Accessing Antibody Reactivities in Serum or Plasma to (Auto-)antigens Using Multiplexed Bead-Based Protein Immunoassays.

Antibody (AB) testing or serotesting for reactive ABs against antigenic proteins is broadly used. Parallel examination of many antigens is of high interest to identify autoantibodies (AAB) or differential antigenic reactivities in many biological settings like allergy and infectious autoimmune, cancerous, or systemic disease. The resulting AAB profiles can be used for diagnosis, prognosis, and monitoring of such conditions. Protein microarrays have been used for AB profiling over the past decade but show some significant limitations which make them unsuitable for clinical applications. Alternative multiplexing platforms such as bead arrays were shown to provide a versatile tool for the confirmation and efficient analysis of high numbers of biological samples. Luminex' bead-based xMAP technology combines advantages such as multiplexing and lower demand for sample volume and at the same time overcomes the challenges of microarrays. It works faster, shows better antigen stability, is more reproducible, and allows the analysis of up to 500 analytes in one sample well. In this chapter we introduce our established workflow for the use of the xMAP technology for AB profiling including an overview of the method principle and protocols for the covalent immobilization of proteins to the MagPlex beads, confirmation of protein coupling, the execution of a multiplexed bead-based protein immunoassay, and subsequent data handling.

Multiplexed Bead-Based Peptide Immunoassays for the Detection of Antibody Reactivities.

Antigenic peptides are commonly used in serological test settings such as enzyme-linked immunosorbent assays (ELISA) to determine reactive antibodies (ABs) from serum or plasma samples. The use of synthetic peptides provides advantages like lower production effort and easier incorporation of specific chemical modifications compared to full-length antigenic proteins. Multiplexed antibody (AB) profiling methods such as microarray technologies enable the simultaneous identification of multiple novel biomarkers for the use in early disease diagnostics, vaccine development, or monitoring of immune responses. Despite various benefits they still show major limitations which can be overcome with bead-based assay technologies like the multi-analyte profiling (xMAP) technology developed by Luminex. In this chapter we introduce our established workflow for AB profiling with a multiplexed bead-based peptide immunoassay. The workflow is based on copper-catalyzed click chemistry to immobilize designed synthetic peptides onto uniquely color-coded paramagnetic beads in an orientation-specific manner. The individual peptide-coupled beads can be distinguished by their unique emission spectra during readout in the xMAP instrument and therefore allow testing of up to 500 different antigenic peptides in one multiplexed reaction. The multistep process described in this chapter is divided into separate sections for peptide design, coupling of functionalized peptides to MagPlex beads via click chemistry, confirmation of successful peptide immobilization, processing of serum or plasma samples, or preferably purified IgG thereof, with the multiplexed bead-based peptide immunoassay and subsequent data export and analysis.

Oxytocin receptor gene methylation as a molecular marker for severity of depressive symptoms in affective disorder patients.

BACKGROUND: Oxytocin (OXT) is a neuropeptide and hormone involved in emotional functioning and also seems to play a role in moderating the stress response. Both preclinical and clinical studies point to an increased methylation status of the Oxytocin receptor (OXTR) promoter region with concomitant deficits in social, cognitive and emotional functioning. We hypothesize that methylation levels (%) of the oxytocin receptor promoter region correlate with the severity of depression symptoms and/or with the severity of childhood trauma within this present sample of affective disorder patients. METHODOLOGY: Eight hundred forty six (846) affective disorder patients of Central European origin were recruited at the Department of Psychiatry and Psychotherapy of the Medical University Vienna, the Karl Landsteiner University for Health and Science and Zentren für seelische Gesundheit, BBRZ-Med Leopoldau. Psychiatric assessment included a semi-structured diagnostic interview (Schedules for Clinical Assessment in Neuropsychiatry), the Hamilton Depression Scale and the Childhood Trauma Questionnaire. Concomitantly DNA samples of peripheral blood cells were collected for Multiplexed and Sensitive DNA Methylation Testing. RESULTS: Our data suggests a positive but not significant association between OXTR promoter Exons 1-3 methylation levels and severity of depression symptoms as well as severity of emotional neglect in affective disorder patients and no association with childhood trauma. CONCLUSIONS: Our findings contribute to elucidate the role of OXTR in affective disorders, but further longitudinal studies in particular are necessary to broaden the current state of knowledge.

Functional Analysis of Autoantibody Signatures in Rheumatoid Arthritis.

For the identification of antigenic protein biomarkers for rheumatoid arthritis (RA), we conducted IgG profiling on high density protein microarrays. Plasma IgG of 96 human samples (healthy controls, osteoarthritis, seropositive and seronegative RA, n = 24 each) and time-series plasma of a pristane-induced arthritis (PIA) rat model (n = 24 total) were probed on AIT's 16k protein microarray. To investigate the analogy of underlying disease pathways, differential reactivity analysis was conducted. A total of n = 602 differentially reactive antigens (DIRAGs) at a significance cutoff of p < 0.05 were identified between seropositive and seronegative RA for the human samples. Correlation with the clinical disease activity index revealed an inverse correlation of antibodies against self-proteins found in pathways relevant for antigen presentation and immune regulation. The PIA model showed n = 1291 significant DIRAGs within acute disease. Significant DIRAGs for (I) seropositive, (II) seronegative and (III) PIA were subjected to the Reactome pathway browser which also revealed pathways relevant for antigen presentation and immune regulation; of these, seven overlapping pathways had high significance. We therefore conclude that the PIA model reflects the biological similarities of the disease pathogenesis. Our data show that protein array analysis can elucidate biological differences and pathways relevant in disease as well be a useful additional layer of omics information.

Impact of Specific N-Glycan Modifications on the Use of Plant-Produced SARS-CoV-2 Antigens in Serological Assays.

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein plays a key role in the virus-host cell interaction, and viral infection. The RBD is a major target for neutralizing antibodies, whilst recombinant RBD is commonly used as an antigen in serological assays. Such assays are essential tools to gain control over the pandemic and detect the extent and durability of an immune response in infected or vaccinated populations. Transient expression in plants can contribute to the fast production of viral antigens, which are required by industry in high amounts. Whilst plant-produced RBDs are glycosylated, N-glycan modifications in plants differ from humans. This can give rise to the formation of carbohydrate epitopes that can be recognized by anti-carbohydrate antibodies present in human sera. For the performance of serological tests using plant-produced recombinant viral antigens, such cross-reactive carbohydrate determinants (CCDs) could result in false positives. Here, we transiently expressed an RBD variant in wild-type and glycoengineered Nicotiana benthamiana leaves and characterized the impact of different plant-specific N-glycans on RBD reactivity in serological assays. While the overall performance of the different RBD glycoforms was comparable to each other and to a human cell line produced RBD, there was a higher tendency toward false positive results with sera containing allergy-related CCD-antibodies when an RBD carrying ß1,2-xylose and core α1,3-fucose was used. These rare events could be further minimized by pre-incubating sera from allergic individuals with a CCD-inhibitor. Thereby, false positive signals obtained from anti-CCD antibodies, could be reduced by 90%, on average.

Multiplexed DNA Methylation Analysis in Colorectal Cancer Using Liquid Biopsy and Its Diagnostic and Predictive Value.

Early diagnosis of colorectal cancer (CRC) is of high importance as prognosis depends on tumour stage at the time of diagnosis. Detection of tumour-specific DNA methylation marks in cfDNA has several advantages over other approaches and has great potential for solving diagnostic needs. We report here the identification of DNA methylation biomarkers for CRC and give insights in our methylation-sensitive restriction enzyme coupled qPCR (MSRE-qPCR) system. Targeted microarrays were used to investigate the DNA methylation status of 360 cancer-associated genes. Validation was done by qPCR-based approaches. A focus was on investigating marker performance in cfDNA from 88 patients (44 CRC, 44 controls). Finally, the workflow was scaled-up to perform 180plex analysis on 110 cfDNA samples, to identify a DNA methylation signature for advanced colonic adenomas (AA). A DNA methylation signature (n = 44) was deduced from microarray experiments and confirmed by quantitative methylation-specific PCR (qMSP) and by MSRE-qPCR, providing for six genes' single areas under the curve (AUC) values of >0.85 (WT1, PENK, SPARC, GDNF, TMEFF2, DCC). A subset of the signatures can be used for patient stratification and therapy monitoring for progressed CRC with liver metastasis using cfDNA. Furthermore, we identified a 35-plex classifier for the identification of AAs with an AUC of 0.80.

Monoamino Oxidase A Gene Single-Nucleotide Polymorphisms and Methylation Status and the Risk of Violent Suicide Attempts in Affective Disorder Patients.

Background: When investigating the neurobiology of suicidal behavior, Monoamino Oxidase A (MAOA) is one of the prime suspects to consider. Interestingly, MAOA dysregulation has also been associated with violent behavior in previous publications. In the present study, we aimed to establish an association between polymorphisms of the MAOA gene and methylation status of the MAOA gene Exon I, and suicide attempts with violent methods in a sample of affective disorder patients. Methods: Eight hundred fourteen Caucasian affective disorder patients were assessed at the Department of Psychiatry and Psychotherapy of the Medical University Vienna, the Karl Landsteiner University for Health and Science and Zentren für seelische Gesundheit, BBRZ-Med Leopoldau. An assemblage of psychiatric interviews was performed (e.g., SCAN, HAMD, SBQ-R, CTQ) and DNA samples of peripheral blood cells were collected for Sequenom MassARRAY® iPLEX Gold genotyping and Multiplexed and Sensitive DNA Methylation Testing. Results: Female affective disorder patients with a history of violent suicide attempt were found to have a significantly increased frequency of the AA genotype in the rs5906957 single nucleotide polymorphism (p = 0.003). Furthermore, the MAOA gene exon I promoter region showed significantly decreased methylation in female violent suicide attempter(s) as opposed to female affective disorder patients who had no history of suicide attempt or no history of suicide attempt with violent method. Limitations: The small sample size hampers to reveal small genetic effects as to be expected in psychiatric disorders. Conclusions: This study offers promising findings about associations between the MAOA gene and violent suicide especially in women.

A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests.

BACKGROUND: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. METHODS: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. FINDINGS: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. INTERPRETATION: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms. FUNDING: WWTF, Project No. COV20-016; BOKU, LBI/LBG.

Balanced Electrolyte Solutions or Normal Saline? Resuscitative Fluid Administration Practice in Swiss Pediatric Acute Care: A Cross-Sectional Study.

INTRODUCTION: The ideal asanguineous intravenous fluid for volume resuscitation in children is controversially debated and clinical practice guidelines are scarce. Administration of large amounts of normal saline has been associated with complications including hyperchloremic acidosis, dysnatremia, neurologic damage, and fatality. AIM: We examined the current practice of intravenous fluid and blood product administration in acutely ill and injured children among pediatric acute care physicians in Switzerland. METHODS: For this descriptive, cross-sectional study, pediatric emergency departments, pediatric and neonatal intensive care units were surveyed by means of an online questionnaire. RESULTS: Sixty of 66 departments and 47 of 87 participants returned the survey. Normal saline (NS) was most commonly administered (n = 42/46, 91.3%) and twice as many times as balanced electrolyte solutions (n = 20/46, 43.5%). The mean fluid volumes ranged from 7.9 to 19.1 mL/kg. Hypertonic saline/NS were selected most often for shock with severe head injury. Half of participants administered colloids (48.9%). Packed red blood cells (97.7%) and fresh frozen plasma (88.4%) were most frequently given blood products. CONCLUSION: There is a distinct practice variation in intravenous fluid and blood product administration in children in Switzerland. Although NS is most frequently given, we observed a trend toward the use of balanced electrolyte solutions. Prospective studies are warranted to compare NS with balanced electrolyte solution (BES) in the pediatric acute care setting. We suggest that pediatric fluid administration guidelines and mass transfusion protocols are implemented to standardize this frequent intervention and minimize complications.

In Planta Production of the Receptor-Binding Domain From SARS-CoV-2 With Human Blood Group A Glycan Structures.

Glycosylation of viral envelope proteins is important for infectivity and immune evasion. The SARS-CoV-2 spike protein is heavily glycosylated and host-derived glycan modifications contribute to the formation of specific immunogenic epitopes, enhance the virus-cell interaction or affect virus transmission. On recombinant viral antigens used as subunit vaccines or for serological assays, distinct glycan structures may enhance the immunogenicity and are recognized by naturally occurring antibodies in human sera. Here, we performed an in vivo glycoengineering approach to produce recombinant variants of the SARS-CoV-2 receptor-binding domain (RBD) with blood group antigens in Nicotiana benthamiana plants. SARS-CoV-2 RBD and human glycosyltransferases for the blood group ABH antigen formation were transiently co-expressed in N. benthamiana leaves. Recombinant RBD was purified and the formation of complex N-glycans carrying blood group A antigens was shown by immunoblotting and MS analysis. Binding to the cellular ACE2 receptor and the conformation-dependent CR3022 antibody showed that the RBD glycosylation variants carrying blood group antigens were functional. Analysis of sera from RBD-positive and RBD-negative individuals revealed further that non-infected RBD-negative blood group O individuals have antibodies that strongly bind to RBD modified with blood group A antigen structures. The binding of IgGs derived from sera of non-infected RBD-negative blood group O individuals to blood group A antigens on SARS-CoV-2 RBD suggests that these antibodies could provide some degree of protection from virus infection.

High similarity of IgG antibody profiles in blood and saliva opens opportunities for saliva based serology.

Saliva based diagnostics is a rapidly evolving field due to the large diagnostic potential and simple sample collection. Currently only few individual molecules were investigated for their diagnostic capabilities in saliva. A systematic comparison of IgG antibody profiles in saliva and plasma is still missing in scientific literature. Our hypothesis is that IgG profiles in plasma and saliva are highly similar for each individual. As a consequence, one could implement practically any plasma based IgG assay (classical serology) as saliva based assay. In other words, the IgG antibodies found in blood are also accessible from saliva. We confirm our hypothesis by comparing IgG reactivities towards protein and peptide antigens. We isolated saliva IgG with high purity and demonstrate that plasma IgG reactivities (classical serology) can be inferred from saliva. As a showcase we perform Hepatitis B virus antibody (plasma-)titer determination from saliva. Additionally we show that plasma and saliva IgG profiles of 20 individuals are highly similar for 256 peptide antigens and match (unsupervised) with high probabilities. Finally, we argue for generalisation to the complete IgG antibody profile. The presented findings could contribute greatly to the development of saliva based diagnostic methods of numerous antibody based tests.