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H. pylori (Hp) is highly causative agent of chronic gastritis, gastric cancer and human death worldwide. To address the challenge of H. pylori infection, numerous immunological assays have been developed for its diagnosis and management. However, the limited availability of these assays in certain laboratories, coupled with their high cost, inconsistent specificity, and sensitivity, has hampered their widespread adoption, particularly in developing countries where H. pylori infection is prevalent. Therefore, this study aimed to develop and validate a competitive enzyme-linked immunosorbent assay (cELISA) assay for detecting H. pylori infections by targeting the Protective Surface Antigen (PSA) and Cytotoxic-Associated Gene Pathogenesis Island (Cag11) proteins in H. pylori stool antigen sample. In the current study, the optimal conditions including the dilution of anti-rPSA D15 and anti-rCag11 antibodies at 1:1000, coating antigens (rPSA D15 and rCag11) at a concentration of 1 μg/well, the dilution of HRP-labelled antibody at 1:5000 and H. pylori stool antigen dilution at 1:1000 with a 1hour incubation and color development time of 30 minutes for cELISA were determined using an ELISA checkerboard titration assay. Based on the optimized conditions, novel rPSA D15-cELISA and rCag11-cELISA assays with a respective optimum cut-off value of 20.80% PI and 24.16% PI were developed. According to the receiver operating characteristic (ROC) curve analysis on the diagnostic performance of the newly developed rPSA D15-cELISA and rCag11-cELISA assays using 60 clinical H. pylori stool samples, the rPSA D15-cELISA test assay established an optimum cut-off point of 20.80% with sensitivity and specificity of 90% (95% confidence of interval (CI) 74.38-96.54), Area under the curve (AUC) of 0.9556 (95% CI = 0.896-1.000) and P value <0.0001. Similarly, the rCag11-cELISA assay revealed optimum cut-off value of 24.16% with sensitivity of 93.33% (95% CI 78.68-98.82), specificity of 90% (95% CI 74.38-96.54), AUC of 0.986 (95% CI = 0.967-1.000) and P <0.0001. Furthermore, the reproducibility assay coefficients of variation (CV) of the newly developed rPSA D15-cELISA and rCag11-cELISA assay were less than 10%, indicating that the two cELISA assays exhibits excellent reproducibility and reliability. To validate their clinical diagnostic application, the comparative study results of rPSA D15-cELISA and rCag11-cELISA showed a high agreement (k = 0.766 and 0.799) with the commercially available H. pylori antigen test immunochromatographic kit and more accurate than the reference kit by detecting stool antigen of H. pylori strain, indicating it is promising for clinical testing. In conclusion, these results indicated that the newly developed rPSA D15-cELISA and rCag11-cELISA H. pylori stool antigen test assays were a potential reliable and a clinically useful assay for rapid, specifically, sensitively and accurately diagnosis and large-scale epidemiological investigation of H. pylori infection., Competing Interests: The authors declare that they have no conflict of interest., (Copyright: © 2025 Eskeziyaw et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pyrogens cause shock symptoms when released into the bloodstream. They are classified into two main categories: endotoxins (lipopolysaccharides [LPS]) and non-endotoxin pyrogens. The monocyte activation test (MAT) is an in vitro assay to detect pyrogens in human monocytes. Cells were incubated in the culture medium, and the cellular response, specifically the production of the inflammatory cytokine interleukin-6 in the culture supernatant, was analyzed using enzyme-linked immunosorbent assay (ELISA). Technical improvements, such as cell acquisition and culture media selection, will be beneficial for the popularization of MAT. The cell freshness was strictly controlled to achieve high MAT sensitivity. However, it is necessary to investigate the usability of older and stored blood samples in the MAT. This study evaluated the effect of cell freshness on MAT using peripheral blood mononuclear cells (PBMCs) isolated from 2- and 5-d-old donated whole blood samples. To mitigate the influence of serum in the culture medium, a serum-free MAT was developed using the LPS-binding protein (LBP) as an enhancer for LPS detection. PBMCs were incubated with a two-fold dilution series of LPS at 0.001-4.096 endotoxin units/mL (EU/mL). Interleukin-6 levels in the culture supernatant were quantified by ELISA in the presence and absence of LBP. In the presence of LBP, the limit of detection (LOD) for LPS was 0.001-0.008 EU/mL. However, in the absence of LBP, the LOD was 0.512 EU/mL. Peripheral PBMCs were 38.6 times more sensitive in the presence of LBP than in its absence. When utilizing the developed serum-free MAT with LBP, 5-d-old PBMCs showed LODs of 0.016-0.064 EU/mL, indicating a 3.1-fold increase in sensitivity compared with 5- to 2-d-old PBMCs. These results suggest that the sensitivity of PBMCs decreased gradually rather than sharply. The study concluded that 2-d-old PBMCs were sufficiently fresh and could be used as serum-free MAT., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hayashi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Diagnosis of equine Lyme borreliosis (LB), an infection caused by members of the Borrelia burgdorferi sensu lato complex (Bbsl), is challenging due to the nonspecific clinical signs of the disease and due to the variety of non-standardized serological tests. Specific vaccine-induced antibodies against LB, providing an effective protection against the infection, complicate the issue further. The standard for the detection of specific antibodies against Bbsl is a two-tier test system based on an enzyme-linked immunosorbent assay (ELISA) or indirect fluorescent antibody test (IFA) for antibody screening combined with a qualitative, highly specific immunoassay (e. g. line immunoassay (LIA)) for confirmation. In this study, three LIAs available for detection of antibodies in equine serum samples were evaluated and compared. A total of 393 serum samples of 131 horses with known serostatus were used. It included groups of non-vaccinated horses, immunized horses (vaccinations against LB on days 0 and 14), and horses that had received an initial immunization plus an additional booster on day 180. Sera were collected on days 0, 135 and 210 of the study. Results were compared considering the tests' sensitivity, specificity, diagnostic outcome, and the operability of each test. Agreements of the diagnostic results among the LIAs were calculated for overall test results and single antigen-antibody-complex signal results. They are presented as inter-rater agreement and statistic reliability, represented by the Fleiss' kappa coefficient. Agreement scores ranged from poor to moderate depending on group and time-point of blood sample collection. Depending on LIA used, deficiencies were observed in the form of non-sufficient sensitivity of antigen signals on the LIA strips (especially for outer surface protein A (OspA) or variable major protein like sequence expressed (VlsE)) or as an inappropriate test interpretation of the OspA signal. Operability of the three LIAs was equally user-friendly with minor variations. In two LIAs, test-evaluation was simplified by a supplied scanner and evaluation software. To improve functionality of available LIAs for equine serum samples it is advisable to adjust sensitivity and specificity of single test antigen signals and establish appropriate evaluation protocols., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Broeckl et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Since the onset of the global COVID-19 pandemic in early 2020, numerous studies have been conducted worldwide to understand our immune response to the virus and to vaccination. This study investigates the humoral response elicited by SARS-CoV-2 infection and by vaccination in the poorly studied population of food and retail workers. These occupations were classified as essential by the Public Health Agency of Canada, potentially placing this population at greater risk of infection. Such a risk requires access to reliable and adaptable serological assays that can be rapidly deployed to guide public health strategies. Here we investigate the benefits and limitations of applying adaptable, decentralized tests for population-level immune surveillance in response to a pandemic, even before centralized testing is available., Methods and Findings: The 1.5-year study period spans from early 2021, when vaccination became available in this region, to mid-2022, following the emergence of the first Omicron variants. The cohort of 304 food and retail workers was recruited in the Québec City area. Participants attended five evenly spaced visits, providing blood samples as well as information on SARS-CoV-2 symptoms or risk factors, prior antigen or PCR test results and vaccination status, as well as work-related risk factors and protective measures. Parallel COVID-19 serological assays were performed using both a standardized chemiluminescent ELISA assay at the centralized platform operated in partnership with the Public Health Agency of Canada, and a semi-automated in-house colorimetric ELISA assay developed at our decentralized site. The YES/NO determination of SARS-CoV-2 vaccine seroconversion and/or infection events using the SARS-CoV-2 ancestral spike protein and nucleocapsid protein validated coherence of the centralized and decentralized assays. The flexibility of the decentralized assays allowed broadening the study to determine cross-reactivity of IgG directed against the spike protein of the SARS-CoV-2 Delta and Omicron VOCs, and IgM directed against the ancestral spike and nucleocapsid proteins. The nature of the data obtained in the decentralized assays allowed treatment with a recently developed mathematical transformation to obtain normal distribution, enabling ANOVA-Welsh statistical analysis. Although no significant differences were observed in humoral response as related to BMI, age, level of education, or chronic illnesses in this cohort of workers, statistically higher levels of vaccine-induced antibodies were observed for restaurant workers and hardware store workers in the early stages of the study, compared to workers in bars and grocery stores and in non-smokers versus smokers., Conclusions: This work highlights the importance of developing adaptable, decentralized tests for population-level immune surveillance in response to a pandemic, even before centralized testing is available. To our knowledge, no other study has reported such an extensive longitudinal investigation during key periods of the COVID-19 pandemic in a cohort of food and retail workers to analyze two types of immunoglobulin, three epitopes and antigens to three VOC. This study will inform strategies and measures to be implemented in the event of a future pandemic., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Djaïleb et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Detection of Strogyloides-specific IgG antibodies in urine and serum has been used in diagnostic and epidemiological studies on strongyloidiasis. However, the usefulness of these assays in assessing responses to anthelmintic treatment is unclear. Thus, we evaluated the diagnostic performance and temporal profiles of Strongyloides-specific IgG antibodies in a cohort of participants at baseline and post-treatment. The participants were prospectively screened for baseline parasitic infections by fecal examination [agar plate culture technique (APCT) and formalin-ethyl acetate concentration technique (FECT)] and digital droplet polymerase reaction (ddPCR) for Strongyloides stercoralis. At each sampling point, Strongyloides-specific IgG in urine and serum were measured by an in-house S. ratti-based enzyme-linked immunosorbent assay (ELISA). At baseline, 169 of 351 participants (48.1%) had S. stercoralis infection by the combined fecal examination and ddPCR. The diagnostic sensitivities of IgG in urine and serum were 91.1% and 88.2%, respectively. The participants were given treatment with a single oral dose of ivermectin (IVM, 200 μg/kg) and were followed up by fecal and immunological diagnosis at 3 to 18 months post-treatment. The cure rate of IVM treatment evaluated by APCT and ddPCR was 88.3% at three months post-treatment. The profiles of IgG in urine in the curative treatment group showed a significant trend of decline with time post-treatment (Kruskal-Wallis test = 113.4-212.6, p value < 0.0001) and the lowest levels were seen 12 months post-treatment. The treatment response (> 50% reduction in urinary IgG antibody units) was 100%, and conversion from positive to negative results was 65.4%. The treatment response and conversion to negative assessed by serum IgG-ELISA were similar to those by urine IgG-ELISA. The results from this long-term diagnostic study highlight the utility of urinary IgG and serum IgG for screening and monitoring treatment outcomes in strongyloidiasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wongphutorn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Evaluation of the effectiveness of vaccination of animals against rabies is not routinely implemented. In cases where it is carried out, the rapid fluorescent focus inhibition test (RFFIT) or the fluorescent antibody virus neutralization (FAVN) test are the recommended tests. However, both of these tests require handling of live rabies virus (RABV), and are cumbersome to perform. In view of this, the enzyme-linked immunosorbent assay (ELISA) has been proposed as a surrogate test; however, availability of appropriate antigen is a major impediment for the development of ELISAs to detect anti-rabies antibodies. The most widely used antigen is the RABV glycoprotein (G) purified from cell culture-propagated virus, which requires a biosafety level 3 containment. The alternative is to use recombinantly expressed G, which needs to be to be properly glycosylated and folded to serve as the best antigen. The most suitable system for its production is the baculovirus expression system (BVES). However, purification of RABV G is challenging. We therefore tested partially purified preparations in the form of extracts of insect cells infected with baculovirus expressing RABV G, against sera from vaccinated dogs in an indirect ELISA. The results showed good concordance against RFFIT, with sensitivity and specificity of 90.48% and 80.00%, respectively. The system may be used for quick screening to determine the presence and an approximate level of antibodies, and can be modified to enable monitoring of mass dog vaccination programs, as well as to facilitate certification of dogs intended for international travel and transportation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Santosh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Viral Load (VL) monitoring is a crucial component of patient care during antiretroviral therapy (ART) but is not routinely available in many resource-constrained settings, where millions of patients will require care for decades to come. We hypothesise a serologic 'recent infection' test (Sedia LAg assay) which has a high dynamic range for detecting antigen-driven antibody response can provide informative proxies for VL trajectories., Methods: A retrospective study where we analysed data linked via specimens in a well-described repository for recent infection test benchmarking (CEPHIA collaboration). Patient panels were comprised of 1) observations straddling ART start; 2) observations from a period of stable viral suppression; 3) observations straddling rebound after a period of viral suppression. We analysed an individual's Sedia LAg ELISA normalised optical density (ODn) trends within these categories. Using groups 2) and 3) we evaluated the specificity and sensitivity of a proposed proxy for "the latest observation is at a time of VL rebound"; proxy was defined as follows: we estimated patient-specific mean-previous-ODn for all observations with at least two preceding virally suppressed observations. We considered various thresholds to define both "VL suppression" and "ODn uptick"., Results: In regression analysis by category: 1) ODn gradients are statistically significantly negative just after ART-start (p = 0.010); 2) During periods of stable viral suppression, ODn tended to decline, but not statistically significantly, for a range of clinically meaningful "VL suppression" thresholds; 3) comparing ODn values just before, versus at, "VL rebound", ODn changes were statistically significantly increasing at rebound (p = 0.001). In the analysis comparing groups 2) and 3), at a Z score threshold of 0.8, the proposed proxy for a first viral rebound had an observed specificity and sensitivity both close to 90%., Conclusion: The high dynamic range of serological tests previously investigated for defining 'recent infection' has potential, as demonstrated using the Sedia LAg ELISA, to provide meaningful information about the success of ART, during treatment initiation, at times of stable suppression, and to flag possible viral rebound. It should be investigated how this can be combined with patient management workflows and (clinical and) other data, to provide efficiencies in long-term monitoring viral control in resource-limited settings., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sempa, Welte. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Valencia-Portillo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Subclinical infection of laboratory animals with one or more of several pathogens affects the results of experiments on animals. Monitoring the health of laboratory animals encompasses routine surveillance for pathogens, including several viruses. This study aimed to explore the development of an alternative assay to the existing ones for detecting infection of mice and rats with the parvoviruses minute virus of mice (MVM) and Kilham rat virus (KRV), respectively. Full-length VP2 and NS1 proteins of these parvoviruses, besides fragments containing multiple predicted epitopes stitched together, were studied for serological detection. The optimal dilution of full-length proteins and antigenic regions containing predicted epitopes for coating, test sera, and conjugate was determined using a checkerboard titration at each step. The assays were evaluated vis-à-vis commercially available ELISA kits. The results showed that an engineered fusion of fragments containing multiple predicted MVM VP2 and NS1 epitopes was better than either of the full-length proteins for detecting antibodies in 90% of the tested sera samples. For KRV ELISA, full-length VP2 was better compared to other individual recombinant protein fragments or combinations thereof for the detection of antibodies in sera. This report is the first description of an ELISA for KRV and an improved assay for MVM. Importantly, our assays could be exploited with small volumes of sera. The results also demonstrate the utility of immunoinformatics-driven polypeptide engineering in the development of diagnostic assays and the potential to develop better tests for monitoring the health status of laboratory animals., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kaur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Detection of infections in wildlife species is increasingly important to reduce the risk of spreading zoonotic and economically important parasites, understand disease epidemiology and promote the conservation of wildlife species. Serological tests are key in disease diagnosis and surveillance by detecting immunoglobulins against infectious agents. However, the need for species-specific reagents has limited the application of serological tests in wildlife species. This study evaluated the serum immunoglobulin-binding capability of polyclonal anti-kangaroo antibody and two non-species-specific reagents, including protein A/G and protein L, with the largest range of Australian marsupial species so far, including 32 species representing three major marsupial orders. Immunoglobulin-binding capability was assessed using immunoblotting, enzyme-linked immunosorbent assay and Western blot techniques. Variation in immunoglobulin-binding capability was observed between the three reagents and across the species tested, both across but also within taxonomic groups. Taxonomic distance was thus not always a good predictor of immunoglobulin-binding affinity, emphasizing the need to validate these reagents for each species separately. However, all three reagents bound with the serum immunoglobulins of most marsupial species tested. The findings of this study provide a valuable reference for species differences in affinity to protein A/G, protein L and anti-kangaroo antibody, assisting in the selection of appropriate reagents and the development of sero-immunological assays in Australian marsupials., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Liyanage et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Serological assays for bovine tuberculosis diagnosis require the use of multiple Mycobacterium bovis specific antigens to ensure the detection of infected animals. In the present study, identification and selection process of antigens, based on data from published proteomic studies and involving the use of bioinformatics tools and an immuno-screening step, was firstly performed for identifying novel antigens that elicit an antibody response in M. bovis infection. Based on this approach, a panel of 10 M. bovis antigens [with known relevance (MPB70, MPB83, MPB70/83, and ESAT6/CFP10) and novel (Mb1961c, Mb1301c, Mb3871, Mb1403, Mb0592, and PE25/PPE41)] were constructed and thenused to develop a new multiplexed serological assay based on Luminex technology. The performance of the Luminex-bTB immunoassay was evaluated using sera from cattle with known tuberculosis status. Among the proteins whose ability to detect bovine tuberculosis was evaluated for the first time, PE25/PPE41 and Mb1403, but not Mb3871, showed good detection capacity. Following multiple antigen combination, the final Luminex-bTB immunoassay included seven antigens (MPB70, MPB83, MPB70/83, ESAT6/CFP10, PE25/PPE41, Mb1403, and Mb0592) and showed better global performance than the immunoassay using the four usual antigens (MPB70, MPB70/83, MPB83 and ESAT6/CFP10). The specificity and sensitivity values were, respectively, of 97.6% and 42.8% when the cut-off of two-positive antigens was used to classify samples as positive. With the use of the more-restrictive criterion of three-positive antigens, the specificity increased to 99.2% but the sensitivity decreased to 23.9%. The analysis of antigen profiles generated with the Luminex-bTB immunoassay showed that mainly serodominant proteins were recognized in samples from infected cattle. The detection of Mb1961c and Mb1301c appeared to be associated with presumed false-positive results. Moreover, sera from cattle originating from bTB-outbreaks but having inconclusive or negative skin test results were identified as positive by the Luminex-bTB immunoassay and showed an antigen pattern associated with M. bovis infection. The Luminex-bTB immunoassay including seven antigens may be useful as adjunct test for the detection of M. bovis-infected herds, and different cut-offs could be applied according to the bovine tuberculosis epidemiological context., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Moens et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). RT-PCR detection of viral RNA represents the gold standard method for diagnosis of COVID-19. However, multiple diagnostic tests are needed for acute disease diagnosis and assessing immunity during the COVID-19 outbreak. Here, we developed in-house anti-RBD IgG and IgA enzyme-linked immunosorbent assays (ELISAs) using a well-defined serum sample panel for screening and identification of human SARS-CoV-2 infection. We found that our in-house anti-SARS-CoV-2 IgG ELISA displayed a 93.5% sensitivity and 98.8% specificity whereas our in-house anti-SARS-CoV-2 IgA ELISA provided assay sensitivity and specificity at 89.5% and 99.4%, respectively. The agreement kappa values of our in-house anti-SARS-CoV-2 IgG and IgA ELISA assays were deemed to be excellent and fair, respectively, when compared to RT-PCR and excellent for both assays when compared to Euroimmun anti-SARS-CoV-2 IgG and IgA ELISAs. These data indicate that our in-house anti-SARS-CoV-2 IgG and IgA ELISAs are compatible performing assays for the detection of SARS-CoV-2 infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: RTS,S/AS01 induced anti-circumsporozoite protein (CSP) IgG antibodies are associated with the vaccine efficacy. There is currently no international standardisation of the assays used in the measurement of anti-CSP IgG antibody concentrations for use in evaluations of the vaccine's immunogenicity and/or efficacy. Here, we compared the levels of RTS,S/AS01 induced anti-CSP IgG antibodies measured using three different enzyme-Linked ImmunoSorbent Assays (ELISA)., Methods: 196 plasma samples were randomly selected from the 447 samples collected during the RTS,S/AS01 phase IIb trial in 2007 from Kenyan children aged between 5-17 months. The vaccine-induced anti-CSP IgG antibodies were then measured using two independently developed ELISA protocols ('Kilifi-RTS,S' and 'Oxford-R21') and compared to the results from the reference 'Ghent-RTS,S' protocol for the same participants. For each pair of protocols, a deming regression model was fitted. Linear equations were then derived to aid in conversions into equivalent ELISA units. The agreement was assessed using Bland and Altman method., Findings: The anti-CSP IgG antibodies measured from the three ELISA protocols were in agreement, and were positively and linearly correlated; 'Oxford' and 'Kilifi' r = 0.93 (95% CI 0.91-0.95), 'Oxford' and 'Ghent' r = 0.94 (95% CI: 0.92-0.96), and 'Kilifi' and 'Ghent' r = 0.97 (95% CI: 0.96-0.98), p<0.0001 for all correlations., Conclusions: With the linearity, agreement and correlations established between the assays, conversion equations can be applied to convert results into equivalent units, enabling comparisons of immunogenicities across different vaccines of the same CSP antigens. This study highlights the need for the international harmonisation of anti-CSP antibody measurements., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Mugo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mycoplasma bovis (Mbovis) was first detected in cattle in New Zealand (NZ) in July 2017. To prevent further spread, NZ launched a world-first National Eradication Programme in May 2018. Existing diagnostic tests for Mbovis have been applied in countries where Mbovis is endemic, for detecting infection following outbreaks of clinical disease. Diagnostic test evaluation (DTE) under NZ conditions was thus required to inform the Programme. We used Bayesian Latent Class Analysis on paired serum ELISA (ID Screen Mycoplasma bovis Indirect from IDvet) and tonsillar swabs (qPCR) for DTE in the absence of a gold standard. Tested samples were collected at slaughter between June 2018 and November 2019, from infected herds depopulated by the Programme. A first set of models evaluated the detection of active infection, i.e. the presence of Mbovis in the host. At a modified serology positivity threshold of SP%> = 90, estimates of animal-level ELISA sensitivity was 72.8% (95% credible interval 68.5%-77.4%), respectively 97.7% (95% credible interval 97.3%-98.1%) for specificity, while the qPCR sensitivity was 45.2% (95% credible interval 41.0%-49.8%), respectively 99.6% (95% credible interval 99.4%-99.8%) for specificity. In a second set of models, prior information about ELISA specificity was obtained from the National Beef Cattle Surveillance Programme, a population theoretically free-or very low prevalence-of Mbovis. These analyses aimed to evaluate the accuracy of the ELISA test targeting prior exposure to Mbovis, rather than active infection. The specificity of the ELISA for detecting exposure to Mbovis was 99.9% (95% credible interval 99.7%-100.0%), hence near perfect at the threshold SP%=90. This specificity estimate, considerably higher than in the first set of models, was equivalent to the manufacturer's estimate. The corresponding ELISA sensitivity estimate was 66.0% (95% credible interval 62.7%-70.7%). These results confirm that the IDvet ELISA test is an appropriate tool for determining exposure and infection status of herds, both to delimit and confirm the absence of Mbovis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Marquetoux et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

ELISA or Western blot is known as a basic technique to be used for measurement of intracellular proteins, but in some cases, they cannot overcome problems such as normalization between samples or extraneous costs for required commercial kits. In order to address this problem, we developed a rapid and effective method (a hybrid of Western blot and ELISA). We use this new hybrid method to detect and normalize trace protein changes in gene expression intracellularly at a lower cost., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Edabashi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: For the proper diagnosis of toxoplasmosis it is essential to determine the stage of the infection, for which the most preferred method is IgG avidity test. The avidity index (AI) should initially be low (AI≤0.3) in the acute phase and increase during the infection. However, persistent low avidity can occur in patients with latent toxoplasmosis, which can complicate the interpretation of the results. The aim of the study is to explain the causes of this phenomenon., Methodology: A retrospective study was carried out with 717 serum samples collected from 442 patients from the categories of pregnant and non-pregnant women, men, and newborns + infants (age < 0.5 year). The trends of AI kinetics were evaluated in repeatedly examined patients. The frequency of cases with low avidity was compared in individual categories of patients and in groups of people with acute and non-acute toxoplasmosis., Results: The proportion of patients with initially low avidity was 42.1% in the acute toxoplasmosis group while it was 13.0% in the non-acute groups. In uninfected newborns with anti-Toxoplasma antibodies transmitted from the mother, a decrease in IgG avidity levels over time was observed, resulting in 29.2% of samples showing low (improper) avidity. While the dynamics of IgG avidity and the frequency of cases of improperly low avidity were similar in men and pregnant and non-pregnant women, the category of newborns and infants differed substantially for these indicators., Conclusions: Due to acceptable specificity and negative predictive value, high avidity can rule out acute toxoplasmosis, but moderate sensitivity complicates the possibility of its confirmation. The results of the avidity test must be interpreted in the context of the results of other methods., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kodym et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Detection of anti-Strongyloides IgG in urine by enzyme-linked immunosorbent assay (ELISA) for diagnosis of strongyloidiasis reportedly has comparable performance to conventional serum assays. Initial comparisons of urine assays using commercial ELISA kits designated for serology have shown its diagnostic potential but sub-optimal accuracy. In the present study, we optimized urine ELISA protocols based on different antigen types and evaluated their accuracies in determining the epidemiology of strongyloidiasis in Northeast Thailand. Paired urine and fecal samples of 966 individuals from the study community were collected for three consecutive days and tested for strongyloidiasis. We compared three ELISA protocols using different antigens including crude S. stercoralis antigen (Ss-ELISA), crude S. ratti antigen (Sr-ELISA) and recombinant NIE antigen (NIE-ELISA) and fecal examination by agar plate-culture (APCT) technique and formalin-ethyl acetate concentration technique (FECT). The optimized ELISA protocols using three different antigen sources yielded significantly higher prevalence rates of strongyloidiasis (58.9-65.1%) than those by fecal examination methods (19.7%). The prevalence of strongyloidiasis determined by ELISA protocols significantly increased with age (p value < 0.0001) and males had higher prevalence than females (p value < 0.0001). Diagnostic agreements between ELISA protocols were moderate (κ = 0.461-0.586) and the agreement between each ELISA protocol and fecal examinations were slight (κ = 0.139-0.210). The results obtained by urine ELISA protocols using three different antigens showed comparable diagnostic performances, provided further supports for the utility of urine as an alternative clinical specimen for diagnosis of strongyloidiasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Eamudomkarn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The main objective of this study was to estimate the performance, under local epidemiological conditions, of two in-house ELISA assays for the combined detection of anti-SARS-CoV-2 IgA, IgM, and IgG immunoglobulins. A total of 94 serum samples were used for the assessment, where 44 corresponded to sera collected before the pandemic (free of SARS-CoV-2 antibodies), and 50 sera were collected from confirmed COVID-19 patients admitted to the main public hospital in the city of Valdivia, southern Chile. The Nucleocapsid (Np) and the receptor-binding domain (RBD) proteins were separately used as antigens (Np and RBD ELISA, respectively) to assess their diagnostic performance. A receiver operating characteristic (ROC) analysis was performed to estimate the optical density (OD) cut-off that maximized the sensitivity (Se) and specificity (Sp) of the ELISA assays. Np ELISA had a mean Se of 94% (95% CI = 83.5-98.8%) and a mean Sp of 100% (95% CI = 92.0-100%), with an OD 450 nm positive cut-off value of 0.88. On the other hand, RBD ELISA presented a mean Se of 96% (95% CI = 86.3-99.5%) and a mean Sp of 90% (95% CI = 78.3-97.5%), with an OD 450 nm positive cut off value of 0.996. Non-significant differences were observed between the Se distributions of Np and RBD ELISAs, but the latter presented a significant lower Sp than Np ELISA. In parallel, collected sera were also analyzed using a commercial lateral flow chromatographic immunoassay (LFCI), to compare the performance of the in-house ELISA assays against a commercial test. The LFCI had a mean sensitivity of 94% (95% CI = 87.4-100%) and a mean specificity of 100% (95% CI = 100-100%). When compared to Np ELISA, non-significant differences were observed on the performance distributions. Conversely, RBD ELISA had a significant lower Sp than the LFCI. Although, Np ELISA presented a similar performance to the commercial test, this was 2.5 times cheaper than the LFCI assay (labor cost not considered). Thus, the in-house Np ELISA could be a suitable alternative tool, in resource limited environments, for the surveillance of SARS-CoV-2 infection, supporting further epidemiological studies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Ramírez-Reveco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fasciolosis is a severe zoonosis responsible for major economic losses in livestock. The enhanced MM3-COPRO test (eMM3-COPRO) and the commercial version BIO K 201 (Bio-X Diagnostics, Rochefort, Belgium) are widely used as immunodiagnostic tools for the specific detection of coproantigens released by Fasciola during the late prepatent and patent stages of infection. However, performance of the eMM3-COPRO has never been evaluated under field conditions. To address this gap, a large number of ovine faecal samples, collected in a region where fasciolosis is endemic (Galicia, NW Spain), were analyzed. Two groups of sheep flocks were selected according to the Fasciola infection status: 'Fasciola-free' and 'Fasciola-infected' flocks. 'Fasciola-free' flocks were seronegative flocks with no history of fasciolosis detected by either coproscopy or necropsy in the last 5 years. Faecal samples from these sheep were used to calculate a cut-off value for infection (OD = 0.021). The cut-off was calculated using a bootstrap resampling method that enables estimation of the sampling distribution of the statistical parameters without making assumptions about the underlying data distribution. 'Fasciola-infected' flocks were characterized by high seroprevalence, a history of fasciolosis and periodical treatment with flukicides. Samples from these flocks were used to estimate the diagnostic accuracy of the eMM3-COPRO relative to coproscopy, which although limited by poor sensitivity is the only reference test available for diagnosing fasciolosis in vivo. To overcome this limitation, all animals classified positive by eMM3-COPRO were treated with triclabendazole and then retested. The eMM3-COPRO displayed higher sensitivity than coproscopy, as it detected coproantigens in all samples with positive coproscopy and in 12% of samples with negative coproscopy. The test also proved highly specific as coproantigens disappeared after the treatment. The eMM3-COPRO was less time consuming than coproscopy, particularly when the procedure involved numerous samples, and showed promise as a tool for monitoring flukicide efficacy., Competing Interests: The authors have declared that no competing interests exist.

Trichinellosis is a zoonotic disease caused by the ingestion of the Trichinella nematode. With a worldwide incidence of approximately 10,000 cases per year, Trichinella spiralis is responsible for most human infections. There are no specific signs or symptoms of this parasitic infection. Muscle biopsy is the gold diagnostic standard for trichinellosis, but the technique is invasive and unable to detect the early stage of infection. Although immunodiagnostics are also available, antibody detection usually occurs after 3 weeks and prolonged up to 19 years after the acute phase. Therefore, additional diagnostic biomarkers must be identified to improve trichinellosis diagnosis. This study aimed to measure concentration changes in mouse serum proteins prior to T. spiralis infection and 2, 4 and 8 weeks after infection, and to identify T. spiralis circulating proteins and antigens using mass spectrometry-based proteomics. Mouse muscle-related proteins including inter-alpha-trypsin inhibitor heavy chain H2, a protein involved in the response to muscle tissue damage, were up-regulated in mouse sera during the T. spiralis larvae invasion. Additionally, 33 circulatory parasite proteins were identified in infected mouse sera. Notably, T. spiralis long-chain fatty acid transport protein 1 could be detected in the early stage of infection and peroxidasin-like protein was identified 2, 4 and 8 weeks after infection. Seventeen T. spiralis circulating antigens were detected in mouse immune complexes, with PX domain protein being found 2, 4 and 8 weeks after infection. Because peroxidasin-like protein and PX domain protein were detected at all post-infection time points, sequence alignments of these proteins were performed, which showed they are conserved among Trichinella spp. and have less similarity to the human and murine sequences. Integrative analysis of T. spiralis biomarkers throughout the course of infection may reveal additional diagnostic targets to improve early diagnosis of trichinellosis., Competing Interests: The authors have declared that no competing interests exist.

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH., Competing Interests: The authors have declared that no competing interests exist.

Background: Early and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to prevent spread of the infection. Understanding of the antibody response to SARS-CoV-2 in patients with coronavirus disease 2019 (COVID-19) is insufficient, particularly in relation to those whose responses persist for more than 1 month after the onset of symptoms. We conducted a SARS-CoV-2 antibody test to identify factors affecting the serological response and to evaluate its diagnostic utility in patients with COVID-19., Methods and Finding: We collected 1,048 residual serum samples from 396 patients with COVID-19 confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2. The samples had been used for routine admission tests in six healthcare institutions in Daegu. Antibody to SARS-CoV-2 was analyzed and the cutoff index (COI) was calculated for quantitative analysis. The patients' information was reviewed to evaluate the relationship between antibody positivity and clinical characteristics. The anti-SARS-CoV-2 antibody positivity rate was 85% and the average COI was 24·3. The positivity rate and COI increased with time elapsed since symptom onset. Anti-SARS-CoV-2 antibody persisted for at least 13 weeks after symptom onset at a high COI. There was a significant difference in anti-SARS-CoV-2 antibody positivity rate between patients with and without symptoms, but not according to sex or disease course. The descending COI pattern at weeks 1 to 5 after symptom onset was significantly more frequent in patients who died than in those who recovered., Conclusions: Anti-SARS-CoV-2 antibody persisted for at least 13 weeks at a high COI in patients with COVID-19. A decreasing COI pattern up to fifth week may be associated with a poor prognosis of COVID-19. As new treatments and vaccines are introduced, it is important to monitor continuously the usefulness of anti-SARS-CoV-2 antibody assays., Competing Interests: The authors have declared that no competing interests exist.

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) of B. anthracis are mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen of B. anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Using in silico analyses, we screened coding sequences encoded on pXO2 plasmid, which is absent in the veterinary vaccine strain Sterne 34F2 but present in virulent strains of B. anthracis. Among the 8 selected antigen candidates, capsule biosynthesis protein CapA (GBAA&#95;RS28240) and peptide ABC transporter substrate-binding protein (GBAA&#95;RS28340) were detected by antibodies in infected horse sera. Of these, CapA has not yet been identified as immunoreactive in other studies to the best of our knowledge. Considering the protein solubility and specificity of B. anthracis, we prepared the C-terminus region of CapA, named CapA322, and developed CapA322-ELISA based on a horse model. Comparative analysis of the CapA322-ELISA and PAD1-ELISA (ELISA uses domain one of the PA) showed that CapA322-ELISA could detect anti-CapA antibodies in sera from infected horses but was non-reactive to sera from vaccinated horses. The CapA322-ELISA could contribute to the anthrax surveillance in endemic areas, and two immunoreactive proteins identified in this study could be additives to the improvement of current or future vaccine development., Competing Interests: The authors have declared that no competing interests exist.

Objectives: A few studies on antibody testing have focused on asymptomatic or mild coronavirus disease 2019 (COVID-19) patients with low initial anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody responses. Anti-SARS-CoV-2 antibody-testing performance was evaluated using blood samples from asymptomatic or mild COVID-19 patients., Methods: Blood samples were collected from 143 COVID-19 patients during an outbreak on a cruise ship 3 weeks after diagnosis. Simultaneously, a follow-up SARS-CoV-2 genetic test was performed. Samples stored before the COVID-19 pandemic were also used to evaluate the lateral flow immunochromatographic assay (LFA) and electrochemiluminescence immunoassay (ECLIA). Titers of anti-SARS-CoV-2 IgM and IgG antibodies against the nucleocapsid and spike proteins were measured using the enzyme-linked immunosorbent assay to confirm which antibodies were influenced on LFA- and ECLIA- false-negative result in crew-member samples., Results: Sensitivity, specificity, positive-predictive, and negative-predictive values of LFA-detected IgM antibodies were 0.231, 1.000, 1.000, and 0.613, respectively; those of LFA-detected IgG antibodies were 0.483, 0.989, 0.972, and 0.601, respectively; and those of ECLIA-detected total antibodies were 0.783, 1.000, 1.000, and 0.848, respectively. All antibody titers measured using ELISA were significantly lower in blood samples with negative results than in those with positive results in both LFA and ECLIA. In the patients with negative results from the follow-up genetic testing, IgM-, IgG-, and total-antibody positivity rates were 22.9%, 47.6%, and 72.4%, respectively., Conclusions: These findings suggest that anti-SARS-CoV-2 antibody testing has lower performance in asymptomatic or mild COVID-19 patients than required in the guidelines., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: We used marketed products of Cellspect Co., Ltd for ELISA in this study; Hironori Sakai is an employee of Cellspect Co., Ltd., and performed ELISA in this study; Norihito Kaku and Kosuke Kosai received research funding from Roche Diagnostics K. K.; Katsunori Yanagihara received research funding from Cellspect Co., Ltd., Roche Diagnostics K. K. and, Abbott Japan LLC. These do not alter our adherence to PLOS ONE policies on sharing data and materials.

Celiac disease (CD) is triggered by ingestion of gluten-containing cereals such as wheat, barley, rye and in some cases oat. The only way for affected individuals to avoid symptoms of this condition is to adopt a gluten-free diet. Thus, gluten-free foodstuffs need to be monitored in order to ensure their innocuity. For this purpose, commercial immunoassays based on recognition of defined linear gluten sequences are currently used. These immunoassays are designed to detect or quantify total gluten regardless of the cereal, and often result in over or underestimation of the exact gluten content. In addition, Canadian regulations require a declaration of the source of gluten on the label of prepackaged foods, which cannot be done due to the limitations of existing methods. In this study, the development of new antibodies targeting discrimination of gluten sources was conducted using synthetic peptides as immunization strategy. Fourteen synthetic peptides selected from unique linear amino acid sequences of gluten were bioconjugated to Concholepas concholepas hemocyanin (CCH) as protein carrier, to elicit antibodies in rabbit. The resulting polyclonal antibodies (pAbs) successfully discriminated wheat, barley and oat prolamins during indirect ELISA assessments. pAbs raised against rye synthetic peptides cross-reacted evenly with wheat and rye prolamins but could still be useful to successfully discriminate gluten sources in combination with the other pAbs. Discrimination of gluten sources can be further refined and enhanced by raising monoclonal antibodies using a similar immunization strategy. A methodology capable of discriminating gluten sources, such as the one proposed in this study, could facilitate compliance with Canadian regulations on this matter. This type of discrimination could also complement current immunoassays by settling the issue of over and underestimation of gluten content, thus improving the safety of food intended to CD and wheat-allergic patients., Competing Interests: Samuel Benrejeb Godefroy’s research activities are funded by the Ministry of Agriculture, Fisheries, and Food, Government of Quebec, Canada; the Ministry of Science, Technology, and Innovation, Government of Quebec; Canada’s Innovation Foundation; the U.S. Department of Agriculture Foreign Agriculture Service; r‐Biopharm GmbH; and r-Biopharm Canada Inc. Samuel Benrejeb Godefroy acts as an expert advisor for members of the food and beverage industry, international organizations (the Food and Agriculture Organization of the United Nations, the United Nations Industrial Development Organization, and the World Bank), and international food regulators such as the China National Centre for Food Safety Risk Assessment and consumer organizations such as Food Allergy Canada. The other authors declare that they have no conflicts of interest.

The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 μg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes., Competing Interests: The authors have declared that no competing interests exist.

COVID-19 is the name of the acute respiratory disease caused by the new coronavirus SARS-CoV-2, a close relative of those that caused the severe outbreaks of SARS and MERS several years ago. Since first appearance on December of 2019, the COVID-19 pandemic has cause extremely high levels of mortality, morbidity, global economic breakdown, and the consequent human suffering. The main diagnostic test for the confirmation of symptomatic individuals is the detection of viral RNA by reverse transcriptase-quantitative real time PCR (RT-PCR). Additionally, serology techniques, such as ELISA are useful to measure the antibodies produced in humans after contact with the virus, as well as the direct presence of viral antigens. In this study we aim to assemble and evaluate four ELISA assays to measure the presence of IgG or IgM specific for the viral Spike protein in COVID-19 patients, using either the full recombinant SARS-CoV-2 Spike protein or the fragment corresponding to the receptor binding domain. As a control, we analyzed a group of pre-pandemic serum samples obtained before 2017. Strong reactivity was observed against both antigens. A few pre-pandemic samples displayed high OD values, suggesting the possibility of some cross reactivity. All four assays show very good repeatability, both intra- and inter-assay. Receiver operating characteristic analysis allowed the definition of cutoffs and evaluation of performance for each ELISA by estimation of the area under the curve. This performance parameter was high for all tests (AUC range: 0.98-0.99). Multiple comparisons between tests revealed no significant difference between each other (P values: 0.24-0.95). Our results show that both antigens are effective to detect both specific IgG and IgM antibodies, with high sensitivity (range 0.92-0.99), specificity (range 0.93-0.97) and congruence with the RT-PCR test (Cohen´s Kappa range 0.87-0.93). These assays will allow health authorities to have a new tool to estimate seroprevalence, in order to manage and improve the severe sanitary situation caused by this virus., Competing Interests: The authors have declared that no competing interests exist.

Paratuberculosis a contagious and chronic disease in domestic and wild ruminants, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Typical clinical signs include intractable diarrhea, progressive emaciation, proliferative enteropathy, and mesenteric lymphadenitis. Paratuberculosis is endemic to many parts of the world and responsible for considerable economic losses. In this study, different types of paratuberculosis and MAP in sheep and goats were investigated in Inner Mongolia, a northern province in China contiguous with two countries and eight other provinces. A total of 4434 serum samples were collected from six cities in the western, central, and eastern regions of Inner Mongolia and analyzed using the ELISA test. In addition, tissue samples were collected from seven animals that were suspected to be infected with MAP. Finally, these tissues samples were analyzed by histopathological examination followed by polymerase chain reaction (PCR), IS1311 PCR-restriction enzyme analysis (PCR-REA), and a sequence analysis of five genes. Among all 4434 ruminant serum samples collected from the six cities in the western, central, and eastern regions of Inner Mongolia, 7.60% (337/4434) measured positive for the MAP antibody. The proportions of positive MAP antibody results for serum samples collected in the western, central, and eastern regions were 5.10% (105/2058), 6.63% (85/1282), and 13.44% (147/1094), respectively. For the seven suspected infected animals selected from the herd with the highest rate of positivity, the gross pathology and histopathology of the necropsied animals were found to be consistent with the pathological features of paratuberculosis. The PCR analysis further confirmed the diagnosis of paratuberculosis. The rest of the results demonstrated that herds of sheep and goats in Inner Mongolia were infected with both MAP type II and type III. To the best of our knowledge, this is the first study of the two subtypes of MAP strains in sheep and goats in Inner Mongolia., Competing Interests: The authors have declared that no competing interests exist.

Mycobacterium tuberculosis (Mtb) infects one-quarter of the world's population. Mtb and HIV coinfections enhance the comorbidity of tuberculosis (TB) and AIDS, accounting for one-third of all AIDS-associated mortalities. Humoral antibody to Mtb correlates with TB susceptibility, and engineering of Mtb antibodies may lead to new diagnostics and therapeutics. The characterization and validation of functional immunoglobulin (Ig) variable chain (IgV) sequences provide a necessary first step towards developing therapeutic antibodies against pathogens. The virulence-associated Mtb antigens SodA (Superoxide Dismutase), KatG (Catalase), PhoS1/PstS1 (regulatory factor), and GroES (heat shock protein) are potential therapeutic targets but lacked IgV sequence characterization. Putative IgV sequences were identified from the mRNA of hybridomas targeting these antigens and isotype-switched into a common immunoglobulin fragment crystallizable region (Fc region) backbone, subclass IgG2aκ. Antibodies were validated by demonstrating recombinant Ig assembly and secretion, followed by the determination of antigen-binding specificity using ELISA and immunoblot assay., Competing Interests: The authors have declared that no competing interests exist.

Zika virus (ZIKV) is a flavivirus that has emerged as a global health threat after the 2015 outbreak in the Americas, where devastating congenital defects were documented. There are currently no vaccines to prevent ZIKV infections nor commercially available clinical diagnostic tests demonstrated to identify ZIKV without cross-reactive interference of related flaviviruses. Early diagnosis is critical when treating symptomatic patients and in preventing ZIKV transmission. In this context, the development of sensitive and accurate diagnostic methods are urgently needed for the detection of ZIKV acute infection. The aim of this study consisted of obtaining monoclonal antibodies (mAbs) against denatured monomeric ZIKV Nonstructural protein 1 (ZNS1), a useful diagnostic marker for flavivirus early detection, in order to develop a highly specific and sensitive ZNS1 indirect competitive ELISA (icELISA). The production of hybridomas secreting ZNS1 mAbs was carried out through immunizations with denatured monomeric ZNS1. We selected 1F5 and 6E2 hybridoma clones, which recognized the heat-denatured ZNS1 hexameric form by indirect ELISA. Cross-reaction studies indicated that these mAbs specifically bind to a ZNS1 linear epitope, and that they do not cross-react with the NS1 protein from other related flaviviruses. The 1F5 mAb enabled the development of a sensitive and reproducible icELISA to detect and quantify small amounts of ZNS1 disease marker in heat-denatured human sera. Here, we establish a reliable 1F5 based-icELISA that constitutes a promising diagnostic tool for control strategies and the prevention of ZIKV propagation., Competing Interests: The authors have declared that no competing interests exist.

This study was aimed at exploring whether latent tuberculosis infection (LTBI) contributes to the pathogenesis of immune-mediated inflammatory diseases in a TB endemic setting. We screened 198 rheumatoid arthritis (RA) patients with tuberculin skin test (TST) and studied 61 (median DAS28-ESR = 6.3) who were positive. Whole blood T cell proliferative responses to Mycobacterium tuberculosis (Mtb) membrane (MtM) antigens, including the latency-induced protein alpha crystallin (Acr), were determined by flow cytometry using Ki67 expression as the marker for nuclear proliferation. Serum antibody levels were determined by ELISA. Follow-up investigations (at 3-6, 9-12 and 15-18 months after baseline) were performed in 41 patients who were classified empirically as 'high' (HR-T/HR-B) or 'low' (LR-T/LR-B) responders based on their dynamic T cell or antibody responses. Significant correlations were seen between baseline T cell responses to MtM and Acr, and between IgG, IgA and IgM antibody responses to MtM. However, no correlation was seen between T and B cell responses. At all time points during the follow-up, T cell responses to both antigens (except for MtM at one point) were significantly higher in HR-T (n = 25) than LR-T (n = 16) patients. Levels of IgA and IgM (but not IgG) antibodies to MtM were also significantly higher in HR-B (n = 13) than LR-B (n = 28) at all time points. Importantly, HR-T patients exhibited significantly higher baseline and follow-up DAS28 scores than LR-T. Ten (of 61) patients had a history of TB and developed RA 6 years (median) after contracting TB. Three new TB cases (1 from TST-positive and 2 from TST-negative groups) emerged during the follow-up. Our results suggest that persistently elevated T cell responses to Mtb antigens may contribute to disease activity in RA., Competing Interests: The authors have declared that no competing interests exist.

Serologic assays developed for SARS-CoV-2 detect different antibody subtypes and are based on different target antigens. Comparison of the performance of a SARS-CoV-2 Spike-Protein ELISA and the nucleocapsid-based Abbott ArchitectTM SARS-CoV-2 IgG assay indicated that the assays had high concordance, with rare paired discordant tests results., Competing Interests: The authors have declared that no competing interests exist.

Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) has been identified as the causative agent for causing the clinical syndrome of COVID -19. Accurate detection of SARS-CoV-2 infection is not only important for management of infected individuals but also to break the chain of transmission. South Africa is the current epicenter of SARS-CoV-2 infection in Africa. To optimize the diagnostic algorithm for SARS-CoV-2 in the South African setting, the study aims to evaluate the diagnostic performance of the EUROIMMUN Anti-SARS-CoV-2 assays. This study reported the performance of EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples targeting the recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Samples were collected from 391 individuals who had tested positive for SARS-CoV-2 and 139 SARS CoV-2 negative controls. Samples were stratified by number of days' post-PCR diagnosis and symptoms. The sensitivity of EUROIMMUN IgG was 64.1% (95% CI: 59.1-69.0%) and 74.3% (95% CI: 69.6-78.6%) for IgA and the specificity was lower for IgA [84.2% (95% CI: 77-89.2%)] than IgG [95.2% (95% CI: 90.8-98.4%)]. The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay sensitivity was higher for IgA but low for IgG and improved for both assays in symptomatic individuals and at later timepoints post PCR diagnosis., Competing Interests: The authors have declared that no competing interests exist.

Bovine pregnancy-associated glycoproteins (boPAG) are expressed by trophoblast cells in the bovine placenta. The multigene family of boPAG belongs to the group of aspartic proteases. The accumulation and circulation in maternal blood and milk has made boPAG very useful and important for pregnancy diagnosis in cattle. The goal of the present study was to develop and validate a new Sandwich-ELISA which allows the detection of boPAG in maternal serum and whole milk. Therefore, 984 serum and 928 milk samples were collected monthly from 231 Holstein Friesian cows (Bos Taurus) from one week after insemination (p.i.) until six weeks postpartum. The ELISA is able to identify a cow as being pregnant at day 30 p.i. in serum and at day 40 p.i in milk with threshold values of 1.0 ng/ml in serum and 0.0165 ng/ml in milk. The postpartum half-life of boPAG was estimated to be 6.4 days in serum and 7.1 days in milk. The boPAG profile established during pregnancy in serum and milk showed a typical pattern. The amount of boPAG found in milk was 1.5 % of the amount of boPAG present in serum. In conclusion, a Sandwich-ELISA has been developed to quantify boPAG in serum and in whole milk simultaneously with the same test procedure. This is time saving for farmers and more efficient for laboratories., Competing Interests: The authors have read the journal’s policy and have the following competing interests: Landwirtschaftliche Rentenbank provided a grant and research materials for the study. MH and DL are employees of TECOdevelopment GmbH. KM and SH are employees of Hessischer Verband für Leistungs- und Qualitätsprüfungen in der Tierzucht e.V. This does not not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Projections of the stage of the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) pandemic and local, regional and national public health policies to limit coronavirus spread as well as "reopen" cities and states, are best informed by serum neutralizing antibody titers measured by reproducible, high throughput, and statically credible antibody (Ab) assays. To date, a myriad of Ab tests, both available and FDA authorized for emergency, has led to confusion rather than insight per se. The present study reports the results of a rapid, point-in-time 1,000-person cohort study using serial blood donors in the New York City metropolitan area (NYC) using multiple serological tests, including enzyme-linked immunosorbent assays (ELISAs) and high throughput serological assays (HTSAs). These were then tested and associated with assays for neutralizing Ab (NAb). Of the 1,000 NYC blood donor samples in late June and early July 2020, 12.1% and 10.9% were seropositive using the Ortho Total Ig and the Abbott IgG HTSA assays, respectively. These serological assays correlated with neutralization activity specific to SARS-CoV-2. The data reported herein suggest that seroconversion in this population occurred in approximately 1 in 8 blood donors from the beginning of the pandemic in NYC (considered March 1, 2020). These findings deviate with an earlier seroprevalence study in NYC showing 13.7% positivity. Collectively however, these data demonstrate that a low number of individuals have serologic evidence of infection during this "first wave" and suggest that the notion of "herd immunity" at rates of ~60% or higher are not near. Furthermore, the data presented herein show that the nature of the Ab-based immunity is not invariably associated with the development of NAb. While the blood donor population may not mimic precisely the NYC population as a whole, rapid assessment of seroprevalence in this cohort and serial reassessment could aid public health decision making., Competing Interests: The authors have read the journal’s policy and declare the following potential competing interests: AB is Senior Vice President and Head of the Regeneron Genetics Center. JH, MJ and MF are employees of Regeneron Genetics Center. TC and SR are employees of Imanis Life Sciences, RV is an employee and phantom stockholder of Imanis Life Sciences, and SJR is a co-founder, officer, and stockholder in both Vyriad, Inc. and Imanis Life Sciences. Vyriad, Inc., Imanis Life Sciences, and Regeneron collaborated in the commercial development of the neutralization assay employed in this study. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity., Competing Interests: The authors have declared that no competing interests exist.

Current methods for detection of mycotoxin in feed are time-consuming and tedious. An up-converting phosphor technology-based lateral flow (UPT-LF) assay system is a new emerging technique for analytes detection. The aim of this study was to compare the performance of UPT-LF, an enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for detecting aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) in feed. The results showed that the use of UPT-LF for AFB1, ZEN and DON detection exhibited the following: limits of detection of 3, 50 and 200 μg/kg; average recoveries of 104.39%, 102.94% and 103.65%; and precision of 13.96%, 13.71% and 12.56%; respectively. UPT-LF required 45 min to determine one mycotoxin and 1.5 h to determine three mycotoxins in a sample, which took the shortest time. Besides, there were positive correlations between the UPT-LF, ELISA and HPLC/MS/MS methods. In conclusion, UPT-LF can be used to detect and quantify AFB1, ZEN and DON in feed samples. Though the sensitivity, accuracy and precision of UPT-LF are inferior to those of HPLC-MS/MS and ELISA, the UPT-LF assay is the most convenient and rapid technique for on-site detection among the three methods., Competing Interests: The authors have declared that no competing interests exist.

Background: In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation., Aim: To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients' biological samples., Methods: In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients. As a capturing agent full-length SARS-CoV2 specific recombinant nucleocapsid was immobilized. Commercial SARS-CoV2 IgG kit based on chemiluminescent assay was used for the selection of samples and optimization of the assay. The threshold cut-off point, inter-assay and intra-assay variations were determined., Results: The incubation/reaction time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2, respectively; with overall 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity was 97.1% (95% CI, 91.9%, 99.4%) with no cross reaction with dengue samples. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%), respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with Elecsys Anti-SARS-CoV-2, with Cohen's kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases., Conclusion: The assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas., Competing Interests: The authors declare that they have no competing interest.

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) causes severe hemorrhagic fever in humans and cats. Clinical symptoms of SFTS-infected cats resemble those of SFTS patients, whereas SFTS-contracted cats have high levels of viral RNA loads in the serum and body fluids. Due to the risk of direct infection from SFTS-infected cats to human, it is important to diagnose SFTS-suspected animals. In this study, a reverse transcription polymerase chain reaction (RT-PCR) was newly developed to diagnose SFTS-suspected animals without non-specific reactions., Methodology/principle Findings: Four primer sets were newly designed from consensus sequences constructed from 108 strains of SFTSV. A RT-PCR with these four primer sets successfully and specifically detected four clades of SFTSV. Their limits of detection are 1-10 copies/reaction. Using this RT-PCR, 5 cat cases among 56 SFTS-suspected animal cases were diagnosed as SFTS. From these cats, IgM or IgG against SFTSV were detected by enzyme-linked immunosorbent assay (ELISA), but not neutralizing antibodies by plaque reduction neutralization titer (PRNT) test. This phenomenon is similar to those of fatal SFTS patients., Conclusion/significance: This newly developed RT-PCR could detect SFTSV RNA of several clades and from SFTS-suspected animals. In addition to ELISA and PRNT test, the useful laboratory diagnosis systems of SFTS-suspected animals has been made in this study., Competing Interests: The authors have declared that no competing interests exist.

Several commercial Zika virus (ZIKV) serology assays have been developed since the recognition of ZIKV outbreaks as a Public Health Emergency of International Concern in 2016. However, test interpretation for ZIKV serology can be challenging due to antibody cross-reactivity with other flaviviruses like dengue virus (DENV). Therefore, we sought to evaluate the performance of eight commercially available ZIKV IgM and IgG assays across three testing platforms, namely, immunochromatographic tests (ICT), ELISAs and immunofluorescence tests (IIFT). The test panel comprised of 278 samples, including acute and convalescent sera or plasma from ZIKV-confirmed, DENV-confirmed, non-ZIKV and non-DENV patients, and residual sera from healthy blood donors. The ZIKV IgM and IgG serology assays yielded higher test sensitivities of 23.5% - 97.1% among ZIKV convalescent samples as compared to 5.6% - 27.8% among ZIKV acute samples; the test specificities were 63.3% - 100% among acute and convalescent DENV, non-DENV samples. Among the ELISAs and IIFTs, the Diapro ZIKV IgM ELISA demonstrated high test sensitivity (96%) and specificity (80%) when tested on early convalescent samples, while the Euroimmun ZIKV IgG ELISA yielded the highest test specificity of 97% - 100% on samples from non-ZIKV patients and healthy blood donors. For rapid ICTs, the LumiQuick IgM rapid ICT yielded low test sensitivity, suggesting its limited utility. We showed that commercial ZIKV IgM and IgG serology assays have differing test performances, with some having moderate to high test sensitivities and specificities when used in a dengue endemic setting, although there were limitations in IgG serology., Competing Interests: The authors have declared that no competing interests exist.

All 4 dengue viruses (DENV) cause sporadic outbreaks of human disease in the Rio Grande Valley along the US-Mexico border. In addition, West Nile virus (WNV) is enzootic in most border communities, and is the only arbovirus known to cause human disease in the El Paso, Texas community. In an effort to determine if DENV were also endemic in the El Paso community, a serosurvey was conducted among mothers at the time of delivery of their babies in selected hospitals. Cord-blood plasma samples obtained from mothers were tested for DENV antibody by an enzyme-linked immuno-sorbent assay (ELISA), plaque reduction neutralization test (PRNT) and a multiplex microsphere immunoassay. All DENV antibody positive plasma samples were also tested for WNV antibody by the same assays to consider the possibility that DENV antibody positive samples reflected WNV cross reactive antibody. The results indicated that 0.74% (11/1,472) of the mothers had a previous DENV infection and that 3.3% (48/1,472) had a previous WNV infection. Of these mothers, 0.20% (3/1,472) had antibody to both DENV and WNV as evidence of infection by both viruses. The results indicated that 0.2% (3/1472) of the mothers were positive for antibody to only WNV envelope, thus suggesting an undetermined flavivirus infection. Although 6 of the 11 DENV antibody positive mothers did not have a history of travel to a DENV endemic country, the findings of this survey provided further evidence of local transmission of WNV and suggested the possibility of focal autochthonous transmission of DENV in the El Paso community., Competing Interests: The authors have declared that no competing interests exist.

Bovine brucellosis is an infectious bacterial disease caused by members of genus Brucella, affecting both animals and humans, and resulting in a serious economic loss in animal production sector and deterioration of public health. A cross-sectional study was conducted from November 2014 to April 2015 to determine the seroprevalence and associated risk factors of bovine brucellosis in Sendafa, Oromia Special Zone, Ethiopia. A total of 503 blood samples were collected using a simple random sampling technique from dairy cattle of above 6 months of age with no history of previous vaccination against brucellosis. All sera samples were subjected to both Rose Bengal Plate Test for screening and Complement Fixation Test for confirmation. Accordingly, the overall seroprevalence of bovine brucellosis in the study area was 0.40%. The result showed that the seroprevalence of bovine brucellosis in the study area was not statistically significant with all proposed risk factors. No reactors were observed in male animals. The seroprevalence was observed in animals without previous history of abortion. Moreover, information was gathered on individual animal and farm-level risk factors and other farm characteristics using a questionnaire. Awareness among society was poor, so the positive animals can be a potential hazard to animals and humans in the study area. Therefore, public education should be done to improve the awareness of the community on bovine brucellosis and its public health impact with due consideration on the safe consumption of food of animal origin., Competing Interests: The authors have declared that no competing interests exist.

Pyrazinamide (PZA) susceptibility testing in Mycobacterium tuberculosis (Mtb) is a current area of development and PZA-resistant strains are increasingly prevalent. Previous studies have demonstrated that the detection of pyrazinoic acid (POA), the metabolite produced by the deamidation of PZA, is a good predictor for PZA resistance since a resistant strain would not convert PZA into POA at a critical required rate, whereas a susceptible strain will do, expelling POA to the extracellular environment at a certain rate, and allowing for quantification of this accumulated analyte. In order to quantify POA, an indirect competitive ELISA (icELISA) test using hyperimmune polyclonal rabbit serum against POA was developed: for this purpose, pure POA was first covalently linked to the highly immunogenic Keyhole Limpet Hemocyanine, and inoculated in rabbits. A construct made of bovine serum albumin (BSA) linked to pure POA and fixed at the bottom of wells was used as a competitor against spiked samples and liquid Mtb culture supernatants. When spiked samples (commercial POA alone) were analyzed, the half maximal inhibitory concentration (IC50) was 1.16 mg/mL, the limit of detection 200 μg/mL and the assay was specific (it did not detect PZA, IC50 > 20 mg/mL). However, culture supernatants (7H9-OADC-PANTA medium) disrupted the competition and a proper icELISA curve was not obtainable. We consider that, although we have shown that it is feasible to induce antibodies against POA, matrix effects could damage its analytical usefulness; multiple, upcoming ways to solve this obstacle are suggested., Competing Interests: The authors have declared that no competing interests exist.

The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01&#95;7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03&#95;17187/T12&#95;7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species., Competing Interests: The authors have declared that no competing interests exist.

An anti-Zaire Ebola virus (EBOV) glycoprotein (GP) immunoglobulin G (IgG) enzyme linked immunosorbent assay (ELISA) was developed to quantify the serum levels of anti-EBOV IgG in human and non-human primate (NHP) serum following vaccination and/or exposure to EBOV. This method was validated for testing human serum samples as previously reported. However, for direct immunobridging comparability between humans and NHPs, additional testing was warranted. First, method feasibility experiments were performed to assess cross-species reactivity and parallelism between human and NHP serum samples. During these preliminary assessments, the goat anti-human IgG secondary antibody conjugate used in the previous human validation was found to be favorably cross-reactive with NHP samples when tested at the same concentrations previously used in the validated assay for human sample testing. Further, NHP serum samples diluted in parallel with human serum when tested side-by-side in the ELISA. A subsequent NHP matrix qualification and partial validation in the anti-GP IgG ELISA were performed based on ICH and FDA guidance, to characterize assay performance for NHP test samples and supplement the previous validation for human sample testing. Based on our assessments, the anti-EBOV GP IgG ELISA method is considered suitable for the intended use of testing with both human and NHP serum samples in the same assay for immunobridging purposes., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Although immune responses to the Japanese Encephalitis virus (JEV), and the dengue viruses (DENV) have a potential to modulate the immune responses to each other, this has been poorly investigated. Therefore, we developed an ELISA to identify JEV specific, DENV non cross-reactive antibody responses by identifying JEV specific, highly conserved regions of the virus and proceeded to investigate if the presence of JEV specific antibodies associate with dengue disease severity., Methodology and Results: 22 JEV specific peptides were identified from highly conserved regions of the virus and the immunogenicity and specificity of these peptides were assessed in individuals who were non-immune to JEV and DENV (JEV-DENV-, N = 30), those who were only immune to the JEV and not DENV (JEV+DENV-, N = 30), those who were only immune to DENV(JEV-DENV+, N = 30) and in those who were immune to both viruses (JEV+DENV+, N = 30). 7/22 peptides were found to be highly immunogenic and specific and these 7 peptides were used as a pool to further evaluate JEV-specific responses. All 30/30 JEV+DENV- and 30/30 JEV+DENV+ individuals, and only 3/30 (10%) JEV-DENV+ individuals responded to this pool. We further evaluated this pool of 7 peptides in patients following primary and secondary dengue infection during the convalescent period and found that the JEV-specific peptides, were unlikely to cross react with DENV IgG antibodies. We further compared this in-house ELISA developed with the peptide pool with an existing commercial JEV IgG assay to identify JEV-specific IgG following vaccination, and our in-house ELISA was found to be more sensitive. We then proceeded to investigate if the presence of JEV-specific antibodies were associated with dengue disease severity, and we found that those who had past severe dengue (n = 175) were significantly more likely (p<0.0001) to have JEV-specific antibodies than those with past non-severe dengue (n = 175) (OR 5.3, 95% CI 3.3 to 8.3)., Conclusions: As our data show that this assay is highly sensitive and specific for detection of JEV-specific antibody responses, it would be an important tool to determine how JEV seropositivity modulate dengue immunity and disease severity when undertaking dengue vaccine trials., Competing Interests: The authors have declared that no competing interests exist.

Background: Coronavirus disease 2019 (COVID-19) is an emerging threat worldwide. This study aims to assess the serologic profiles and time kinetics of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with COVID-19 using two immunoassays., Methods: A total of 97 samples serially collected from 17 patients with COVID-19 and 137 negative control samples were analyzed for IgM and IgG against SARS-CoV-2 using the AFIAS COVID-19 Ab (Boditech Med Inc., Chuncheon, Republic of Korea) and the EDI™ Novel Coronavirus COVID-19 ELISA Kit (Epitope Diagnostics, Inc., San Diego, CA)., Results: With both assays, IgM and IgG rapidly increased after 7 days post symptom onset (PSO). IgM antibody levels reached a peak at 15-35 d PSO and gradually decreased. IgG levels gradually increased and remained at similar levels after 22-35 d. The diagnostic sensitivities of IgM/IgG for ≤14d PSO were 21.4%/35.7~57.1% and increased to 41.2~52.9%/88.2~94.1% at >14 d PSO with specificities of 98.5%/94.2% for AFIAS COVID-19 Ab and 100.0%/96.4% for EDI™ Novel Coronavirus COVID-19 ELISA Kit. Among 137 negative controls, 12 samples (8.8%) showed positive or indeterminate results., Conclusions: The antibody kinetics against SARS-CoV-2 are similar to common findings of acute viral infectious diseases. Antibody testing is useful for ruling out SARS-CoV-2 infection after 14 d PSO, detecting past infection, and epidemiologic surveys., Competing Interests: The authors have declared that no competing interests exist.

Public health emergency of SARS-CoV-2 has facilitated diagnostic testing as a related medical countermeasure against COVID-19 outbreak. Numerous serologic antibody tests have become available through an expedited federal emergency use only process. This paper highlights the analytical characteristic of an ELISA based assay by AnshLabs and three random access immunoassay (RAIA) by DiaSorin, Roche, and Abbott that have been approved for emergency use authorization (EUA), at a tertiary academic center in a low disease-prevalence area. The AnshLabs gave higher estimates of sero-prevalence, over the three RAIA methods. For positive results, AnshLabs had 93.3% and 100% agreement with DiaSorin or Abbott and Roche respectively. For negative results, AnshLabs had 74.3% and 78.3% agreement with DiaSorin and Roche or Abbott respectively. All discrepant samples that were positive by AnshLabs and negative by RAIA tested positive by all-in-one step SARS-CoV-2 Total (COV2T) assay performed on the automated Siemens Advia Centaur XPT analyzer. None of these methods, however, are useful in early diagnosis of SARS-CoV-2., Competing Interests: Baylor Scott and White Health provided support for the study in the form of salaries for all authors. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare. All authors have no other potential competing financial, non-financial, professional, or personal interests.

Introduction: Nucleosomes consist of small fragments of DNA wrapped around a histone octamer core. Diseases such as cancer or inflammation lead to cell death, which causes fragmentation and release of nucleosomes into the blood. The Nu.Q™ technology measures circulating nucleosome levels and exploits the different compositions of cancer derived nucleosomes in blood to detect and identify cancer even at early stages. The objectives of this study are to identify the optimal sample type for the Nu.Q™ H3.1 assay and to determine if it can accurately detect nucleosomes in the blood of healthy canines as well as those with cancer., Materials and Methods: Blood samples from healthy canine volunteers as well as dogs newly diagnosed with lymphoma were used. The blood was processed at a variety of times under a variety of conditions to determine the most reliable sample type and conditions, and to develop an appropriate processing strategy to ensure reliably accurate results., Results: Nucleosomes could be detected using a variety of sample collection and processing protocols. Nucleosome signals were highest in EDTA plasma and serum samples and most consistent in plasma. Samples should be processed within an hour of collection. Experiments showed that samples were able to withstand several freeze thaw cycles. Processing time and tcollection tube type did affect nucleosome detection levels. Finally, significantly elevated concentrations of nucleosomes were seen in a small cohort of dogs that had been newly diagnosed with lymphoma., Conclusions: When samples are collected and processed appropriately, the Nu.Q™ platform can reliably detect nucleosomes in the plasma of dogs. Further testing is underway to validate and optimize the Nu.Q™ platform for veterinary use., Competing Interests: I have read the Journal's policy and the authors of this manuscript have the following competing interests: JT, ND, TK, MH, TB NH and GM are employees of Belgian Volition & Volition America, which have patents covering Nu.Q technology and are developers of Nu.Q™ assays. Volition Veterinary is a joint venture between Belgian Volition and Texas A&M University. HWR is a paid consultant of Volition Veterinary. TM and JJ have no conflicts of interest to declare. Additional salary support for TM was provided by the Fred and Vola Palmer Chair in Comparative Oncology held by HWR. The Palmers did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support for the authors’ salaries (T.M.). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Acidovorax citrulli, a seedborne bacterium and quarantine pest, causes the devastating bacterial fruit blotch disease in cucurbit plants. Immunological assays such as ELISA are widely used in routine field inspections for this bacterium. However, to the best of our knowledge, none of the currently available monoclonal antibodies (MAbs) can detect all common A. citrulli strains. We therefore aimed to produce a panel of MAbs and to develop an ELISA-based method capable of detecting all A. citrulli strains. We used a high-throughput bead array technique to screen and characterize A. citrulli-specific MAbs produced from hybridoma clones. The hybridoma library was simultaneously screened against five A. citrulli strains (PSA, KK9, SQA, SQB and P) and the closely related bacterium, Delftia acidovorans. Three MAbs exhibiting different binding patterns to A. citrulli were used to develop an ELISA-based method called "double antibody pairs sandwich ELISA" (DAPS-ELISA). DAPS-ELISA employing mixtures of MAbs was able to specifically detect all 16 A. citrulli strains tested without cross-reactivity with other bacteria. By contrast, our previously developed MAb capture-sandwich ELISA (MC-sELISA) and a commercial test kit detected only 15 and 14 of 16 strains, respectively. The sensitivity of the DAPS-ELISA ranged from 5×105 to 1×106 CFU/mL, while those of the MC-sELISA and the commercial test kit ranged from 5×104 to 1×107 CFU/mL and 5×104 to 5×105 CFU/mL, respectively. DAPS-ELISA thus represents an alternative method enabling rapid, accurate, and inexpensive detection of all A. citrulli strains. The method can be applied to seed testing prior to planting as well as to routine field inspections., Competing Interests: The authors have declared that no competing interests exist.