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Background: Dermatophagoides pteronyssinus is one of the most important perennial allergen sources worldwide. Molecular diagnostics using the commercially available major allergens (Der p 1 and Der p 2) in combination with Der p 10 do not detect house dust mite (HDM) sensitization in a number of cases when used alone. The objective was to evaluate the IgE reactivity profiles of these patients using an experimental immunoassay biochip. Methods: Sera of HDM-allergic patients (positive skin prick test, CAP class = 1 for allergen extract, and positive intranasal provocation) were tested for IgE antibodies against Der p 1, Der p 2, and Der p 10 by ImmunoCAP fluorescence enzyme immunoassay. Negatively tested sera were examined by an experimental chip containing 13 microarrayed HDM allergens. Results: Of 97 patients tested, 16 showed negative results to Der p 1, Der p 2, and Der p 10. MeDALL chip evaluation revealed 5 patients mono-sensitized to Der p 23, and 11 patients were negative for all HDM MeDALL chip components. Seven sera were available for further testing, and 3 of them showed IgE reactivity to dot-blotted nDer p 1, and 2 reacted with high-molecular weight components (>100 kDa) in nitrocellulose-blotted HDM extract when tested with 125 I-labeled anti-IgE in a RASTbased assay. The HDM extract-specific IgE levels of the 11 patients were <3.9 kU/l. Conclusions: Recombinant allergen- based IgE serology is of great value when conventional IgE diagnostics fails. Der p 23 is an important HDM allergen, especially when major allergens are negative. Therefore, it would be desirable to have Der p 23 commercially available. Further research concerning the prevalence and clinical significance of different HDM allergens is needed. [ABSTRACT FROM AUTHOR]

Background: Molecular antibody reactivity profiles have not yet been studied in depth in patients treated by sublingual house dust mite (HDM) tablet immunotherapy. Humoral immune responses to a large panel of HDM mite allergens were studied using allergen microarray technology in a subset of clinically defined high and low responder patients from a double‐blind placebo‐controlled allergen‐specific immunotherapy (AIT) trial using sublingual 300 IR HDM tablets. Methods: Serum levels of IgE, IgG and IgG4 to 13 Dermatophagoides pteronyssinus molecules were measured at baseline and after 1‐year AIT, using allergen microarrays in 100 subjects exhibiting high or low clinical benefit. Results: Der p 1, Der p 2 and Der p 23 were the most frequently recognized allergens in the study population. Patients with HDM‐related asthma had significantly higher allergen‐specific IgE levels to Der p 1 and Der p 23. No significant difference in the distribution of allergen sensitization pattern was observed between high and low responders. An increase in serum allergen‐specific IgG and IgG4 occurred upon AIT, in particular to allergens Der p 1, Der p 2 and Der p 23 (p < 0.0001). Conclusions: We confirm for our study population that Der p 1‐ and Der p 23‐specific IgE levels are associated with asthma. IgE reactivity profiles were not predicitive of sublingual AIT outcomes, with 300 IR tablets as efficacious in pauci‐ and multi‐sensitized subjects. Our study is the first to demonstrate the induction of IgG and IgG4 specific for the HDM allergens Der p 1, Der p 2 and Der p 23 by sublingual AIT. [ABSTRACT FROM AUTHOR]

Rhinoviruses (RV) account for a significant number of asthma exacerbations, and RV species C may be associated with a severe course in vulnerable patient groups. Despite important evidence on the role of RV reported by clinicians and life scientists, there are still unanswered questions regarding their influence on asthma exacerbation in young patients. Thus, we measured the RVspecies-specific IgG titers in our German pediatric exacerbation cohort using a microarray-based technology. For this approach, human sera of patients with exacerbated asthma and wheeze, as well as healthy control subjects (n = 136) were included, and correlation analyses were performed. Concordantly with previously published results, we observed significantly higher cumulative levels of RV species A-specific IgG (p = 0.011) and RV-C-specific IgG (p = 0.051) in exacerbated asthma group compared to age-matched controls. Moreover, atopic wheezers had increased RV-specific IgG levels for species A (p = 0.0011) and species C (p = 0.0009) compared to non-atopic wheezers. Hypothesizing that bacterial infection positively correlates with immune memory against RV, we included nasopharyngeal swab results in our analyses and detected limited correlations. Interestingly, the eosinophil blood titer positively correlated with RV-specific IgG levels. With these observations, we add important observations to the existing data regarding exacerbation in pediatric and adolescent medicine. We propose that scientists and clinicians should pay more attention to the relevance of RV species in susceptible pediatric patients. [ABSTRACT FROM AUTHOR]

Background: While children usually experience a mild course of COVID‐19, and a severe disease is more common in adults, the features, specificities, and functionality of the SARS‐CoV‐2‐specific antibody response in the pediatric population are of interest. Methods: We performed a detailed analysis of IgG antibodies specific for SARS‐CoV‐2‐derived antigens S and RBD by ELISA in 26 SARS‐CoV‐2 seropositive schoolchildren with mild or asymptomatic disease course, and in an equally sized, age‐ and gender‐matched control group. Furthermore, a detailed mapping of IgG reactivity to a panel of microarrayed SARS‐CoV‐2 proteins and S‐derived peptides was performed by microarray technology. The capacity of the antibody response to block RBD‐ACE2 binding and virus neutralization were assessed. Results were compared with those obtained in an adult COVID‐19 convalescent population. Results: After mild COVID‐19, anti‐S and RBD‐specific IgG antibodies were developed by 100% and 84.6% of pediatric subjects, respectively. No difference was observed in regards to symptoms and gender. Mounted antibodies recognized conformational epitopes of the spike protein and were capable to neutralize the virus up to a titer of ≥80 and to inhibit the ACE2‐RBD interaction by up to 65%. SARS‐CoV‐2‐specific IgG responses in children were comparable to mildly affected adult patients. Conclusion: SARS‐CoV‐2 asymptomatic and mildly affected pediatric patients develop a SARS‐CoV‐2‐specific antibody response, which is comparable regarding antigen, epitope recognition, and the ability to inhibit the RBD‐ACE2 interaction to that observed in adult patients after mild COVID‐19. [ABSTRACT FROM AUTHOR]

Background: The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important. Methods: In this study, we analyzed the SARS‐CoV‐2 polyclonal antibody response in a large population of clinically well‐characterized patients after mild and severe COVID‐19 using a panel of microarrayed structurally folded and unfolded SARS‐CoV‐2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor‐binding domain (RBD) of the virus. Results: S‐ and RBD‐specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1, and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin‐converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD‐specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus‐neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants. Conclusion: These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS‐CoV‐2–neutralizing antibodies conferring sterilizing immunity. [ABSTRACT FROM AUTHOR]

Background: Sensitization to house dust mite (HDM) is a leading cause of allergic rhinitis and asthma. Despite more than 30 HDM‐derived allergens having been identified to date, specific therapeutic approaches do not yet take into account the local sensitization profiles of patients. This study aimed to identify patterns of HDM sensitization in HDM‐allergic adults living in distinct geographic areas, to inform the development of targeted diagnostic and therapeutic tools. Methods: Serum samples from 685 HDM‐allergic subjects from Canada, Europe, South Africa, and the USA were tested for levels of IgE specific for 17 micro‐arrayed HDM allergens by ImmunoCAP Immuno Solid‐phase Allergen Chip (ISAC) technology. Results: The results confirmed significant geographical variability in sensitization patterns and levels of IgE. In all areas, the major sensitizers were the group 1 and group 2 allergens and Der p 23. Der p 23 was a frequent sensitizer: 64% of the subjects had IgE specific for Der p 23, and 2.3% were monosensitized to it. In South Africa, Der p 23 was the dominant HDM allergen (86% prevalence) and Der p 7 achieved major allergen status (56%). IgE sensitization to HDM was influenced by asthmatic status, levels of allergen exposure, age, race‐ethnicity and smoking status, but not by BMI. Conclusion: Sensitization profiles to HDM allergens differ considerably among distinct geographic areas, with Der p 7 and Der p 23 being major sensitizers in South Africa. Such heterogeneity should be taken into account in the diagnosis and treatment of HDM‐allergic patients. [ABSTRACT FROM AUTHOR]

SANSSERIFWe used the microarray technology to develop chips containing a comprehensive set of p/SANSSERIFroteins and peptides covering the proteome of HIV-1 clade C, which is the HIV-1 subtype that causes the majority of infections worldwide. We demonstrate that the HIV microarray allows simultaneous, sensitive and specific detection of antibody responses for the major immunoglobulin classes (IgG, IgA, IgM, IgE) and subclasses (IgG1–4) with minute amounts of serum samples towards a large number of HIV antigens and peptides. Furthermore, we show that the HIV chip can be used for the monitoring of antibody responses during the course of the disease and during treatment. The HIV microarray should be useful to study antibody responses to multiple HIV antigens and epitopes in HIV-infected patients to explore pathomechanisms of the disease, for diagnosis and for monitoring of treatment and of vaccine trials. [ABSTRACT FROM AUTHOR]

Abstract: Two monoclonal antibody-mediated immunomagnetic separation PCR kits (AnDiaTec ParaTub-S® IMS-PCR-ELISA and ParaTub-SL® IMS-real time PCR) were developed and evaluated for the automated high-throughput detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in bulk milk of naturally infected dairy herds and made commercially available. M. paratuberculosis are first isolated from milk by high-throughput immunomagnetic bead separation using a completely automated magnetic particle pipetting robot within 45 min and released subsequently for analysis directly into PCR amplification mixtures for real time PCR or for PCR-ELISA. The threshold detection level and specificity of the tests were evaluated first with different M. paratuberculosis pure cultures and artificially contaminated (spiked) bulk milk samples. Both experiments proved a good detection limit, specificity and reliability of the tests that consistently detected 20 or less M. paratuberculosis organisms from cattle, deer and mufflon in 1 ml milk. Experiments with more than 200 bulk milk samples that were tested in parallel with the PCR methods and with the cultural method in a second evaluation study demonstrated that both PCR tests are superior to culture and sufficiently sensitive to detect single shedders in pooled milk samples. The experiments proved that the newly developed tests are sensitive, specific and fast, and thus for the first time allow the standardized large-scale routine M. paratuberculosis screening of bulk milk samples at acceptable costs. [Copyright &y& Elsevier]

Correction for ‘HIV microarray for the mapping and characterization of HIV-specific antibody responses’ by Daniela Gallerano et al., Lab Chip, 2015, 15, 1574–1589. [ABSTRACT FROM AUTHOR]