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.

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.

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.

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.

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.

Recent trends in electrochemical biosensors of superoxide dismutases.

Superoxide dismutases (SODs), a family of ubiquitous enzymes, provide essential protection to biological systems against uncontrolled reactions with oxygen- and nitrogen- based radical species. We review first the role of SODs in oxidative stress and the other biological functions such as peroxidase, nitrite oxidase, thiol oxidase activities etc., implicating its role in neurodegenerative, cardiovascular diseases, and ageing. Also, this review focuses on the development of electrochemical label-free immunosensor for SOD1 and the recent advances in biosensing assay methods based on their catalytic and biological functions with various substrates including reactive oxygen species (superoxide anion radical, hydrogen peroxide), nitric oxide metabolites (nitrite, nitrate) and thiols using thiol oxidase activity. Furthermore, we emphasize the progress made in improving the detection performance through incorporation of the SOD into conducting polymers and nanocomposite matrices. In addition, we address the potential opportunities, challenges, advances in electrochemical-sensing platforms and development of portable analyzer for point-of-care applications.

Trends in testing algorithms used to diagnose HIV infection, 2011-2015, United States and 6 dependent areas.

BACKGROUND: In 2014 the Centers for Disease Control and Prevention (CDC) and the Association of Public Health Laboratories (APHL) issued updated laboratory testing recommendations for the diagnosis of HIV infection. OBJECTIVES: To examine trends in the use of HIV diagnostic testing algorithms, and determine whether the use of different algorithms is associated with selected patient characteristics and linkage to HIV medical care. STUDY DESIGN: Analysis of HIV infection diagnoses during 2011-2015 reported to the National HIV Surveillance System through December 2016. Algorithm classification: traditional = initial HIV antibody immunoassay followed by a Western blot or immunofluorescence antibody test; recommended = initial HIV antibody IA followed by HIV-1/2 type-differentiating antibody test; rapid = two CLIA-waived rapid tests on same date. RESULTS: During 2011-2015, the percentage of HIV diagnoses made using the traditional algorithm decreased from 84% to 16%, the percentage using the recommended algorithm increased from 0.1% to 64%, and the percentage using the rapid testing algorithm increased from 0.1% to 2%. The percentage of persons linked to care within 30 days after HIV diagnosis in 2015 was higher for diagnoses using the recommended algorithm (59%) than for diagnoses using the traditional algorithm (55%) (p < 0.05). CONCLUSIONS: During 2011-2015, the percentage of HIV diagnoses reported using the recommended and rapid testing algorithms increased while the use of the traditional algorithm decreased. In 2015, persons with HIV diagnosed using the recommended algorithm were more promptly linked to care than those with diagnosis using the traditional algorithm.

Electrochemical immunosensors - A powerful tool for analytical applications.

Immunosensors are biosensors based on interactions between an antibody and antigen on a transducer surface. Either antibody or antigen can be the species immobilized on the transducer to detect antigen or antibody, respectively. Because of the strong binding forces between these biomolecules, immunosensors present high selectivity and very high sensitivity, making them very attractive for many applications in different science fields. Electrochemical immunosensors explore measurements of an electrical signal produced on an electrochemical transductor. This signal can be voltammetric, potentiometric, conductometric or impedimetric. Immunosensors utilizing electrochemical detection have been explored in several analyses since they are specific, simple, portable, and generally disposable and can carry out in situ or automated detection. This review addresses the potential of immunosensors destined for application in food and environmental analysis, and cancer biomarker diagnosis. Emphasis is given to the approaches that have been used for construction of electrochemical immunosensors. Additionally, the fundamentals of immunosensors, technology of transducers and nanomaterials and a general overview of the possible applications of electrochemical immunosensors to the food, environmental and diseases analysis fields are described.

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.

Tests used in the diagnostic evaluation of infertility: from ubiquitous to obsolete.

The only true wisdom is in knowing you know nothing. -Socrates.

Emerging Human Fetuin A Assays for Biomedical Diagnostics.

Human fetuin A (HFA) plays a prominent pathophysiological role in numerous diseases and pathophysiological conditions with considerable biomedical significance; one example is the formation of calciprotein particles in osteoporosis and impaired calcium metabolisms. With impressive advances in in vitro diagnostic assays during the last decade, ELISAs have become a workhorse in routine clinical diagnostics. Recent diagnostic formats involve high-sensitivity immunoassay procedures, surface plasmon resonance, rapid immunoassay chemistries, signal enhancement, and smartphone detection. The current trend is toward fully integrated lab-on-chip platforms with smartphone readouts, enabling health-care practitioners and even patients to monitor pathological changes in biomarker levels. This review provides a critical analysis of advances made in HFA assays along with the challenges and future prospects.

[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.

Development of nanostructures in the diagnosis of drug hypersensitivity reactions.

PURPOSE OF REVIEW: This article provides an overview of novel nanoscale structures potentially applicable to the field of allergy, and to discuss the required properties, advantages, and disadvantages of those nanostructures for clinical application focusing on diagnosis of drug hypersensitivity reactions. RECENT FINDINGS: Advances in the development of different nanostructures are favoring their biomedical applications. One area of interest is the interaction between nanostructures and the immune system, including their ability to emulate carrier molecules and their potential use for the diagnosis of allergic reactions. SUMMARY: Immunoassays are the most widely used in-vitro test for evaluating immunoglobulin E (IgE)-mediated drug hypersensitivity reactions. However, they have important technical limitations affecting their sensitivity. A wide variety of nanostructures have been designed to quantify specific IgE, with the aim of diagnosing different kinds of allergies. Nanoparticles-based colloidal immunoassay employed in microdevices and/or miniaturized systems are improving IgE detection sensitivity. Dendrimers have shown immense potential for the design and development of sensor platforms for evaluating IgE-mediated drug hypersensitivity reactions, due to the increase in hapten density and IgE accessibility. In this sense, a variety of dendritic structures as well as their hybridization to different solid supports have been shown to be successful when applied in the diagnosis of drug allergy. Moreover, the knowledge of the complete antigenic determinants would allow their inclusion and therefore further improvement of the sensitivity.

Human Immunodeficiency Virus Diagnostic Testing: 30 Years of Evolution.

A concern during the early AIDS epidemic was the lack of a test to identify individuals who carried the virus. The first HIV antibody test, developed in 1985, was designed to screen blood products, not to diagnose AIDS. The first-generation assays detected IgG antibody and became positive 6 to 12 weeks postinfection. False-positive results occurred; thus, a two-test algorithm was developed using a Western blot or immunofluorescence test as a confirmatory procedure. The second-generation HIV test added recombinant antigens, and the third-generation HIV tests included IgM detection, reducing the test-negative window to approximately 3 weeks postinfection. Fourth- and fifth-generation HIV assays added p24 antigen detection to the screening assay, reducing the test-negative window to 11 to 14 days. A new algorithm addressed the fourth-generation assay's ability to detect both antibody and antigen and yet not differentiate between them. The fifth-generation HIV assay provides separate antigen and antibody results and will require yet another algorithm. HIV infection may now be detected approximately 2 weeks postexposure, with a reduced number of false-positive results.

Mass spectrometry and immunoassay: how to measure steroid hormones today and tomorrow.

The recent onslaught of mass spectrometry (MS) to measurements of steroid hormones, including demands that they should be the only acceptable method, has confused clinicians and scientists who have relied for more than 40 years on a variety of immunoassay (IA) methods in steroid hormone measurements. There is little doubt that MS methods with their superior specificity will be the future method of choice in many clinical and research applications of steroid hormone measurement. However, the majority of steroid measurements are currently, and will continue to be, carried out using various types of IAs for several reasons, including their technical ease, cost and availability of commercial reagents. Speedy replacement of all IAs with MS is an unrealistic and unnecessary goal, because the availability of MS measurements is limited by cost, need of expensive equipment, technical demands and lack of commercial applications. Furthermore, IAs have multiple well-known advantages that vindicate their continuing use. The purpose of this article is to elucidate the advantages and limitations of the MS and IA techniques from two angles, i.e. promotion of MS and defence of IA. The purpose of the text is to give the reader an unbiased view about the current state and future trends of steroid analysis and to help him/her choose the correct assay method to serve his/her diagnostic and research needs.

Next generation ligand binding assays-review of emerging technologies' capabilities to enhance throughput and multiplexing.

The purpose of this manuscript is to provide a summary of the evaluation done by the Throughput and Multiplexing subteam on five emerging technologies: Single molecule array (Simoa™), Optimiser™, CyTOF® (Mass cytometry), SQIDLite™, and iLite™. Most of the information is presented with a minimum amount of published data and much is based on discussions with users and the vendor, to help provide the reader with an unbiased assessment of where the subteam sees each technology fitting best in the bioanalysis of large molecules. The evaluation focuses on technologies with advantages in throughput and multiplexing, but is wide enough to capture their strengths in other areas. While all platforms may be suited to support bioanalysis in the discovery space, because of their emergent nature, only Optimiser and SQIDLite are currently ready to be used in the regulated space. With the exception of Optimiser, each instrument/technology requires an up-front investment from the bioanalytical lab that will need justification during capital budget discussions. Ultimately, the platform choice should be driven by the quality of data, project needs, and the intended use of the data generated. In a time- and resource-constrained environment, it is not possible to evaluate all emergent technologies available in the market; we hope that this review gives the reader some of the information needed to decide which technology he/she may want to consider evaluating to support their drug development program in comparison to the options they already have in their hands.

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.