Evolution Toward Chip-Based Arrays in the Laboratory Diagnosis of Human Allergic Disease.

Multiplex-based specific IgE antibody assays have emerged into the clinical immunology laboratory through the combined use of pure, recombinant allergenic molecules and new methods to simultaneously and accurately analyze specific IgE antibodies to hundreds of allergens. This review traces the historical development and examines outstanding questions related to the strengths and limitations of these new molecular allergen multipex technologies for the assessment of human allergic sensitization. Multiplexed technologies are poised to provide the most cost-effective and comprehensive evaluation of patients with suspected allergy as compared with the commonly used singleplex autoanalyzers. How analytically sensitive and quantitative are the multiplex technologies, down to 0.1 kUA/L? Because each allergen is viewed as a unique assay, how will analytical and clinical performance be documented at the manufacturing and clinical laboratory levels to guarantee reproducibility and obtain government regulatory clearance? Will interference by naturally occurring allergen-specific IgG compromise analytical performance? Successful resolution of these and other questions covered in this review will position multiplex technologies to become the single most-effective means of screening patients for allergic sensitization, assessing IgE antibody cross-reactivity, and planning therapy directed at the patient with allergy.

Clinical Effectiveness of Liraglutide vs Sitagliptin on Glycemic Control and Body Weight in Patients with Type 2 Diabetes: A Retrospective Assessment in Sweden.

INTRODUCTION: The aim of the present study was to use real-world data from Swedish primary-care and national registries to understand clinical outcomes in patients with Type 2 diabetes (T2D) treated with liraglutide in clinical practice, and to compare with data from those treated with sitagliptin. METHODS: This was a non-interventional, retrospective study conducted between February 2014 and September 2014 using T2D patient data from Swedish primary-care centers and national healthcare registries. The primary objective was to assess the effectiveness of liraglutide in control of glycemia and body weight in clinical practice (stage 1). The secondary objective was to compare the clinical effectiveness of liraglutide with sitagliptin on glycemic control and body weight in clinical practice in a propensity-score-matched population (stage 2). RESULTS: In stage 1 (n = 402), 39.4% of patients treated with liraglutide achieved ≥1.0% (10.9 mmol/mol) reduction in glycated hemoglobin (HbA1c) after 180 days of treatment and 54.9% achieved the target HbA1c of <7.0% (53.0 mmol/mol). Moreover, compared with baseline, 22.5% of patients treated with liraglutide achieved both ≥1.0% reduction in HbA1c and ≥3.0% reduction in body weight. In stage 2, a significantly greater proportion of patients receiving liraglutide (n = 180) than sitagliptin (n = 208) achieved ≥1.0% reduction in HbA1c [52.9% vs 33.5%, respectively (P = 0.0002)]. Mean body-weight loss was also significantly greater in patients receiving liraglutide vs sitagliptin [-3.5 vs -1.3 kg, respectively (P < 0.0001)]. CONCLUSION: This study provides real-world evidence from Sweden corroborating previous clinical trials that demonstrate greater efficacy of liraglutide over sitagliptin on glycemic control and body-weight reduction in patients with T2D. FUNDING: Novo Nordisk A/S. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02077946.

Validation of a multiplex chip-based assay for the detection of autoantibodies against citrullinated peptides.

INTRODUCTION: Autoantibodies directed against citrullinated proteins/peptides (ACPAs) are highly specific and predictive for the development of rheumatoid arthritis (RA). Different subgroups of RA patients, which have different prognoses and may require different treatments, are characterized by different autoantibody profiles. The objective of this study was to develop a microarray for the detection of multiple RA-associated autoantibodies, initially focusing on responses against citrullinated epitopes on candidate autoantigens in RA. METHODS: The microarray is based on Phadia's ImmunoCAP ISAC system, with which reactivity to more than 100 antigens can be analyzed simultaneously, by using minute serum volumes (< 10 µl). Twelve citrullinated peptides, and the corresponding native arginine-containing control peptides, were immobilized in an arrayed fashion onto a chemically modified glass slide, allowing a three-dimensional layer with high binding capacity. The assay was optimized concerning serum dilution and glass surface, whereas each individual antigen was optimized concerning coupling chemistry, antigen concentration, and selection of spotting buffer. The performance of each peptide in the ImmunoCAP ISAC system was compared with the performance in enzyme-linked immunosorbent assays (ELISAs). Serum from 927 RA patients and 461 healthy controls from a matched case-control study were applied onto reaction sites on glass slides, followed by fluorescent-labeled anti-human immunoglobulin G (IgG) antibody. Fluorescence intensities were detected with a laser scanner, and the results analyzed by using image-analysis software. RESULTS: Strong correlations between the ImmunoCAP ISAC system and ELISA results were found for individual citrullinated peptides (Spearman ρ typically between 0.75 and 0.90). Reactivity of RA sera with the peptides was seen mainly in the anticyclic citrullinated peptide 2 (CCP2)-positive subset, but some additional reactivity with single citrullinated peptides was seen in the anti-CCP2-negative subset. Adjusting for reactivity against arginine-containing control peptides did not uniformly change the diagnostic performance for antibodies against the individual citrullinated peptides. CONCLUSIONS: The multiplexed array, for detection of autoantibodies against multiple citrullinated epitopes on candidate RA autoantigens, will be of benefit in studies of RA pathogenesis, diagnosis, and potentially as a guide to individualized treatment.

Population-based study of multiplexed IgE sensitization in relation to asthma, exhaled nitric oxide, and bronchial responsiveness.

BACKGROUND: IgE sensitization is an important risk factor for the development of asthma. OBJECTIVE: The aim of this study was to investigate the IgE antibody profile for a broad spectrum of allergen molecules in asthmatic patients. METHODS: Participants from the European Community Respiratory Health Survey II (n=467) were tested with ImmunoCAP ISAC against 103 allergen molecules. The presence of bronchial hyperresponsiveness was measured with a methacholine challenge test and bronchial inflammation with fraction of exhaled nitric oxide (Feno). RESULTS: A total of 38% of the controls and 72% of the asthmatic patients were sensitized against at least 1 of the allergen components (P<.0001). Asthma was independently related to having IgE antibodies against pollen (odds ratio=2.2) and perennial airway allergens (odds ratio=5.6), increased Feno was independently related to having IgE antibodies against food allergens and perennial allergens, while bronchial responsiveness was independently associated with having IgE antibodies against only perennial allergens. Sensitization to food allergens was related to asthma and increased Feno if IgE antibody against pollen allergens was present. Simultaneous sensitization to perennial, pollen, and food allergens involves the highest risk of asthma (odds ratio=18.3), bronchial inflammation, and responsiveness. CONCLUSIONS: Feno, bronchial responsiveness, and the risk of asthma increase with multiple sensitizations to different allergen groups. We show for the first time that the presence of IgE antibodies against food allergens is independently associated with increased Feno and increases the risk of asthma in subjects with simultaneous sensitization to pollen allergens.

Multivariate statistical analysis of large-scale IgE antibody measurements reveals allergen extract relationships in sensitized individuals.

BACKGROUND: Many allergenic sources are reportedly cross-reactive because of protein structural similarities. Although several aggregations are well characterized, no holistic mapping of IgE reactivity has hitherto been reported. OBJECTIVE: The aim of this study was to disclose relevant associations within a large set of allergen preparations, as revealed by specific IgE antibody levels in blood sera of multireactive human donors. METHODS: A dataset of recorded IgE antibody serum concentrations of 1011 nonidentifiable multireactive individuals (devoid of clinical records) to 89 allergen extracts was compiled for in silico analysis. Various algorithms were used to identify specific multivariate dependencies between the IgE antibody levels. RESULTS: Exhaustive cluster analysis demonstrates that IgE antibody responses to the 89 extracts can be aggregated into 12 stable formations. These clusters hold both well-known relationships, unexpected patterns, and unknown patterns, the latter categories being exemplified by the coclustering of wasp and certain seafood and a clear differentiation among pollen allergens. CONCLUSION: Identified relationships within several well-known groups of cross-reactive allergen extracts confirm the applicability of dedicated multivariate data analysis within the allergology field. Moreover, some of the unexpected IgE reactivity associations in sensitized human subjects might help in identifying new relationships with potential importance to allergy. CLINICAL IMPLICATIONS: Although clinical implications from this study should be validated in subsequent investigations with documentation on symptoms included, we believe this seminal approach is a key step toward the development of new analysis tools for interpretation of allergy data generated by using high-throughput recording systems.