IgG Reactivity Research Articles

Neurological disorders-associated anti-glycosphingolipid IgG-antibodies display differentially restricted IgG subclass distribution

Antibodies against several self-glycans on glycosphingolipids are frequently detected in different neurological disorders. Their pathogenic role is profusely documented, but the keys for their origin remain elusive. Additionally, antibodies recognizing non-self glycans appear in normal human serum during immune response to bacteria. Using HPTLC-immunostaining we aimed to characterize IgM and IgG subclass antibody responses against glycosphingolipids carrying self glycans (GM1/GM2/GM3/GD1a/GD1b/GD3/GT1b/GQ1b) and non-self glycans (Forssman/GA1/“A” blood group/Nt7) in sera from 27 randomly selected neurological disorder patients presenting IgG reactivity towards any of these antigens. Presence of IgG2 (p = 0.0001) and IgG1 (p = 0.0078) was more frequent for IgG antibodies against non-self glycans, along with less restricted antibody response (two or more simultaneous IgG subclasses). Contrariwise, IgG subclass distribution against self glycans showed clear dominance for IgG3 presence (p = 0.0017) and more restricted IgG-subclass distributions (i.e. a single IgG subclass, p = 0.0133). Interestingly, anti-self glycan IgG antibodies with simultaneous IgM presence had higher proportion of IgG2 (p = 0.0295). IgG subclass frequencies were skewed towards IgG1 (p = 0.0266) for “anti-self glycan A” subgroup (GM2/GM1/GD1b) and to IgG3 (p = 0.0007) for “anti-self glycan B” subgroup (GM3/GD1a/GD3/GT1b/GQ1b). Variations in players and/or antigenic presentation pathways supporting isotype (M-G) and IgG-subclass pattern differences in the humoral immune response against glycosphingolipids carrying non-self versus self-glycans are discussed.

Qualitative assessment of SARS-CoV-2-specific antibody avidity by lateral flow immunochromatographic IgG/IgM antibody assay.

Knowledge of the precise timing of SARS‐CoV‐2 infection may be of clinical and epidemiological relevance. The presence of low‐avidity IgGs has conventionally been considered an indicator of recent infection. Here, we carried out qualitative assessment of SARS‐CoV‐2‐specific antibody avidity using an urea (6M) dissociation test performed on a lateral flow immunochromatographic IgG/IgM device. We included a total of 76 serum specimens collected from 57 COVID‐19 patients, of which 39 tested positive for both IgG and IgM and 37 only for IgG. Sera losing IgG reactivity after urea treatment (n = 28) were drawn significantly earlier (P = .04) after onset of symptoms than those which preserved it (n = 48). This assay may be helpful to estimate the time of acquisition of infection in patients with mild to severe COVID‐19.

Increased Antibody Response to Fucosylated Oligosaccharides and Fucose-Carrying Bacteroides Species in Crohn's Disease.

Inflammatory bowel disease is associated with intestinal dysbiosis and with elevated antibody production toward microbial epitopes. The underlying processes linking the gut microbiota with inflammation are still unclear. Considering the constant induction of antibodies by gut microbial glycans, the aim of this study was to address whether the repertoire of carbohydrate-specific antibodies is altered in Crohn’s disease or ulcerative colitis. IgG and IgM reactivities to oligosaccharides representative of mucosal glycans were tested in blood serum from 20 healthy control subjects, 17 ulcerative colitis patients, and 23 Crohn’s disease patients using glycan arrays. An increased IgG and IgM reactivity toward fucosylated oligosaccharides was detected in Crohn’s disease but not in ulcerative colitis. To address the antibody reactivity to the gut microbiota, IgG binding to members of a complex intestinal microbiota was measured and observed to be increased in sera of patients with Crohn’s disease. Based on the elevated reactivity to fucosylated oligosaccharides, gut bacteria were tested for recognition by the fucose-binding Aleuria aurantia lectin. Bacteroides stercoris was detected in IgG- and lectin-positive fractions and reactivity of A. aurantia lectin was demonstrated for additional Bacteroides species. IgG reactivity to these Bacteroides species was significantly increased in inflammatory bowel disease patients, indicating that the increased reactivity to fucosylated oligosaccharides detected in Crohn’s disease may be induced by fucose-carrying intestinal bacteria. Enhanced antibody response to fucosylated epitopes may have systemic effects by altering the binding of circulating antibodies to endogenous glycoproteins.

Mimotope-based antigens as potential vaccine candidates in experimental murine cysticercosis.

Human cysticercosis is a public health problem caused by Taenia solium metacestodes; thus, eradication of T. solium transmission by vaccination is an urgent requirement. The Cc48 mimotope from T. solium cysticerci was tested expressed in phage particles (mCc48) and chemically synthesized (sCc48) as a vaccine candidate in experimental murine cysticercosis. For this, BALB/c mice were immunized with mCc48 (G1; n = 40), sCc48 (G2; n = 40) and phosphate-buffered saline (PBS) (G3; n = 40, positive control) and challenged with Taenia crassiceps metacestodes. Another PBS group without parasite challenge was used as a negative control (G4; n = 40). Mice were sacrificed 15, 30, 45 and 60 days post-infection for cysticerci and serum collection. Immunization efficacy was determined by cysticerci counting. Serum samples were tested by ELISA to verify antibody (IgM, IgG, IgA and IgE) and cytokine (IFNγ and IL-4) levels. The sCc48 achieved the highest rates of protection and efficacy (90 and 98%, respectively). The group immunized with mCc48 presented the highest reactivity for IgM, IgG and IgE. All groups presented IL-4, but IFNγ was quite variable among groups. The protection induced by sCc48 synthetic peptide supports further studies of this mimotope as a potential vaccine candidate against cysticercosis.

Complement activation by human IgG antibodies to galactose-α-1,3-galactose.

Some human antibodies may paradoxically inhibit complement activation on bacteria and enhance pathogen survival in humans. This property was also claimed for IgG antibodies reacting with terminal galactose-α-1,3-galactose (Galα3Gal; IgG anti-αGal), a naturally occurring and abundant antibody in human plasma that targets numerous different pathogens. To reinvestigate these effects, we used IgG anti-αGal affinity isolated from a pool of normal human IgG and human hypogammaglobulinaemia serum as a complement source. Flow cytometry was performed to examine antibody binding and complement deposition on pig erythrocytes, Escherichia coli O86 and Streptococcus pneumoniae serotype 9V. Specific nanobodies were used to block the effect of single complement factors and to delineate the complement pathways involved. IgG anti-αGal was capable of activating the classical complement pathway on all the tested target cells. The degree of activation was exponentially related to the density of bound antibody on E.coli O86 and pig erythrocytes, but more linearly on S.pneumoniae 9V. The alternative pathway of complement amplified complement deposition. Deposited C3 fragments covered the activating IgG anti-αGal, obstructing its detection and highlighting this as a likely general caveat in studies of antibody density and complement deposition. The inherent capacity for complement activation by the purified carbohydrate reactive IgG anti-αGal was similar to that of normal human IgG. We propose that the previously reported complement inhibition by IgG anti-αGal relates to suboptimal assay configurations, in contrast to the complement activating property of the antibodies demonstrated in this paper.

Antibody profiling of a Borreliella burgdorferi (Lyme disease) C6 antibody positive, symptomatic Rottweiler and her pups

Lyme disease (LD) is a tick-transmitted disease caused by Borreliella burgdorferi (Bb). Temporal studies of maternal antibody (Ab) profiles in Bb infected pregnant dogs and their pups have not been conducted. In this study, Ab profiles of a client-owned Bb C6 Ab positive Rottweiler and her nine pups were assessed. The dam presented with lameness 12 days prior to parturition and was C6 Ab positive with a Quant C6 Ab concentration of 237U/mL. Treatment with amoxicillin was initiated and 11 days later nine pups were delivered.Screening of the sera from the dam and pups against Bb cell lysates and a panel of antigens revealed similar immunoreactivity profiles. While antigen-specific IgG and IgM reactivity persisted in the dam for at least 7 months, a rapid decline in IgG specific for BBA36, BBK53, BB0238, BBA73 and outer surface protein (Osp) E in the pups occurred between days 29 and 52 post-parturition. In contrast, Ab specific for DbpA and the diagnostic antigens VlsE (C6) and OspF, remained elevated in the pups. Sera from the dam displayed potent complement-dependent bactericidal activity against Bb. Sera from the pups was also bactericidal but primarily through a complement-independent mechanism. Lastly, single dose vaccination of the dam at day 51 post-parturition with a LD subunit vaccine consisting of OspA and an OspC chimeritope triggered a broad anti-OspC Ab response indicative of an anamnestic response. Although this study focused on a single case, these findings add to our knowledge of maternal Ab profiles and will aid the interpretation of serological assays in pups delivered by a Bb C6 Ab positive dog.

Evaluation of ViroTrack Sero Zika IgG/IgM, a New Rapid and Quantitative Zika Serological Diagnostic Assay.

Dengue virus (DENV) and Zika virus (ZIKV) belong to the flavivirus genus and are antigenically closely related. They also share the same mosquito vector and can cause similar symptoms upon infection. However, DENV and ZIKV infections lead to different clinical sequelae and treatments; therefore, clinicians need rapid and accurate diagnostics capable of distinguishing between the two diseases. Methods: We employed the immuno-magnetic assay technology on a microfluidic cartridge (ViroTrack Sero Zika IgG/IgM) for diagnosis of ZIKV infection based on the aggregation of magnetic nanoparticles. We carried out three serological studies including samples from the Dominican Republic, USA, and Nicaragua, aimed at detecting ZIKV-specific IgG and IgM using the ViroTrack Sero Zika IgG/IgM test. Results: The seroconversion results were comparable with ZIKV IgG and IgM reactivity measured by the commercial ZIKV ELISA kit. The sensitivity and specificity for both ZIKV IgG and IgM tested by the ViroTrack Sero Zika IgG/IgM was approximately 98% and 93%, respectively. Conclusion: Serological detection of ZIKV infection by the new ViroTrack Sero Zika IgG/IgM test shows promising performance and limited cross-reactivity with DENV.

Toxoplasmosis congénita en gemelos

Congenital toxoplasmosis in twin pregnancies is infrequent. We present the case of a monozygotic and monocorial twin pregnancy with maternal toxoplasmosis infection diagnosed at 33 weeks gestation by detecting reactive IgM and IgG and low avidity test for IgG. The detection of the parasite in amniotic fluid could not be performed because the amniocentesis was not done. The term newborns presented cerebral calcifications and bilateral active chorioretinitis without other associated clinical manifestations. They had IgG and IgM reactive for toxoplasmosis. The detection of Toxoplasma gondii was not performed by placental PCR or cord blood. They received treatment during the first year of life with pyrimethamine, sulfadiazine and folinic acid. They had no adverse events associated with the therapy. The diagnostic assessment and treatment does not differ from pregnancies with a single fetus. One or both newborns may be compromised. Multidisciplinary monitoring is essential for the early detection of reactivations or progression of lesions.

Longitudinal Monitoring of SARS-CoV-2 IgM and IgG Seropositivity to Detect COVID-19.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a novel beta-coronavirus that has recently emerged as the cause of the 2019 coronavirus pandemic (COVID-19). Polymerase chain reaction (PCR) based tests are optimal and recommended for the diagnosis of an acute SARS-CoV-2 infection. Serology tests for viral antibodies provide an important tool to diagnose previous exposure to the virus. Here we evaluate the analytical performance parameters of the Diazyme SARS-CoV-2 IgM/IgG serology assays and describe the kinetics of IgM and IgG seroconversion observed in patients with PCR confirmed COVID-19 who were admitted to our hospital.MethodsWe validated the performance of the Diazyme assay in 235 subjects to determine specificity. Subsequently, we evaluated the SARS-CoV-2 IgM and IgG seroconversion of 54 PCR confirmed COVID-19 patients and determined sensitivity of the assay at three different timeframes.ResultSensitivity and specificity for detecting seropositivity at ≥ 15 days following a positive SARS-CoV-2 PCR result, was 100.0% and 98.7% when assaying for the panel of IgM and IgG. The median time to seropositivity observed for a reactive IgM and IgG result from the date of a positive PCR was 5 days (IQR: 2.75-9 days) and 4 days (IQR: 2.75-6.75 days), respectively.ConclusionsOur data demonstrates that the Diazyme IgM/IgG assays are suited for the purpose of detecting SARS-CoV-2 IgG and IgM in patients with suspected SARS-CoV-2 infections. For the first time, we report longitudinal data showing the evolution of seroconversion for both IgG and IgM in a cohort of acutely ill patients in the United States. We also demonstrate a low false positive rate in patients who were presumed to be disease free.

Serum and cervicovaginal IgG immune responses against α7 and α9 HPV in non-vaccinated women at risk for cervical cancer: Implication for catch-up prophylactic HPV vaccination.

BackgroundCervical cancer associated with high risk-human papillomavirus (HR-HPV) infection is becoming the one of the most common female cancer in many sub-Saharan African countries. First-generation immigrant African women living in Europe are at-risk for cervical cancer, in a context of social vulnerability, with frequent lack of cervical cancer screening and HPV vaccination.ObjectiveOur objective was to address immunologically the issue of catch-up prophylactic HPV vaccination in first-generation African immigrant women living in France.MethodsIgG immune responses and cross-reactivities to α7 (HPV-18, -45 and -68) and α9 (HPV-16, -31, -33, -35, -52 and -58) HPV types, including 7 HR-HPV targeted by the Gardasil-9® prophylactic vaccine, were evaluated in paired serum and cervicovaginal secretions (CVS) by HPV L1-virus-like particles-based ELISA. Genital HPV were detected by multiplex real time PCR (Seegene, Seoul, South Korea).ResultsFifty-one immigrant women (mean age, 41.7 years; 72.5% HIV-infected) were prospectively included. More than two-third (68.6%) of them carried genital HPV (group I) while 31.4% were negative (group II). The majority (90.2%) exhibited serum IgG to at least one α7/α9 HR-HPV. Serum HPV-specific IgG were more frequently detected in group I than group II (100% versus 68.7%; P = 0.002). The distribution of serum and genital HPV-specific IgG was similar, but mean number of IgG reactivities to α7/α9 HR-HPV was higher in serum than CVS (5.6 IgG per woman in serum versus 3.2 in CVS; P<0.001). Rates of IgG cross-reactivities against HPV different from detected cervicovaginal HPV were higher in serum and CVS in group I than group II. Finally, the majority of groups I and II women (68.6% and 68.7%, respectively) exhibited serum or cervicovaginal IgG to Gardasil-9® HR-HPV, with higher mean rates in group I than group II (6.1 Gardasil-9® HR-HPV per woman versus 1.4; P<0.01). One-third (31.2%) of group II women did not show any serum and genital HPV-specific IgG.ConclusionsAround two-third of first-generation African immigrant women living in France showed frequent ongoing genital HPV infection and high rates of circulating and genital IgG to α7/α9 HPV, generally cross-reacting, avoiding the possibility of catch-up vaccination. Nevertheless, about one-third of women had no evidence of previous HPV infection, or showed only low levels of genital and circulating HR-HPV-specific IgG and could therefore be eligible for catch-up vaccination.

Lyme Disease: Diversity of Borrelia Species in California and Mexico Detected Using a Novel Immunoblot Assay.

Background: With more than 300,000 new cases reported each year in the United States of America (USA), Lyme disease is a major public health concern. Borrelia burgdorferi sensu stricto (Bbss) is considered the primary agent of Lyme disease in North America. However, multiple genetically diverse Borrelia species encompassing the Borrelia burgdorferi sensu lato (Bbsl) complex and the Relapsing Fever Borrelia (RFB) group are capable of causing tickborne disease. We report preliminary results of a serological survey of previously undetected species of Bbsl and RFB in California and Mexico using a novel immunoblot technique. Methods: Serum samples were tested for seroreactivity to specific species of Bbsl and RFB using an immunoblot method based on recombinant Borrelia membrane proteins, as previously described. A sample was recorded as seropositive if it showed immunoglobulin M (IgM) and/or IgG reactivity with at least two proteins from a specific Borrelia species. Results: The patient cohort consisted of 90 patients residing in California or Mexico who met the clinical case definition of chronic Lyme disease. Immunoblot testing revealed that 42 patients were seropositive for Bbsl (Group 1), while 56 patients were seropositive for RFB (Group 2). Eight patients were seropositive for both Bbsl and RFB species. Group 1 included patients who were seropositive for Bbss (14), B. californiensis (eight), B. spielmanii (10), B. afzelii/B. garinii (10), and mixed infections that included B. mayonii (three). Group 2 included patients who were seropositive for B. hermsii (nine), B. miyamotoi (seven), B. turicatae (nine), and B. turcica (two). In the remaining Group 1 and Group 2 patients, the exact Borrelia species could not be identified using the immunoblot technique. Conclusions: Lyme disease is associated with a diverse group of Borrelia species in California and Mexico. Current testing for Lyme disease focuses on detection of Bbss, possibly resulting in missed diagnoses and failure to administer appropriate antibiotic therapy in a timely manner. The genetic diversity of Borrelia spirochetes must be considered in future Lyme disease test development.

Molecular profiling of allergen-specific antibody responses may enhance success of specific immunotherapy

House dust mites (HDMs) are among the most important allergen sources containing many different allergenic molecules. Analysis of patients from a double-blind, placebo-controlled allergen-specific immunotherapy (AIT) study indicated that patients may benefit from AIT to different extents depending on their molecular sensitization profiles. Our aim was to investigate in a real-life setting whether stratification of patients with HDM allergy according to molecular analysis may enhance AIT success. Serum and nasal secretion samples from patients with HDM allergy (n= 24) (at baseline, 7, 15, 33, and 52 weeks) who had received 1 year of treatment with a well-defined subcutaneous AIT form (Alutard SQ 510) were tested for IgE and IgG reactivity to 15 microarrayed HDM allergen molecules with ImmunoCAP Immuno-solid-phase Allergen Chip technology. IgG subclass levels to allergens and peptides were determined by ELISA, and IgG blocking was assessed by basophil activation. Invitro parameters were related to reduction of symptoms determined by combined symptom medication score and visual analog scale score. Alutard SQ 510 induced protective IgG mainly against Dermatophagoides pteronyssinus (Der p) 1 and Der p 2 and to a lesser extent to Der p 23, but not to the other important allergens such as Der p 5, Der p 7, and Der p 21, showing better clinical efficacy in patients sensitized only to Der p 1 and/or Der p 2 as compared with patients having additional IgE specificities. Stratification of patients with HDM allergy according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance the success of AIT.

Metabolic Dysfunction in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Not Due to Anti-mitochondrial Antibodies.

Metabolic profiling studies have recently indicated dysfunctional mitochondria in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). This includes an impaired function of pyruvate dehydrogenase complex (PDC), possibly driven by serum factor(s), which leads to inadequate adenosine triphosphate generation and excessive lactate accumulation. A reminiscent energy blockade is likely to occur in primary biliary cholangitis (PBC), caused by anti-PDC autoantibodies, as recently proposed. PBC is associated with fatigue and post-exertional malaise, also signifying ME/CFS. We herein have investigated whether ME/CFS patients have autoreactive antibodies that could interfere with mitochondrial function. We found that only 1 of 161 examined ME/CFS patients was positive for anti-PDC, while all PBC patients (15/15) presented significant IgM, IgG, and IgA anti-PDC reactivity, as previously shown. None of fibromyalgia patients (0/14), multiple sclerosis patients (0/29), and healthy blood donors (0/44) controls showed reactivities. Anti-mitochondrial autoantibodies (inner and outer membrane) were negative in ME/CFS cohort. Anti-cardiolipin antibody levels in patients did not differ significantly from healthy blood donors. In conclusion, the impaired mitochondrial/metabolic dysfunction, observed in ME/CFS, cannot be explained by presence of circulating autoantibodies against the tested mitochondrial epitopes.

Natural Antibodies and Left Ventricular Assist Device Complications

Left ventricular assist devices (LVAD) are widely used as a support strategy for advanced heart failure. Complications such as thrombosis and bleeding have been linked to LVAD. We observed that LVAD implantation was followed by a sharp increase in serum levels of IgG natural antibodies (Nabs) recognizing oxidation-specific epitopes (OSE) and apoptotic cells. Nabs have been implicated in inflammatory reactions related to atherosclerosis, ischemic stroke and primary graft dysfunction following heart transplantation. However, the consequence of IgG Nabs production following LVAD is not known. We hypothesized that Nabs generated following implant may be associated with LVAD complications, promoting thrombosis, stroke or bleeding. We studied a multicenter cohort (n=71) of adult patients who received a continuous flow LVAD at Columbia University Irving Medical Center (CUIMC), Virginia Commonwealth University (VCU) and Baylor University Medical Center (BUMC). Nabs were measured in pre- and longitudinal post-LVAD sera by assessing IgG reactivity to the OSE malondialdehyde (MDA) by ELISA and to apoptotic Jurkat cells by flow cytometry. Wilcoxon's signed rank test for matched pairs was used to compare measurements. Mean IgG Nabs levels were significantly increased in post-LVAD sera at 1 (0.41±0.05 Nabs units, p<0.0001), 6 (0.63±0.074, p<0.0001) and 12 months (0.49±0.69, p=0.0004) compared to pre-LVAD (0.19±0.021). Kinetics of Nabs development was found to differ between centers with 90.9% of CUIMC patients developing elevated Nabs at 6 months post-implantation compared to 30% and 29.7% for BUMC and VCU, respectively. Preliminary analysis revealed elevated Nabs in 2/3 VCU patients with pump thrombosis and 1/2 with stroke within the first year post-LVAD. Our studies revealed that serum IgG Nabs reactive to oxidized epitopes and apoptotic cells are elevated following LVAD implant. The appearance of Nabs differed between the centers. and we are investigating the possible contribution of differing perioperative procedures on Nabs development. Preliminary findings suggest that IgG Nabs may be associated with thromboembolism. Analysis of Nabs and adverse events in the other centers are forthcoming.

Reducing the Immunogenic Potential of Wheat Flour: Silencing of Alpha Gliadin Genes in a U.S. Wheat Cultivar.

The alpha gliadins are a group of more than 20 proteins with very similar sequences that comprise about 15%–20% of the total flour protein and contribute to the functional properties of wheat flour dough. Some alpha gliadins also contain immunodominant epitopes that trigger celiac disease, a chronic autoimmune disease that affects approximately 1% of the worldwide population. In an attempt to reduce the immunogenic potential of wheat flour from the U.S. spring wheat cultivar Butte 86, RNA interference was used to silence a subset of alpha gliadin genes encoding proteins containing celiac disease epitopes. Two of the resulting transgenic lines were analyzed in detail by quantitative two-dimensional gel electrophoresis combined with tandem mass spectrometry. Although the RNA interference construct was designed to target only some alpha gliadin genes, all alpha gliadins were effectively silenced in the transgenic plants. In addition, some off-target silencing of high molecular weight glutenin subunits was detected in both transgenic lines. Compensatory effects were not observed within other gluten protein classes. Reactivities of IgG and IgA antibodies from a cohort of patients with celiac disease toward proteins from the transgenic lines were reduced significantly relative to the nontransgenic line. Both mixing properties and SDS sedimentation volumes suggested a decrease in dough strength in the transgenic lines when compared to the control. The data suggest that it will be difficult to selectively silence specific genes within families as complex as the wheat alpha gliadins. Nonetheless, it may be possible to reduce the immunogenic potential of the flour and still retain many of the functional properties essential for the utilization of wheat.

Hepatitis E diagnostics: immunocompetent and IgM-negative implies PCR negative

Hepatitis E virus has, over the past few years, been increasingly recognised as a cause of acute hepatitis in the western world. The first hepatitis E Standard for Microbiology Investigations (SMI) was issued in November 2018 and recommends cascading testing in immunocompetent individuals, with RNA assays clarifying results of antibody assays. We investigated all clinical samples tested for hepatitis E in our regional laboratory between October 2014 and October 2018 (12671 IgM, 10738 IgG and 2290 RNA assays). In 495 cases, samples collected within 24 hours from a patient underwent IgM assays (DS2 platform) and PCR assays (in-house). 7 pairs, corresponding to 5 patients, of 404 pairs with negative or low-positive IgM results, had detectable RNA. 4 of those 5 patients were demonstrably immunocompromised; the final sample came from general practice without further details. 3/5 patients had reactive IgG assays, and one of the two remaining patients had a rise in IgM titre between two successive samples. We conclude that, in our sample population, hepatitis E RNA PCR testing adds minimal diagnostic information in immunocompetent individuals with negative or low-positive IgM results. This finding supports current testing cascade recommendations. We discuss our sample population’s demographics and external validity of findings: in particular, we saw few tests sent by neurologists. Finally, we briefly discuss other IgM/IgG/PCR result scenarios and our findings in relation to current SMI recommendations, and speculate how our findings affect understanding of antibody response and viraemia in hepatitis E infection.

DOP09 IgG responses to multiple bacterial flagellins identify a subgroup of paediatric therapy-naive Crohn’s disease patients with increased microbiota-specific T-cell reactivity

Abstract Background inflammatory bowel disease (IBD), comprising Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic intestinal disease driven by inflammatory T-cell responses thought to target intestinal bacteria. However, evidence for such microbiota-specific T-cell responses is scarce. Immunoglobulin (Ig) titers to bacterial flagellins are highly elevated in a subgroup of CD patients and associate with a more severe disease course, namely penetrating and stricturing disease. This prompted us to hypothesise that flagellin-specific humoral responses could help uncover the inflammatory T-cell responses underlying disease pathogenesis. Methods Plasma IgG and IgA reactivity for 12 flagellins from pathogenic and commensal human intestinal bacteria was assessed by antigen microarray in paediatric CD patients (n = 49), UC patients (n = 12), and age-matched paediatric healthy controls (n = 22) of the PIBD-SET Quality and TISKids cohorts before start of therapy and 10 weeks after the start of therapy. Positivity was defined as values above the mean + 2SD of the paediatric healthy control group. Peripheral blood CD4+ T cells of a subgroup of ‘flagellin high IgG responder’ (IgG positivity to 8 flagellins) and ‘flagellin low IgG responder’ (IgG positivity to 0 flagellin) therapy-naïve paediatric CD patients were cultured with recombinant flagellins. Frequency of flagellin-reactive T cells was assessed by measuring the expression of the activation marker CD154 by flow cytometry. Results As previously shown, IgG positivity to at least one flagellin was elevated in virtually all CD and UC patients (73% and 83%, respectively) compared with age-matched healthy controls. Crucially, a third of CD patients showed IgG reactivity to a high diversity of flagellins (8 or more) while UC patients showed IgG reactivity to a maximum of 3 flagellins. Notably, IgG levels specific for the Escherichia coli outer membrane porine (anti-OmpC IgG) were only increased in 8% of patients, demonstrating that a specific anti-flagellin response is mounted in IBD patients. Anti-flagellin IgA reactivity was not increased either in CD or UC patients compared with controls, suggesting that anti-flagellin CD4+ T-cell responses are required for these anti-flagellin humoral responses. In agreement, increased frequencies of CD4+CD154+ flagellin-reactive T cells were detected in ‘flagellin high IgG responder’ CD patients when compared with both controls and ‘flagellin low IgG responder’ CD patients. Conclusion Our study demonstrates that humoral responses to bacterial flagellins distinguish subgroups of paediatric CD patients. In particular, high diversity in anti-flagellin IgG reactivity is indicative of increased frequencies of anti-flagellin T cells.

Microbial transglutaminase alters the immunogenic potential and cross-reactivity of horse and cow milk proteins

Horse milk is a valuable raw material and a very attractive alternative for scientific research to address the issue of cow milk (CM) allergy due to its protein profile. A decrease in immunoreactive properties can be achieved by thermal, enzymatic, and hydrolytic processing. Therefore, the aim of this study was to explore the possibility of reducing the immunoreactivity of horse milk proteins by microbial transglutaminase (TG) polymerization. To determine how TG linking alters immunoreactivity under simulated digestion of the examined milk, analyses were performed before, during, and after digestion. The dose-dependent (1, 10, and 100 U) effects of microbial TG on horse and cow milk were analyzed. A consecutive 3-stage digestion was simulated with salivary, gastric, and intestinal fluids. The effects of digestion were analyzed by SDS-PAGE, particle size analysis, and size-exclusion chromatography. Immunoreactivity was assessed using competitive ELISA (β-lactoglobulin and α-casein) and immunodot (sera from 7 patients aged 3 to 13 years who are allergic to CM proteins). Horse milk contained almost half of the amount of total proteins in CM. The dose 1 U/g of total milk protein changed the immunoreactivity of both cow and horse milk. With increasing TG doses, α-casein immunoreactivity increased, and β-lactoglobulin decreased. After total digestion, horse milk was characterized by 2.4-fold lower average IgE and 4.8-fold lower IgG reactivity than CM. We found that TG alters the IgE and IgG reactivity of CM after in vitro digestion. Horse milk was less reactive to IgE and IgG than was CM, with animal and patient sera. The effect of TG on immunoreactivity depends on enzyme quantity and milk protein type. The diet based on modified horse milk proteins could be an alternative for some patients with CM protein allergy; however, confirmation through clinical trials is needed.

Evaluation of ViroTrack Sero Zika IgG/IgM, a New Rapid and Quantitative Zika Serological Diagnostic Assay

Background: Dengue virus (DENV) and Zika virus (ZIKV) belong to the flavivirus genus and are antigenically closely related. They also share the same mosquito vector and can cause similar symptoms upon infection. However, DENV and ZIKV infections lead to different clinical sequelae and treatments; therefore, clinicians need rapid and accurate diagnostics capable of distinguishing between the two diseases. Methods: We employed the Immuno-Magnetic Assay technology on a microfluidic cartridge (ViroTrack Sero Zika IgG/IgM) for diagnosis of DENV and ZIKV infection based on aggregation of magnetic nanoparticles. We carried out three serological studies including samples from Dominican Republic, USA, and Nicaragua, aimed at detecting ZIKV-specific IgG and IgM using the ViroTrack Sero Zika IgG/IgM test. Findings: The seroconversion results were comparable with ZIKV IgG and IgM reactivity measured by commercial ZIKV ELISA kit. The sensitivity and specificity for both ZIKV IgG and IgM tested by the ViroTrack Sero Zika IgG/IgM was approximately 98% and 93%, respectively. Interpretation: Serological detection of ZIKV infection by the new ViroTrack Sero Zika IgG/IgM test shows promising performance and limited cross-reactivity with DENV. Funding Statement: This work was financially supported in part by grant AID-OAA-F-16-0089 from USAID to MD and grants from the Ministry of Science and Technology, Taiwan (MOST-106-2622-B-182-002-CC2 &107-2622-B-182-002-CC2) and the Chang Gung Memorial Hospital Research Fund (CMRPD1E0411-3, CMRPD1F0281-2 and CMRPD1H0321-2) to RW. The Nicaraguan studies as well as sample collection and characterization were supported by grants P01 AI106695 and U19 AI118610 to EH and R01 AI099631 to AB from the US National Institutes of Health. This project received support from NIH grant P01. AI106695 to EH and AI107731. Declaration of Interests: The authors have no financial conflicts to declare. Ethics Approval Statement: All patients’ samples were collected from different sources as indicated below and these serum experimentations were under the approval of Research Ethics Board of Chang Gung Memorial Hospital in Taiwan (IRB 201601701B0). The Nicaraguan Pediatric Dengue Cohort Study was approved by the Institutional Review Boards of the University of California, Berkeley, and the Nicaraguan Ministry of Health. Parents or legal guardians of the subjects provided written informed consent, and participants 6 years of age and older provided assent.

Chembio DPP COVID-19 IgM/IgG Serological Testing: Laboratory Validation, Guidelines, and Recommendations.

On January 30, 2020, WHO declared COVID-19 a pandemic. In this article we describe our experience at Richmond University Medical Center with Chembio serological IgM, IgG testing. In this prospective cohort study of patients and hospital employees, we utilized Chembio COVID-19 IgM/IgG serological testing in addition to Cepheid RT-PCR analysis. We evaluated the performance of Chembio serological test for IgM and IgG as an employee screening tool in a community hospital setting. The total number of currently asymptomatic employees screened was 1,866 from the Richmond University Medical Center. The non-exposed group included 1,253 (67.1%) employees with no significant clinical history and non-reactive IgM and IgG antibodies. The convalescent group included 255 (13.7%) of the employees with elevation of IgG only, 18 (1%) employees with past history of positive PCR and COVID-19 who currently have non-reactive IgM and IgG antibodies or demonstrate elevated IgG only, followed by 3 employees (< 1%) with no past clinical history who demonstrated reactive IgM and IgG antibodies and negative follow up by PCR. The reported 14.9% exposure/convalescent rate is lower than the reported 20% by the Department of Health and Governor Andrew Cuomo and may represent a better utilization of personal protective equipment, better hand washing techniques, and better disinfection procedures combined with strict social distancing. Chembio's performance is satisfactory; however, hospitals must design their own policies addressing: who needs to be screened and who will interpret the results as well as constructing management algorithms for employees with no previous history and current double positive antibodies.