Inhibition ELISA Research Articles

Serotype-specific epidemiological patterns of inapparent versus symptomatic primary dengue virus infections: a 17-year cohort study in Nicaragua.

Dengue is the most prevalent mosquito-borne viral disease and a major public health problem worldwide. Most primary infections with the four dengue virus serotypes (DENV1-4) are inapparent; nonetheless, whether the distribution of symptomatic versus inapparent infections by serotype varies remains unknown. Here, we present (1) the evaluation of a DENV1-4 envelope domain III multiplex microsphere-based assay (EDIII-MMBA) to serotype inapparent primary infections and (2) its application leveraging 17 years of prospective sample collection from the Nicaraguan Pediatric Dengue Cohort Study (PDCS). We analysed primary DENV infections in the PDCS from 2004 to 2022 detected by inhibition ELISA (iELISA) or RT-PCR. First, we evaluated the performance of the EDIII-MMBA for serotyping with samples characterised by RT-PCR or focus reduction neutralisation test. Next, we analysed a subset of inapparent primary DENV infections in the PDCS with the EDIII-MMBA to evaluate the epidemiology of inapparent infections. Remaining infections were inferred using stochastic imputation, taking year and neighbourhood into account. Infection incidence and percentage of inapparent, symptomatic, and severe infections were analysed by serotype. Between Aug 30, 2004, and March 10, 2022, a total of 5931 DENV-naive participants were followed in the PDCS. There were 1626 primary infections (382 symptomatic, 1244 inapparent) detected by iELISA or RT-PCR over the study period. The EDIII-MMBA demonstrated excellent overall accuracy (100%, 95% CI 95·8-100) for serotyping inapparent primary DENV infections when evaluated against gold-standard serotyping methods. Of the 1244 inapparent infections, we analysed 574 (46%) using the EDIII-MMBA. We found that the majority of primary infections were inapparent, with DENV3 exhibiting the highest likelihood of symptomatic (pooled odds ratio compared with DENV1: 2·13, 95% CI 1·28-3·56) and severe (6·75, 2·01-22·62) primary infections, whereas DENV2 was similar to DENV1 in both analyses. Considerable within-year and between-year variation in serotype distribution between symptomatic and inapparent infections and circulation of serotypes undetected in symptomatic cases were observed in multiple years. Our study indicates that case surveillance skews the perceived epidemiological footprint of DENV. We reveal a more complex and intricate pattern of serotype distribution in inapparent infections. The substantial differences in infection outcomes by serotype emphasises the need for vaccines with balanced immunogenicity and efficacy across serotypes. National Institute of Allergy and Infectious Diseases (National Institutes of Health) and Bill & Melinda Gates Foundation. For the Spanish translation of the abstract see Supplementary Materials section.

CDR identification, epitope mapping and binding affinity determination of novel monoclonal antibodies generated against human apolipoprotein B-100

In-house generated mAbs to apolipoprotein B-100 (apoB-100) clones hLDL-E8, hLDL-2D8 and hLDL-F5 were extensively studied to determine their complementarity-determining regions (CDRs), binding epitopes and affinity. RT-PCR revealed that all mAbs consisted of kappa light chains and gamma heavy chains. DNA sequencing and bioinformatic analysis showed that the variable gene and protein sequences of their CDRs shared over 50 % identity with the existing databases. The 3D structures of the mAb variable fragments (Fv) with a QSQE score above 0.7 were constructed using the SWISS-MODEL platform. The structural accuracy was confirmed by Ramachandran plots, with 99 % of amino acid residues falling within acceptable regions. Thrombolytic cleavage of apoB-100 and Western blot analysis demonstrated that hLDL-E8 and hLDL-F5 specifically bind to the T3 fragment (aa 1297–3249), whereas hLDL-2D8 binds to the T4 fragment (aa 1–1297). These findings were supported with epitope-binding assays using inhibition ELISA, which indicated that hLDL-E8 binds at different epitopes from hLDL-2D8 and has some overlap with hLDL-F5. Lastly, the binding affinity of the mAbs was examined by indirect ELISA. The average affinity constants (Kaff) for mAbs hLDL-2D8, hLDL-E8 and hLDL-F5 are 1.51 ± 0.69 × 109 Mol−1, 7.25 ± 3.56 × 108 Mol−1 and 4.39 ± 2.63 × 106 Mol−1, respectively. Additionally, the behavior of the antibodies in the dose-response curve revealed that hLDL-F5 may recognize two epitopes of apoB-100 or have very low binding affinity. In contrast, hLDL-2D8 and hLDL-E8 each recognize a single epitope. These findings provide information that will be useful when selecting mAbs for both laboratory and clinical research purposes.

New benzophenone analogs from Nigrospora sphaerica and their inhibitory activity against PD-1/PD-L1 interactions

Four newly identified benzophenone analogs [nigrophenone A–D (1–4)] and a pyrrolidinone analog [nigropyrrolidinone (5)], alongside thirteen known congeners (6–18), were isolated from Nigrospora sphaerica. Transcriptome analysis revealed that 6 might have the potential to modulate T-cell immunity. Quantitative measurements of the binding affinities between eighteen natural molecules and the immunological checkpoint receptors PD-1 and PD-L1 were performed using Surface Plasmon Resonance (SPR). The results of SPR analysis showed that 1–18 have KD values ranging from 1.8 to 99.5 μM for PD-1 and from 10.6 to 99.5 μM for PD-L1. Competitive inhibition studies, employing SPR and ELISA assays, have indicated that compounds 6, 10, 15, and 18 are capable of inhibiting the PD-1/PD-L1 interaction. Additionally, compound 6 exhibited notable in vitro anticancer potency through the augmentation of activating signals and the upregulation of PD-1 expression on CD8+ T cells, concurrently elevating the secretion of IFN-γ and IL-2, thereby inhibiting the proliferation of LLC and MC38 cells and promoting MC38 apoptosis. Moreover, compound 6 modulates the PI3K/Akt pathway, which is a key downstream effector of the PD-1/PD-L1 axis. These compounds are considered promising candidates for more in-depth exploration because they could significantly inhibit PD-1/PD-L1 interactions in tumor immunotherapy.

Newborns with microcephaly in Brazil and potential vertical transmission of Oropouche virus: a case series

Oropouche fever, an orthobunyavirus disease endemic in Brazilian Amazon, has caused many febrile epidemics. In 2024, an epidemic of Oropouche fever spread in Brazil, with more than 7930 cases reported between Jan 1 and Aug 31. Infections in pregnant people have suggested the possibility of negative fetal consequences, therefore we tested newborns with microcephaly for known congenital pathogens and Oropouche virus (OROV). In this case series, we assessed historical cases of infants born with microcephaly, arthrogryposis, and other congenital malformations without a confirmed cause and their mothers for potential OROV congenital infections. The study population consisted of infants born in Brazil with samples from 2015-21 and 2024. Serum and cerebrospinal fluid (CSF) from this case series were analysed for: syphilis, toxoplasmosis, rubella, cytomegalovirus, herpes simplex, HIV, Zika, dengue, and chikungunya. Individuals that were negative for these pathogens were then tested for OROV. Pathogen testing included ELISA and haemagglutination inhibition testing for antibodies and RT-PCR for virus RNA. We tested 68 samples from 65 historical cases of congential malformations and three cases from 2024. All cases were from ten states in Brazil. Three historical cases tested positive for OROV and 62 historical cases tested negative. The three cases from 2024 all tested positive for OROV. Of the positive cases, five were female and one was male. Not all pathogens were tested for each case, and some did not have maternal samples available. One of the newborns (case 6) died aged 47 days and tissue samples were tested by real-time RT-PCR, histopathology, and immunohistochemistry assays. One other newborn died in 2016 but no post-mortem samples were available. OROV IgM was detected in five of five newborn CSF samples, and five of five newborn serum samples. Four of five maternal serum samples were positive for OROV IgM. One of four newborn CSF samples (case 6 at age 44 days) was OROV positive by real-time RT-quantitative PCR and 0 of four newborn serum samples were positive, as were 0 of three maternal serum samples. Case 6 had major tissue changes of the brain macroscopically and microscopically, including necrotic and apoptotic changes of neurons, microglia and astrocytes, vacuolisation, and tissue atrophy. OROV RNA was detected in brain, lungs, kidney, CSF, and pleural fluid; OROV antigens were found in CNS, liver, kidney, heart, and lung, mainly in neurons and microglia and also in endothelial cells, suggesting vasculitis. We detected OROV IgM in six of 68 newborns with microcephaly of unknown cause. One infant who died had OROV RNA and antigen in several tissues, including the brain. The possibility of OROV vertical transmission and potential fetal harm must be investigated with urgency. The evidence presented here does not completely confirm vertical transmission or congenital malformations due to OROV, but thorough case finding and detailed investigation of maternal or fetal OROV infection is a priority. Evandro Chagas Institute, Secretaria de Vigilância em Saúde e Ambiente, and Ministry of Health and National Institute of Science and Technology for Emerging and Reemerging Viruses.

Walnut Jug r 1 is Responsible for Primary Sensitization among Patients Suffering Walnut-Hazelnut 2S Albumin Cross-Reactivity.

Walnut and hazelnut coallergy is a frequent manifestation in clinical practice whose molecular basis remains unclear. For this purpose, walnut-hazelnut cross-reactivity was evaluated in 20 patients allergic to one or both tree nuts and sensitized to their 2S albumins. Immunoblotting assays showed that 85% of patients recognized Jug r 1, walnut 2S albumin, which was associated with the development of severe symptoms; 50% of them corecognized hazelnut 2S albumin, Cor a 14. Both allergens were isolated using chromatographic techniques. Inhibition ELISAs revealed that Jug r 1 strongly inhibited the binding of Cor a 14-specific IgE, but Cor a 14 only partially inhibited Jug r 1-specific IgE binding. Our results showed that patients sensitized to walnut/hazelnut 2S albumins were not a homogeneous population. There were patients sensitized to specific epitopes of walnut 2S albumins and patients sensitized to cross-reactive epitopes between walnut and hazelnut, with Jug r 1 being the primary sensitizer.

A homogeneous bioluminescent inhibition immunoassay to detect anti-interferon gamma antibodies.

Adult-onset immunodeficiency with antibodies to interferon-γ (AOID with AIGA), is a rare, acquired immunodeficiency causing susceptibility to disseminated non-tuberculous mycobacteria and other intracellular opportunistic infections. The diagnosis depends on demonstrating the presence of endogenous anti-interferon-γ antibodies (AIGA) that suppress Th1 cell mediated immunity. Bioluminescent immunoassays are a newly emerging immunoassay format which utilise the action of bioluminescent enzymes on a substrate for specific analyte detection. In-short, detecting antibodies are conjugated with a bioluminescent enzyme. The detecting antibodies bind the analyte of interest and produce light (luminescence) after addition of a substrate. The purpose of this study was to evaluate two newly developed bioluminescent immunoassays using Lumit® (Promega) technology as a diagnostic test for AOID with AIGA. Specific aims included the clinical validation of a new inhibition bioluminescent immunoassay technique to detect AIGA which block detection of interferon-γ (IFN-γ) in-vitro and correlation of inhibition bioluminescent immunoassay results with AOID with AIGA disease status. Two bioluminescent inhibition immunoassays were developed. One which adapted an existing kit from Promega (Lumit® Human IFN-γ Immunoassay) and one which was developed in-house. 87 healthy controls and 48 patients with previously diagnosed AOID with AIGA were recruited and tested using these two methods. Results showed both bioluminescent inhibition immunoassays were able to clearly discriminate between AOID with AIGA patients and healthy controls. The mean inhibition percentage between patient groups correlated with disease activity. Both assays appeared to be more sensitive when compared to the existing inhibition ELISA.

Specificity of serological screening tests and reference laboratory tests to diagnose gambiense human African trypanosomiasis: a prospective clinical performance study

BackgroundSerological screening tests play a crucial role to diagnose gambiense human African trypanosomiasis (gHAT). Presently, they preselect individuals for microscopic confirmation, but in future “screen and treat” strategies they will identify individuals for treatment. Variability in reported specificities, the development of new rapid diagnostic tests (RDT) and the hypothesis that malaria infection may decrease RDT specificity led us to evaluate the specificity of 5 gHAT screening tests.MethodsDuring active screening, venous blood samples from 1095 individuals from Côte d’Ivoire and Guinea were tested consecutively with commercial (CATT, HAT Sero-K-SeT, Abbott Bioline HAT 2.0) and prototype (DCN HAT RDT, HAT Sero-K-SeT 2.0) gHAT screening tests and with a malaria RDT. Individuals with ≥ 1 positive gHAT screening test underwent microscopy and further immunological (trypanolysis with T.b. gambiense LiTat 1.3, 1.5 and 1.6; indirect ELISA/T.b. gambiense; T.b. gambiense inhibition ELISA with T.b. gambiense LiTat 1.3 and 1.5 VSG) and molecular reference laboratory tests (PCR TBRN3, 18S and TgsGP; SHERLOCK 18S Tids, 7SL Zoon, and TgsGP; Trypanozoon S2-RT-qPCR 18S2, 177T, GPI-PLC and TgsGP in multiplex; RT-qPCR DT8, DT9 and TgsGP in multiplex). Microscopic trypanosome detection confirmed gHAT, while other individuals were considered gHAT free. Differences in fractions between groups were assessed by Chi square and differences in specificity between 2 tests on the same individuals by McNemar.ResultsOne gHAT case was diagnosed. Overall test specificities (n = 1094) were: CATT 98.9% (95% CI: 98.1–99.4%); HAT Sero-K-SeT 86.7% (95% CI: 84.5–88.5%); Bioline HAT 2.0 82.1% (95% CI: 79.7–84.2%); DCN HAT RDT 78.2% (95% CI: 75.7–80.6%); and HAT Sero-K-SeT 2.0 78.4% (95% CI: 75.9–80.8%). In malaria positives, gHAT screening tests appeared less specific, but the difference was significant only in Guinea for Abbott Bioline HAT 2.0 (P = 0.03) and HAT Sero-K-Set 2.0 (P = 0.0006). The specificities of immunological and molecular laboratory tests in gHAT seropositives were 98.7–100% (n = 399) and 93.0–100% (n = 302), respectively. Among 44 reference laboratory test positives, only the confirmed gHAT patient and one screening test seropositive combined immunological and molecular reference laboratory test positivity.ConclusionsAlthough a minor effect of malaria cannot be excluded, gHAT RDT specificities are far below the 95% minimal specificity stipulated by the WHO target product profile for a simple diagnostic tool to identify individuals eligible for treatment. Unless specificity is improved, an RDT-based “screen and treat” strategy would result in massive overtreatment. In view of their inconsistent results, additional comparative evaluations of the diagnostic performance of reference laboratory tests are indicated for better identifying, among screening test positives, those at increased suspicion for gHAT.Trial registrationThe trial was retrospectively registered under NCT05466630 in clinicaltrials.gov on July 15 2022.Graphical

Development and Bayesian validation of a competitive inhibition ELISA for detection of antibodies against Brucella abortus in cattle

Bovine brucellosis is a zoonotic disease caused by Brucella abortus, responsible for abortions in cows. It is endemic in low- and middle-income countries, where the brucellosis control and eradication programs are based on compulsory vaccination, detection of infected cattle through serologic assays, and culling of infected animals at slaughterhouses. The development of high sensitivity and specificity, and low-cost serologic assays guarantee their implementation in countries where the disease is endemic. The aim of the present study was to develop and validate a competitive inhibition enzyme-linked immune assay (ciELISA) to detect anti-B. abortus antibodies in sera from cattle. The developed ciELISA was validated using 2833 serum samples from dairy and beef cattle. From these, 1515 sera were from uninfected cows that belonged to free of brucellosis herds and 1318 were from infected cows that belonged positive to brucellosis herds. Sera were analyzed with the developed ciELISA, the buffer plate antigen (BPA) test, and the complement fixation test (CFT). The brucellosis status of the herds was officially established according to the country legislation and consistent for at least 5 years and was defined for each cow using the CFT as gold standard. The cutoff for the ciELISA was calculated using a ROC curve and its sensitivity and specificity were analyzed using the Bayesian Latent Class Model (BLCM) approach. The agreement among tests was calculated using the kappa (κ) value. In addition, 15 calves were vaccinated with 3 × 1010 viable cells of B. abortus Strain 19 vaccine, and the dynamics of antibodies were measured by CFT, buffered plate antigen (BPA) test, and the developed ciELISA. The obtained cutoff for ciELISA was ≥ 47 percentage of inhibition (% I), at the BLCM approach the sensitivity was 99.01 % (95 % CI: 97.55–100) and the specificity 98.74 % (95 % CI: 97.68–99.8). The κ between the ciELISA and BPA was κ = 0.88 and between the ciELISA and CFT κ = 0.95. Antibodies against B. abortus were detected in all the vaccinated calves 7 days after vaccination (AV) by the three assays, at day 135 AV all the calves were negative to CFT (15/15), 93.3 % (14/15) to ciELISA and 73.3 % (11/15) to BPA, and at day 190 AV all the calves were negative to the three assays. The developed ciELISA showed a very good performance, could detect the majority of vaccinated animals as negative after 135 days and could be used for the detection of anti-B. abortus antibodies in serum samples for the brucellosis control and eradication program.

Chondroitin sulfate liposome: clustering toward high functional efficiency.

Chondroitin sulfate (CS) is a linear polysaccharide chain of alternating residues of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc), modified with sulfate groups. Based on the structure, CS chains bind to bioactive molecules specifically and regulate their functions. For example, CS whose GalNAc is sulfated at the C4 position, termed CSA, and CS whose GalNAc is sulfated at both C4 and C6 positions, termed CSE, bind to a malaria protein VAR2CSA and receptor type of protein tyrosine phosphatase sigma (RPTPσ), respectively, in a specific manner. Here, we modified CSA and CSE chains with phosphatidylethanolamine (PE) at a reducing end, attached them to liposomes containing phospholipids and generated CSA and CSE liposomes. The CS-PE was incorporated into the liposome particles efficiently. Inhibition ELISA revealed specific interaction of CSA and CSE with recombinant VAR2CSA and RPTPσ, respectively, more efficiently than CS chains alone. Furthermore, CSE liposome was specifically incorporated into RPTPσ-expressing HEK293T cells. These results indicate CS liposome as a novel and efficient drug delivery system, especially for CS-binding molecules.

Heterogenous Induction of Blocking Antibodies against Ragweed Allergen Molecules by Allergen Extract-Based Immunotherapy Vaccines.

Currently, allergen-specific immunotherapy (AIT) for ragweed allergy is still based on natural allergen extracts. This study aimed to analyse the ability of four commercially available AIT vaccines (CLUSTOID, TYRO-SIT, POLLINEX Quattro Plus and Diater Depot) regarding their ability to induce IgG antibodies against ragweed pollen allergens in rabbits. Accordingly, the IgG reactivity of AIT-induced rabbit sera was tested for ten different ragweed pollen allergens (Amb a 1, 3, 4, 5, 6, 8, 9, 10, 11 and 12) by an ELISA. Furthermore, the ability of rabbit AIT-specific sera to block allergic patients' IgE binding to relevant ragweed allergens (Amb a 1, 4, 6, 8 and 11) and to inhibit allergen-induced basophil activation was evaluated by an IgE inhibition ELISA and a mediator release assay. Only two AIT vaccines (Diater Depot > CLUSTOID) induced relevant IgG antibody levels to the major ragweed allergen Amb a 1. The IgG responses induced by the AIT vaccines against the other ragweed allergens were low and highly heterogeneous. Interestingly, the kinetics of IgG responses were different among the AIT vaccines and even within one AIT vaccine (Diater Depot) for Amb a 1 (long-lasting) versus Amb a 8 and Amb a 11 (short-lived). This could be due to variations in allergen contents, the immunogenicity of the allergens, and different immunization protocols. The IgE inhibition experiments showed that rabbit AIT-specific sera containing high allergen-specific IgG levels were able to inhibit patients' IgE binding and prevent the mediator release with Diater Depot. The high levels of allergen-specific IgG levels were associated with their ability to prevent the recognition of allergens by patients' IgE and allergen-induced basophil activation, indicating that the measurement of allergen-induced IgG could be a useful surrogate marker for the immunological efficacy of vaccines. Accordingly, the results of our study may be helpful for the selection of personalized AIT vaccination strategies for ragweed-allergic patients.

Occurrence of antimicrobial residues in broiler chickens from selected farms and markets in Kano Metropolis, Nigeria

The indiscriminate use of antibiotics in animal meant for human and animal’s consumption lead to occurrence of antibiotic residues which threaten the health of both animals and humans. The aim of the study was to detect the presence of antimicrobial residues in broiler chicken in Kano state, Nigeria. Samples of fresh breast muscle, thigh muscle, liver and kidney were collected from 100 birds and examined using microbial inhibition test and ELISA test to determine the occurrence of antimicrobial residues and Enrofloxacin residues. A questionnaire was also administered to 33 workers and managers of the selected bird’s farms to assess their knowledge on the use of antibiotics and presence of antimicrobial residues. The result of the study revealed that 43% of the studied birds tested positive to antibiotic residue. Kidney samples recorded the highest (43%) residue present while thigh muscles (35%) recorded the least. The result indicated that among the birds detected with positive antibiotic residues, 36.4% were also positive for Enrofloxacin residue. All the respondents were using one or more antibiotic for treatment, prophylaxis or both and to a lesser extent as growth promoters. Majority (55.5%) of the respondents were not aware of the presence of residues in poultry. Most of the respondents (63.7%) have knowledge of withdrawal period but all of them were not observing withdrawal practice before disposing their birds. Doxycycline antibiotic was found to be the most indiscriminately use antibiotic, while erythromycin and chloramphenicol are the least. The study advocates the need for awareness campaigns for the farmers on the correct way of administering the antibiotics with special emphasis on withdrawal periods as well as continuous monitoring of the residues for public health reasons.

Duration of maternally derived antibodies of porcine parvovirus in growing pigs and presence of antibodies in gilts and sows vaccinated with three different parvovirus vaccines

While gilts and sows are regularly vaccinated against the porcine parvovirus (PPV), little is known on the presence of antibodies in vaccinated sows nor the decline of maternally derived antibodies (MDA) in their offspring. On twelve farms serum samples were taken from 180 gilts and sows vaccinated at least twice with one of three different commercial PPV vaccines. On nine farms, additional 270 serum samples were collected from growing pigs of three different age categories. All 450 samples were examined for PPV antibodies (Abs) by ELISA and haemagglutination inhibition (HI) assay. In total, 65% of all gilts vaccinated twice with either vaccine 1 or vaccine 3 were seronegative by HI assay. In each farm, there were at least three animals with high Ab titres (≥ 1:1280) indicating the presence of PPV in all twelve study farms. However, PPV DNA could not be detected in collected faecal samples. While low to moderately high Ab titres (1:10–1:640) were measured in 98% of twelve-weeks-old pigs, ELISA was only positive in 30% of the same pigs. Though, the statement on the duration of MDA may depend on the applied test, we could confirm an exponential decay of MDA. In addition, we could demonstrate that applied serological tools are insufficient for the confirmation of successful vaccination.

Assessment of the Adsorption Activity of Bacterial Cells Using Yersinia pseudotuberculosis Model

Currently, the Russian Anti-Plague Institute “Microbe” produces diagnostic immunoglobulins used in laboratory diagnostics of plague pathogen. One of the important stages in obtaining this category of drugs is the process of adsorption and removal of cross-reacting antibodies to increase the specificity of the drug. For this purpose, inactivated cells of Yersinia pseudotuberculosis strains are used.The aim of the work was to assess the possibility of using immunochemical methods to analyze the adsorption properties of bacterial cells, followed by an assessment of the impact of cultivation method and conditions, and the variant of the strain used on the adsorption properties of bacterial cells.Materials and methods. Cultivation was carried out on solid and liquid nutrient media; cells were inactivated by temperature and chemical exposure. Adsorption activity was assessed using immunoblotting and inhibitory enzyme-linked immunosorbent assay. Statistical processing of the results was performed using two-factor analysis of variance.Results and discussion. The suitability of immunoblotting and inhibitory ELISA methods for qualitative and quantitative assessment of the adsorption properties of bacterial cells has been demonstrated. It has been established that the adsorption properties of bacterial cells are influenced by the method of inactivation and the application of substrate feeding; the method of cultivation does not affect the adsorption properties of cells.

Early administration of canine parvovirus monoclonal antibody prevented mortality after experimental challenge.

To evaluate the effectiveness of canine parvovirus monoclonal antibody (CPMA) as a treatment against canine parvovirus (CPV-2)-induced mortality and to support USDA product licensure. 28 purpose-bred Beagle dogs aged 8 weeks were randomized to the treated (n = 21) or control (7) group. Dogs were challenged intranasally with 104.2 TCID50 virulent CPV-2b on Day 0 and monitored for 14 days for fecal viral shed and clinical disease. All dogs began shedding CPV-2 on Day 4 and were treated intravenously with a single dose of either CPMA (0.2 mL/kg) or saline (equal volume). No additional treatments were given to either group. Feces and sera were collected for quantitative analysis of fecal viral shed (hemagglutination) and antibody responses (hemagglutination inhibition and dot-blot ELISA), respectively. Dogs were monitored twice daily for parameters including lymphopenia, fever, vomiting, abnormal feces, inappetence, and lethargy. Humane endpoints triggered euthanasia by a veterinarian masked to treatment groups. The primary outcome variable was prevention of mortality as compared to controls. Mortality was prevented in all CPMA-treated dogs compared to 57% mortality in the control group (P = .0017, Fisher exact test). Canine parvovirus monoclonal antibody-treated dogs also experienced less severe and/or shorter durations of diarrhea, fever, vomiting, CPV-2 shedding in feces, and lymphopenia. Both groups showed similar immunoglobulin M responses as measured by semiquantitative analysis. Intravenous administration of CPMA can effectively improve clinical outcome when administered early in CPV-2 disease. Canine parvovirus monoclonal antibody treatment after proven infection does not interfere with adaptive immunity.

Exploring cell-free assays for COVID-19 serosurvey

Serosurveys to monitor immunity toward COVID-19 in the population are primarily performed using an ELISA to screen samples for SARS-CoV-2 antibodies, followed by confirmation by a virus neutralization test, which is considered the Gold Standard. However, virus neutralization test may not be feasible for some laboratories because of the requirement for specific facilities and trained personnel. In an attempt to address this limitation, we evaluated three cell-free methods as potential alternatives for assessing SARS-CoV-2 seroprevalence in human population from plasma. We report the establishment of two inhibition ELISAs designed to detect anti-Spike RBD IgG antibodies and a microsphere quantitative suspension array technology assay, based on the Luminex xMAP platform, to measure the presence of antibodies against various SARS-CoV-2 antigens, including anti-RBD. These methods were also compared to a commercial chemiluminescent immunoassay designed for anti-RBD antibodies detection and to the combined ELISA + virus neutralization test strategy. These cell-free assays performed equally to estimate the percentage of positive and negative samples and could be used to determine the prevalence of SARS-CoV-2 antibodies in human population, at least in cohort with high-expected prevalence, without the use of seroneutralization assay.

Neutralizing IgG4 antibodies are a biomarker of sustained efficacy after peanut oral immunotherapy

BackgroundClinical efficacy of oral immunotherapy (OIT) has been associated with the induction of blocking antibodies, particularly those capable of disrupting IgE-allergen interactions. Previously, we identified monoclonal antibodies (mAb) to Ara h 2 and structurally characterized their epitopes. ObjectiveWe investigated longitudinal changes during OIT in antibody binding to conformational epitopes and correlated the results with isotype and clinical efficacy. MethodsWe developed an indirect inhibitory ELISA using mAbs to block conformational epitopes on immobilized Ara h 2 from binding to serum immunoglobulins from peanut-allergic patients undergoing OIT. We tested the functional blocking ability of mAbs using passive cutaneous anaphylaxis (PCA) in mice with humanized FcεRI receptors. ResultsDiverse serum IgE recognition of Ara h 2 conformational epitopes are similar before and after OIT. Optimal inhibition of serum IgE occurs with the combination of two neutralizing mAbs (nAbs) recognizing epitopes 1.2 and 3, compared to two non-neutralizing mAbs (non-nAbs). Post-OIT, IgG4 nAbs, but not IgG1 or IgG2 nAbs, increased in sustained compared to transient outcomes. Induction of IgG4 nAbs occurs after OIT only in those with sustained efficacy. Murine PCA after sensitization with pooled human sera is significantly inhibited by nAbs compared to non-nAbs. ConclusionsSerum IgE conformational epitope diversity remains unchanged during OIT. However, IgG4 nAbs capable of uniquely disrupting IgE-allergen interactions to prevent effector cell activation are selectively induced in OIT-treated individuals with sustained clinical efficacy. Therefore, the induction of neutralizing IgG4 antibodies to Ara h 2 are clinically relevant biomarkers of durable efficacy in OIT.

Host genetics drives differences in cecal microbiota composition and immune traits of laying hens raised in the same environment

Vaccination is one of the most effective strategies for preventing infectious diseases but individual vaccine responses are highly heterogeneous. Host genetics and gut microbiota composition are 2 likely drivers of this heterogeneity. We studied 94 animals belonging to 4 lines of laying hens: a White Leghorn experimental line genetically selected for a high antibody response against the Newcastle Disease Virus (NDV) vaccine (ND3) and its unselected control line (CTR), and 2 commercial lines (White Leghorn [LEG] and Rhode Island Red [RIR]). Animals were reared in the same conditions from hatching to 42 d of age, and animals from different genetic lines were mixed. Animals were vaccinated at 22 d of age and their humoral vaccine response against NDV was assessed by hemagglutination inhibition assay and ELISA from blood samples collected at 15, 19, and 21 d after vaccination. The immune parameters studied were the 3 immunoglobulins subtypes A, M, and Y and the blood cell composition was assessed by flow cytometry. The composition of the cecal microbiota was assessed at the end of the experiment by analyzing amplified 16S rRNA gene sequences to obtain amplicon sequence variants (ASV). The 4 lines showed significantly different levels of NDV vaccine response at the 3 measured points, with, logically, a higher response of the genetically selected ND3 line, and intermediate and low responses for the unselected CTR control line and for the 2 commercial lines, respectively. The ND3 line displayed also a higher proportion of immunoglobulins (IgA, IgM, and IgY). The RIR line showed the most different blood cell composition. The 4 lines showed significantly different microbiota characteristics: composition, abundances at all taxonomic levels, and correlations between genera and vaccine response. The tested genetic lines differ for immune parameters and gut microbiota composition and functions. These phenotypic differences can be attributed to genetic differences between lines. Causal relationships between both types of parameters are discussed and will be investigated in further studies.

Defining the cross-reactivity between peanut allergens Ara h 2 and Ara h 6 using monoclonal antibodies.

In peanut allergy, Arachis hypogaea 2 (Ara h 2) and Arachis hypogaea 6 (Ara h 6) are two clinically relevant peanut allergens with known structural and sequence homology and demonstrated cross-reactivity. We have previously utilized X-ray crystallography and epitope binning to define the epitopes on Ara h 2. We aimed to quantitatively characterize the cross-reactivity between Ara h 2 and Ara h 6 on a molecular level using human monoclonal antibodies (mAbs) and structural characterization of allergenic epitopes. We utilized mAbs cloned from Ara h 2 positive single B cells isolated from peanut-allergic, oral immunotherapy-treated patients to quantitatively analyze cross-reactivity between recombinant Ara h 2 (rAra h 2) and Ara h 6 (rAra h 6) proteins using biolayer interferometry and indirect inhibitory ELISA. Molecular dynamics simulations assessed time-dependent motions and interactions in the antibody-antigen complexes. Three epitopes-conformational epitopes 1.1 and 3, and the sequential epitope KRELRNL/KRELMNL-are conserved between Ara h 2 and Ara h 6, while two more conformational and three sequential epitopes are not. Overall, mAb affinity was significantly lower to rAra h 6 than it was to rAra h 2. This difference in affinity was primarily due to increased dissociation of the antibodies from rAra h 6, a phenomenon explained by the higher conformational flexibility of the Ara h 6-antibody complexes in comparison to Ara h 2-antibody complexes. Our results further elucidate the cross-reactivity of peanut 2S albumins on a molecular level and support the clinical immunodominance of Ara h 2.

Generation of Autoantibodies in Metal-catalyzed Oxidatively Damaged DNA in Various Cancer Subjects.

Free radicals exist as unstable and highly reactive substances, occurring both in and outside the body. Free radicals are labeled as electron-hungry molecules formed from metabolism and endogenous burning of oxygen. They are transported in cells, upsetting the arrangement of molecules and instigating cellular injury. Hydroxyl radical (•OH) is one of the highly reactive free radicals, which damages the biomolecules in its close vicinity. In the present study, DNA was modified by the hydroxyl radical generated via the Fenton reaction. The •OH-oxidized/-modified DNA (Ox-DNA) was characterized by UV-visible and fluorescence spectroscopy. Thermal denaturation was performed to reveal the susceptibility of modified DNA toward heat. The role of Ox-DNA was also established in probing the presence of autoantibodies against Ox-DNA in the sera of cancer patients by direct binding ELISA. The specificity of autoantibodies was also checked by inhibition ELISA. In biophysical characterization, an increase in hyperchromicity and relative reduction of fluorescence intensity for Ox-DNA was reported compared to the native DNA analog. A thermal denaturation study revealed that Ox-DNA was highly susceptible to heat in comparison to the native conformers. The direct binding ELISA showed the prevalence of autoantibodies from cancer patient sera separated for immunoassay detection against the Ox-DNA. The generated autoantibodies against the Ox-DNA were detected as highly specific against bladder, head, neck, and lung cancer, which was further confirmed by the inhibition ELISA for the serum and IgG antibodies. The generated neoepitopes on DNA molecules are recognized as nonself by the immune system, which leads to the formation of autoantibodies in cancer patients. Therefore, our study confirmed that oxidative stress plays a role in the structural perturbation of DNA and makes it immunogenic.

Long-term immunity induced by SPBN GASGAS in orally vaccinated dogs is non-inferior to inactivated rabies vaccines

In a long-term immunogenicity study (1100 days post vaccination) in local Thai dogs the immune response of the oral rabies vaccine SPBN GASGAS was compared to those elicited by a commercial inactivated vaccine using immunobridging. Based on the detection of rabies virus binding (rVBA) and rabies virus neutralizing antibodies (rVNA) as measured by ELISA and Rapid Fluorescent Focus Inhibition Test (RFFIT) the long-term immune response in dogs vaccinated orally with the SPBNA GASGAS strain of rabies vaccine in a bait was non-inferior to a conventional inactivated rabies vaccine. The outcome of this study supports extending the originally claimed duration of immunity (DOI) of SPBN GASGAS after oral vaccination for dogs from 6 to 30 months.