ABSTRACT
Because of their superior properties for certain biological applications small antibody derivatives like fragment of antigen binding (Fab) have found widespread use in basic research and as therapeutics. However, generation of Fab-fragments is still a rather complex matter, reflected by the fact that a variety of methods and purification techniques are necessary for the production of all the different classes of Fab-fragments (kappa/lambda light chains, type of species). Here we demonstrate that Fab-fragments derived from six different antibodies of human or murine origin produced by transient expression in HEK cells can be purified in a single step to a high degree of purity by standard protein G affinity chromatography. This is most likely due to alternative contact sites for protein G located in the CH1 domain of the Fab heavy chain. Our data demonstrate that protein G affinity chromatography as for whole antibodies is a robust method for the purification of tag-less Fab-fragments independent of species, significantly simplifying the process of Fab-fragment purification.
Subject(s)
Chromatography, Affinity , Immunoglobulin Fab Fragments , Immunoglobulin Fab Fragments/isolation & purification , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/chemistry , Chromatography, Affinity/methods , Humans , Animals , Mice , HEK293 Cells , Bacterial Proteins/isolation & purification , Bacterial Proteins/immunology , Bacterial Proteins/chemistryABSTRACT
In the recent mpox outbreak, people living with HIV (PLWH) were at high risk both for contracting infection and for suffering a more severe disease course. We studied cellular and humoral immune responses elicited by mpox infection (n = 5; n = 3 PLWH) or smallpox vaccination (n = 17; all PLWH) in a cohort of men who have sex with men. All PLWH were successfully treated, with stable CD4 counts and undetectable HIV viral loads. 11/17 vaccinated individuals had received childhood smallpox vaccination. In this group of individuals, both two-dose MVA-vaccination and natural infection evoked mpox-specific immune responses mediated by B cells as well as CD4 and CD8 T cells. This study improves our understanding of smallpox vaccination mediated cross-reactivity to other orthopox viruses, and the long-lasting durability of childhood smallpox vaccination mediated immune responses including in PLWH.
ABSTRACT
Measles is a highly contagious airborne viral disease. It can lead to serious complications and death and is preventable by vaccination. The live-attenuated measles vaccine (LAMV) derived from a measles virus (MV) isolated in 1954 has been in use globally for six decades and protects effectively by providing a durable humoral and cell-mediated immunity. Our study addresses the temporal stability of epitopes on the viral surface glycoprotein hemagglutinin (H) which is the major target of MV-neutralizing antibodies. We investigated the binding of seven vaccine-induced MV-H-specific monoclonal antibodies (mAbs) to cell-free synthesized MV-H proteins derived from the H gene sequences obtained from a lung specimen of a fatal case of measles pneumonia in 1912 and an isolate from a current case. The binding of four out of seven mAbs to the H protein of both MV strains provides evidence of epitopes that are stable for more than 100 years. The binding of the universally neutralizing mAbs RKI-MV-12b and RKI-MV-34c to the H protein of the 1912â¯MV suggests the long-term stability of highly conserved epitopes on the MV surface.
Subject(s)
Measles virus , Measles , Humans , Measles virus/genetics , Antibodies, Neutralizing , Neutralization Tests , Measles Vaccine/genetics , Measles/prevention & control , Antibodies, Viral , Epitopes/genetics , Hemagglutinins, Viral/genetics , Antibodies, MonoclonalABSTRACT
The monkeypox virus (MPXV) outbreak in 2022 has renewed interest in the detection of antibodies against orthopox viruses (OPXV) and MPXV, as serological methods can aid diagnostics and are key to epidemiological studies. Here three complementary serological methods are described with different strengths to aid the development and evaluation of in-house assays: An immunofluorescence assay (IFA) for specific detection of IgG and IgM, an enzyme-linked immunosorbent assay for higher sample throughput to aid epidemiological studies and a neutralization test to detect virus neutralizing antibodies. As implementation of MPXV-specific diagnostics is often hampered by the requirement for a dedicated biosafety level 3 laboratory (BSL-3), the focus of this study is on biosafety aspects to facilitate safe testing also under BSL-2 conditions. To this aim, it was analyzed whether OPXV, which can be handled under BSL-2 conditions, could be used as less virulent alternatives to MPXV. Furthermore, an inactivation method was established to remove up to five log-steps of infectious virus particles from viraemic sera without compromising antibody detection. The results show that immunological cross-reactivity between OPXV provides an opportunity for the interchangeable usage of different OPXV species in serological assays, enabling MPXV serology outside of BSL-3 facilities.
Subject(s)
Containment of Biohazards , Monkeypox virus , Humans , Laboratories , Antibodies, Viral , Neutralization TestsABSTRACT
Serological assays measuring antibodies against SARS-CoV-2 are key to describe the epidemiology, pathobiology or induction of immunity after infection or vaccination. Of those, multiplex assays targeting multiple antigens are especially helpful as closely related coronaviruses or other antigens can be analysed simultaneously from small sample volumes, hereby shedding light on patterns in the immune response that would otherwise remain undetected. We established a bead-based 17-plex assay detecting antibodies targeting antigens from all coronaviruses pathogenic for humans: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV strains 229E, OC43, HKU1, and NL63. The assay was validated against five commercial serological immunoassays, a commercial surrogate virus neutralisation test, and a virus neutralisation assay, all targeting SARS-CoV-2. It was found to be highly versatile as shown by antibody detection from both serum and dried blot spots and as shown in three case studies. First, we followed seroconversion for all four endemic HCoV strains and SARS-CoV-2 in an outbreak study in day-care centres for children. Second, we were able to link a more severe clinical course to a stronger IgG response with this 17-plex-assay, which was IgG1 and IgG3 dominated. Finally, our assay was able to discriminate recent from previous SARS-CoV-2 infections by calculating the IgG/IgM ratio on the N antigen targeting antibodies. In conclusion, due to the comprehensive method comparison, thorough validation, and the proven versatility, our multiplex assay is a valuable tool for studies on coronavirus serology.
Subject(s)
COVID-19 , Coronavirus OC43, Human , Middle East Respiratory Syndrome Coronavirus , Child , Humans , SARS-CoV-2 , Immunity, Humoral , COVID-19/diagnosis , COVID-19/epidemiology , Immunoglobulin G , Antibodies, ViralABSTRACT
The intercalated cells of the amygdala (ITCs) are a fundamental processing structure in the amygdala that remain relatively understudied. They are phylogenetically conserved from insectivores through primates, inhibitory, and project to several of the main processing and output stations of the amygdala and basal forebrain. Through these connections, the ITCs are best known for their role in conditioned fear, where they are required for fear extinction learning and recall. Prior work on ITC connectivity is limited, and thus holistic characterization of their afferent and efferent connectivity in a genetically defined manner is incomplete. The ITCs express the FoxP2 transcription factor, affording genetic access to these neurons for viral input-output mapping. To fully characterize the anatomic connectivity of the ITCs, we used cre-dependent viral strategies in FoxP2-cre mice to reveal the projections of the main (mITC), caudal (cITC), and lateral (lITC) clusters along with their presynaptic sources of innervation. Broadly, the results confirm many known pathways, reveal previously unknown ones, and demonstrate important novel insights about each nucleus's unique connectivity profile and relative distributions. We show that the ITCs receive information from a wide range of cortical, subcortical, basal, amygdalar, hippocampal, and thalamic structures, and project broadly to areas of the basal forebrain, hypothalamus, and entire extent of the amygdala. The results provide a comprehensive map of their connectivity and suggest that the ITCs could potentially influence a broad range of behaviors by integrating information from a wide array of sources throughout the brain.
Subject(s)
Extinction, Psychological , Fear , Mice , Animals , Extinction, Psychological/physiology , Fear/physiology , Amygdala/physiology , Neurons/physiology , Transcription Factors/metabolismABSTRACT
Type Ia supernovae (SNe Ia) are thermonuclear explosions of degenerate white dwarf stars destabilized by mass accretion from a companion star1, but the nature of their progenitors remains poorly understood. A way to discriminate between progenitor systems is through radio observations; a non-degenerate companion star is expected to lose material through winds2 or binary interaction3 before explosion, and the supernova ejecta crashing into this nearby circumstellar material should result in radio synchrotron emission. However, despite extensive efforts, no type Ia supernova (SN Ia) has ever been detected at radio wavelengths, which suggests a clean environment and a companion star that is itself a degenerate white dwarf star4,5. Here we report on the study of SN 2020eyj, a SN Ia showing helium-rich circumstellar material, as demonstrated by its spectral features, infrared emission and, for the first time in a SN Ia to our knowledge, a radio counterpart. On the basis of our modelling, we conclude that the circumstellar material probably originates from a single-degenerate binary system in which a white dwarf accretes material from a helium donor star, an often proposed formation channel for SNe Ia (refs. 6,7). We describe how comprehensive radio follow-up of SN 2020eyj-like SNe Ia can improve the constraints on their progenitor systems.
ABSTRACT
PURPOSE: COViK, a prospective hospital-based multicenter case-control study in Germany, aims to assess the effectiveness of COVID-19 vaccines against severe disease. Here, we report vaccine effectiveness (VE) against COVID-19-caused hospitalization and intensive care treatment during the Omicron wave. METHODS: We analyzed data from 276 cases with COVID-19 and 494 control patients recruited in 13 hospitals from 1 December 2021 to 5 September 2022. We calculated crude and confounder-adjusted VE estimates. RESULTS: 21% of cases (57/276) were not vaccinated, compared to 5% of controls (26/494; p < 0.001). Confounder-adjusted VE against COVID-19-caused hospitalization was 55.4% (95% CI: 12-78%), 81.5% (95% CI: 68-90%) and 95.6% (95%CI: 88-99%) after two, three and four vaccine doses, respectively. VE against hospitalization due to COVID-19 remained stable up to one year after three vaccine doses. CONCLUSION: Three vaccine doses remained highly effective in preventing severe disease and this protection was sustained; a fourth dose further increased protection.
Subject(s)
COVID-19 , Humans , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19 Vaccines , Case-Control Studies , Prospective Studies , Vaccine Efficacy , Germany/epidemiologyABSTRACT
We included 852 patients in a prospectively recruiting multicenter matched case-control study in Germany to assess vaccine effectiveness (VE) in preventing COVID-19-associated hospitalization during the Delta-variant dominance. The two-dose VE was 89 % (95 % CI 84-93 %) overall, 79 % in patients with more than two comorbidities and 77 % in adults aged 60-75 years. A third dose increased the VE to more than 93 % in all patient-subgroups.
Subject(s)
COVID-19 , Vaccines , Adult , Humans , Case-Control Studies , COVID-19/prevention & control , Hospitalization , Hospitals , Germany/epidemiologyABSTRACT
Gram-positive bacteria are protected by a thick mesh of peptidoglycan (PG) completely engulfing their cells. This PG network is the main component of the bacterial cell wall, it provides rigidity and acts as foundation for the attachment of other surface molecules. Biosynthesis of PG consumes a high amount of cellular resources and therefore requires careful adjustments to environmental conditions. An important switch in the control of PG biosynthesis of Listeria monocytogenes, a Gram-positive pathogen with a high infection fatality rate, is the serine/threonine protein kinase PrkA. A key substrate of this kinase is the small cytosolic protein ReoM. We have shown previously that ReoM phosphorylation regulates PG formation through control of MurA stability. MurA catalyzes the first step in PG biosynthesis and the current model suggests that phosphorylated ReoM prevents MurA degradation by the ClpCP protease. In contrast, conditions leading to ReoM dephosphorylation stimulate MurA degradation. How ReoM controls degradation of MurA and potential other substrates is not understood. Also, the individual contribution of the ~20 other known PrkA targets to PG biosynthesis regulation is unknown. We here present murA mutants which escape proteolytic degradation. The release of MurA from ClpCP-dependent proteolysis was able to activate PG biosynthesis and further enhanced the intrinsic cephalosporin resistance of L. monocytogenes. This latter effect required the RodA3/PBP B3 transglycosylase/transpeptidase pair. One murA escape mutation not only fully rescued an otherwise non-viable prkA mutant during growth in batch culture and inside macrophages but also overcompensated cephalosporin hypersensitivity. Our data collectively indicate that the main purpose of PrkA-mediated signaling in L. monocytogenes is control of MurA stability during standard laboratory growth conditions and intracellular growth in macrophages. These findings have important implications for the understanding of PG biosynthesis regulation and ß-lactam resistance of L. monocytogenes and related Gram-positive bacteria.
Subject(s)
Listeria monocytogenes , Peptidoglycan , Cell Wall/metabolism , Listeria monocytogenes/genetics , Listeria monocytogenes/metabolism , Mutation , Peptidoglycan/metabolism , Protein Serine-Threonine Kinases/geneticsABSTRACT
The collection of data on SARS-CoV2 tests is central to the assessment of the infection rate in the context of the COVID-19 pandemic. At the Robert Koch Institute (RKI), data collected from various laboratory data recording systems are consolidated. First, this article aims to exemplify significant aspects regarding test procedures. Subsequently the different systems for recording laboratory tests are described and test numbers from the RKI test laboratory query and the laboratory-based SARS-CoV2 surveillance as well as accounting data from the Association of Statutory Health Insurance Physicians for SARS-CoV2 laboratory tests are shown.Early in the pandemic, the RKI test laboratory query and the laboratory-based SARS-CoV2 surveillance became available and able to evaluate data on performed tests and test capacities. By recording the positive and negative test results, statements about the total number of tests and the proportion of positive test rates can be made. While the aggregate test numbers are largely representative nationwide, they are not always representative at the state and district level. The billing data of the Association of Statutory Health Insurance Physicians can complement the laboratory data afterwards. In addition, it can provide a retrospective assessment of the total number of SARS-CoV2 numbers in Germany, because the services provided by statutory health insurers (around 85% of the population in Germany) are included. The various laboratory data recording systems complement one another and the evaluations flow into the recommended measures for the pandemic response.
Subject(s)
COVID-19 , Pandemics , COVID-19 Testing , Germany/epidemiology , Humans , Retrospective Studies , SARS-CoV-2ABSTRACT
Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.
Subject(s)
Antibodies, Monoclonal , Claudin-4/metabolism , Clostridium Infections/diagnosis , Clostridium perfringens/metabolism , Enterotoxins/analysis , Enzyme-Linked Immunosorbent Assay , Foodborne Diseases/diagnosis , Animals , Antibody Affinity , Antibody Specificity , Automation, Laboratory , Claudin-4/genetics , Claudin-4/immunology , Clostridium Infections/microbiology , Clostridium perfringens/genetics , Clostridium perfringens/immunology , Enterotoxins/genetics , Enterotoxins/immunology , Enterotoxins/metabolism , Epitope Mapping , Epitopes , Feces , Foodborne Diseases/microbiology , Humans , Limit of Detection , Mice , Predictive Value of Tests , Protein Binding , Reproducibility of Results , WorkflowABSTRACT
Abrin, the toxic lectin from the rosary pea plant Abrus precatorius, has gained considerable interest in the recent past due to its potential malevolent use. However, reliable and easy-to-use assays for the detection and discrimination of abrin from related plant proteins such as Abrus precatorius agglutinin or the homologous toxin ricin from Ricinus communis are sparse. To address this gap, a panel of highly specific monoclonal antibodies was generated against abrin and the related Abrus precatorius agglutinin. These antibodies were used to establish two sandwich ELISAs to preferentially detect abrin or A. precatorius agglutinin (limit of detection 22 pg/mL for abrin; 35 pg/mL for A. precatorius agglutinin). Furthermore, an abrin-specific lateral flow assay was developed for rapid on-site detection (limit of detection ~1 ng/mL abrin). Assays were validated for complex food, environmental and clinical matrices illustrating broad applicability in different threat scenarios. Additionally, the antibodies turned out to be suitable for immuno-enrichment strategies in combination with mass spectrometry-based approaches for unambiguous identification. Finally, we were able to demonstrate for the first time how the developed assays can be applied to detect, identify and quantify abrin from a clinical sample derived from an attempted suicide case involving A. precatorius.
Subject(s)
Abrin/analysis , Abrus/chemistry , Antibodies, Monoclonal/immunology , Enzyme-Linked Immunosorbent Assay , Plant Lectins/analysis , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Abrin/immunology , Abrin/poisoning , Abrus/immunology , Antibody Specificity , Feces/chemistry , Humans , Limit of Detection , Plant Lectins/immunology , Reproducibility of Results , Suicide, AttemptedABSTRACT
BACKGROUND: Few bead-based multiplex assays have been described that detect antibodies against the protozoan parasite Toxoplasma gondii in large-scale seroepidemiological surveys. Moreover, each multiplex assay has specific variations or limitations, such as the use of truncated or fusion proteins as antigens, potentially masking important epitopes. Consequently, such an assay must be developed by interested groups as none is commercially available. RESULTS: We report the bacterial expression and use of N-terminal fusion-free, soluble, in vivo biotinylated recombinant surface antigens SAG1 and SAG2A for the detection of anti-T. gondii IgG antibodies. The expression system relies on three compatible plasmids. An expression construct produces a fusion of maltose-binding protein with SAG1 (or SAG2A), separated by a TEV protease cleavage site, followed by a peptide sequence recognized by E. coli biotin ligase BirA (AviTag), and a terminal six histidine tag for affinity purification. TEV protease and BirA are encoded on a second plasmid, and their expression leads to proteolytic cleavage of the fusion protein and a single biotinylated lysine within the AviTag by BirA. Correct folding of the parasite proteins is dependent on proper disulfide bonding, which is facilitated by a sulfhydryl oxidase and a protein disulfide isomerase, encoded on the third plasmid. The C-terminal biotinylation allowed the oriented, reproducible coupling of the purified surface antigens to magnetic Luminex beads, requiring only minute amounts of protein per determination. We showed that an N-terminal fusion partner such as maltose-binding protein negatively influenced antibody binding, confirming that access to SAG1's N-terminal epitopes is important for antibody recognition. We validated our bead-based multiplex assay with human sera previously tested with commercial diagnostic assays and found concordance of 98-100% regarding both, sensitivity and specificity, even when only biotinylated SAG1 was used as antigen. CONCLUSIONS: Our recombinant in vivo-biotinylated T. gondii antigens offer distinct advantages compared to previously described proteins used in multiplex serological assays for T. gondii. They offer a cheap, specific and sensitive alternative to either parasite lysates or eukaryotic-cell expressed SAG1/SAG2A for BBMA and other formats. The described general expression strategy can also be used for other antigens where oriented immobilization is key for sensitive recognition by antibodies and ligands.
Subject(s)
Antigens, Protozoan/genetics , Membrane Glycoproteins/genetics , Protozoan Proteins/genetics , Toxoplasma/genetics , Toxoplasma/immunology , Animals , Antibodies, Protozoan/blood , Antibodies, Protozoan/immunology , Antigens, Protozoan/chemistry , Antigens, Protozoan/immunology , Biotinylation , Carbon-Nitrogen Ligases , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Escherichia coli Proteins , Gene Expression Regulation, Bacterial , Humans , Immunoglobulin G , Membrane Glycoproteins/chemistry , Protozoan Proteins/chemistry , Protozoan Proteins/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Repressor Proteins , Sequence Analysis , Seroepidemiologic Studies , Toxoplasmosis/diagnosisABSTRACT
Surveillance of highly pathogenic viruses circulating in both human and animal populations is crucial to unveil endemic infections and potential zoonotic reservoirs. Monitoring the burden of disease by serological assay could be used as an early warning system for imminent outbreaks as an increased seroprevalance often precedes larger outbreaks. However, the multitude of highly pathogenic viruses necessitates the need to identify specific antibodies against several targets from both humans as well as from potential reservoir animals such as bats. In order to address this, we have developed a broadly reactive multiplex microsphere immunoassay (MMIA) for the detection of antibodies against several highly pathogenic viruses from both humans and animals. To this aim, nucleoproteins (NP) of Ebola virus (EBOV), Marburg virus (MARV) and nucleocapsid proteins (NP) of Crimean-Congo haemorrhagic fever virus, Rift Valley fever virus and Dobrava-Belgrade hantavirus were employed in a 5-plex assay for IgG detection. After optimisation, specific binding to each respective NP was shown by testing sera from humans and non-human primates with known infection status. The usefulness of our assay for serosurveillance was shown by determining the immune response against the NP antigens in a panel of 129 human serum samples collected in Guinea between 2011 and 2012 in comparison to a panel of 88 sera from the German blood bank. We found good agreement between our MMIA and commercial or in-house reference methods by ELISA or IIFT with statistically significant higher binding to both EBOV NP and MARV NP coupled microspheres in the Guinea panel. Finally, the MMIA was successfully adapted to detect antibodies from bats that had been inoculated with EBOV- and MARV- virus-like particles, highlighting the versatility of this technique and potentially enabling the monitoring of wildlife as well as human populations with this assay. We were thus able to develop and validate a sensitive and broadly reactive high-throughput serological assay which could be used as a screening tool to detect antibodies against several highly pathogenic viruses.
Subject(s)
Antibodies, Viral/blood , Immunoassay/methods , Microspheres , Nucleocapsid Proteins/immunology , Virus Diseases/veterinary , Animals , Chiroptera , Humans , Primates , Virus Diseases/diagnosis , Virus Diseases/virologyABSTRACT
PURPOSE: The purpose of this study was to describe the microbiology of recurrent acute exacerbations of chronic rhinosinusitis over time. METHODS: Retrospective review of patients with recurrent acute exacerbations of chronic rhinosinusitis who underwent endoscopic-guided cultures during acute exacerbations of chronic rhinosinusitis. RESULTS: 386 cultures were obtained from 112 patients during recurrent acute exacerbations of CRS. A change of bacterial isolates during the course of recurrent exacerbations was observed in 68% (76/112) of patients, necessitating a change of treatment in 40% (45/112). The main risk factor for the subsequent change in cultures was polymicrobial growth. Sinus surgery was not associated with subsequent change in cultured isolates. Resistant strains developed in 11.6% (13/112) of patients, of whom those with abnormal mucociliary clearance being at the highest risk. CONCLUSION: Repeated middle meatal cultures should be considered in patients with recurrent exacerbations of CRS, particularly in cases not responding to standard therapy.
Subject(s)
Bacteriology , Rhinitis , Sinusitis , Chronic Disease , Endoscopy , Humans , Retrospective StudiesABSTRACT
The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins' light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A-F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A-F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.
Subject(s)
Botulinum Toxins/pharmacology , Synaptosomal-Associated Protein 25/metabolism , Vesicle-Associated Membrane Protein 2/metabolism , Buffers , Escherichia coli/genetics , Recombinant Proteins/metabolism , Serogroup , Synaptosomal-Associated Protein 25/genetics , Vesicle-Associated Membrane Protein 2/geneticsABSTRACT
Toxoplasmosis, while often an asymptomatic parasitic disease in healthy individuals, can cause severe complications in immunocompromised persons and during pregnancy. The most common method to diagnose Toxoplasma gondii infections is the serological determination of antibodies directed against parasite protein antigens. Here we report the use of a bead-based multiplex assay containing a synthetic phosphoglycan portion of the Toxoplasma gondii glycosylphosphatidylinositol (GPI1) for the detection of GPI1-specific antibodies in human sera. The glycan was conjugated to beads at the lipid site to retain its natural orientation and its immunogenic groups. We compared the response against GPI1 with that against the protein antigen SAG1, a common component of commercial serological assays, via the detection of parasite-specific human IgG and IgM antibodies, respectively. The GPI1-based test is in excellent agreement with the results for the commercial ELISA, as the ROC analysis of the GPI1 test shows 97% specificity and 98% sensitivity for the assay. GPI1 was a more reliable predictor for a parasite-specific IgM response compared to SAG1, indicating that a bead-based multiplex assay using GPI1 in combination with SAG1 may strengthen Toxoplasma gondii serology, in particular in seroepidemiological studies.
Subject(s)
Antibodies, Protozoan/blood , Glycosylphosphatidylinositols/chemistry , Polysaccharides/chemistry , Toxoplasma/chemistry , Antibodies, Protozoan/immunology , Antibody Specificity , Antigens, Protozoan/blood , Antigens, Protozoan/immunology , Enzyme-Linked Immunosorbent Assay , Glycosylphosphatidylinositols/immunology , Humans , Polysaccharides/immunology , ROC Curve , Toxoplasma/immunology , Toxoplasmosis/blood , Toxoplasmosis/immunologyABSTRACT
BACKGROUND: The subcutaneous implantable cardioverter-defibrillator (S-ICD) has established its role in the prevention of sudden cardiac death in a defined population. Inappropriate shocks and device malfunction in S-ICD therapy may be caused by myopotential (MP) oversensing. OBJECTIVE: The purpose of this study was to systematically evaluate a cohort of consecutive S-ICD patients for MP inducibility. METHODS: After S-ICD implantation, all vectors (primary [PrimV], secondary [SecV], alternative [AltV]) were analyzed during isometric chest press (ICP), lifting and holding a 20-kg weight, and side plank exercise (SPE), supporting the body weight on the left arm. When MPs were induced, signal classification was assessed: adequate noise detection, induced undersensing (R waves classified as noise), and oversensing (noise annotated as R waves). In case of noise induction in the current vector, device reprogramming to a noise-free vector was done. RESULTS: We systematically assessed 41 patients. In nearly all patients (90.2%), MPs were inducible. ICP was the most potent inductor of MPs. Whereas SecV (70.7%) and AltV (75.6%) were most vulnerable during ICP, PrimV was most affected during SPE (51.2%). In only a few cases did the S-ICD software distinguish correctly between MPs and QRS. MPs predominantly led to undersensing (up to 65.9%), but in up to 22% of patients MP-induced oversensing occurred but did not lead to tachycardia detection. No relation was seen between S-ICD lead and generator position and MP inducibility. CONCLUSION: Induction of MPs during physical exercise was observed frequently. Although in most cases MP noise led to undersensing, oversensing events were commonly observed.