valley" of autoantibody discovery and become "orphan" autoantibodies. Those that achieve regulatory approval are further threatened by the business world's "Darwinian Sea of Survival". As one example of the trappings of autoantibody progression or failure, it is reported that more than 200 different autoantibodies have been described in systemic lupus erythematosus (SLE), a small handful (~10%) of these have achieved regulatory approval and are widely available as commercial diagnostic kits, while a few others may be available as RUO or LDT assays. However, the vast majority (90%) are orphaned and languish in an autoantibody 'death valley'. This review proposes that it is important to keep an inventory of these "orphan autoantibodies" in 'death valley' because, with the increasing availability of multi-analyte arrays and artificial intelligence (MAAI), some can be rescued to achieve a useful role in clinical diagnostic especially in light of patient stratification and precision medicine.

Multiplexed microbead immunoassays by flow cytometry for molecular profiling: Basic concepts and proteomics applications.

Flow cytometry was originally established as an automated method for measuring optical or fluorescence characteristics of cells or particles in suspension. With the enormous increase in development of reliable electronics, lasers, micro-fluidics, as well as many advances in immunology and other fields, flow cytometers have become user-friendlier, less-expensive instruments with an increasing importance for both basic research and clinical applications. Conventional uses of flow cytometry include immunophenotyping of blood cells and the analysis of the cell cycle. Importantly, methods for labeling microbeads with unique combinations of fluorescent spectral signatures have made multiplex analysis of soluble analytes (i.e. the ability to detect multiple targets in a single test sample) feasible by flow cytometry. The result is a rapid, high-throughput, sensitive, and reproducible detection technology for a wide range of biomedical applications requiring detection of proteins (in cells and biofluids) and nucleic acids. Thus, novel methods of flow cytometry are becoming important for diagnostic purposes (e.g. identifying multiple clinical biomarkers for a wide range of diseases) as well as for developing novel therapies (e.g. elucidating drug mechanisms and potential toxicities). In addition, flow cytometry for multiplex analysis, coupled with automated sample handling devices, has the potential to significantly enhance proteomics research, particularly analysis of post-translational modifications of proteins, on a large scale. Inherently, flow cytometry methods are strongly rooted in the laws of the physics of optics, fluidics, and electromagnetism. This review article describes principles and early sources of flow cytometry, provides an introduction to the multiplex microbead technology, and discusses its applications and advantages in comparison to other methods. Anticipated future directions, particularly for translational research in medicine, are also discussed.