Antigen Preparations Research Articles

Preparation of Deoxynivalenol Complete Antigen and Its Application in Microchannel Resistance Immune Sensing Platform with Electrical Signal Analysis

Preparation of Deoxynivalenol Complete Antigen and Its Application in Microchannel Resistance Immune Sensing Platform with Electrical Signal Analysis

Differences of in vitro immune responses between patent and pre-patent Litomosoides sigmodontis-infected mice are independent of the filarial antigenic stimulus used.

Lymphatic filariasis and onchocerciasis are neglected tropical diseases and cause significant public health problems in endemic countries, especially in sub-Saharan Africa. Since the human parasites are not viable in immune-competent mice, animal models have been developed, among them Litomosoides sigmodontis which permits a complete life cycle in BALB/c mice, including the development of patent infections with circulating microfilariae (Mf, the worm's offspring). To investigate the immunomodulatory properties of helminths in vitro, antigenic extracts can be prepared from different life cycle stages of the L. sigmodontis model, including adult worms, but the methods to prepare these antigens differ between research groups. This study analyzed whether different centrifugation methods during the preparation of an antigenic extract, the gender of used worms, or the different fractions (soluble or pellet) altered filarial-specific CD4+ Tcell responses. These cells were isolated from pre-patent or patent/chronic infected mice, hence those without and those with Mf, respectively. Ex vivo immune responses were compared at these two different time points of the infection as well as the parasitic parameters. Worm burden and cell infiltration were elevated in the thoracic cavity (TC) and draining mediastinal lymph nodes at the pre-patent stage. Within the TC, eosinophils were significantly up-regulated at the earlier time point of infection which was further reflected by the eosinophil-related eotaxin-1 levels. Regarding the production of cytokines by re-stimulated CD4+ T cells in the presence of different antigen preparations, cytokine levels were comparable for all used extracts. Our data show that immune responses differ between pre-patent and patent filarial infection, but not in response to the different antigenic extracts themselves.

Optimizing antigen preparation for oxalyl-CoA decarboxylase enzyme diagnostic kit and ELISA system cutoff determination.

The prevalence of kidney stone disease is increasing globally, with calcium oxalate stones being the most common type. Oxalyl-CoA decarboxylase (OXC), an enzyme produced by the gut bacterium Oxalobacter formigenes, plays a crucial role in oxalate metabolism. Deficiencies in OXC activity can lead to the accumulation of oxalate, contributing to kidney stone formation. This study aimed to develop a reliable diagnostic assay for OXC by optimizing antigen production and establishing a cutoff value for an enzyme-linked immunosorbent assay (ELISA). We cloned, expressed, and purified recombinant OXC protein in Escherichia coli BL21(DE3), and generated specific polyclonal antibodies in rabbits. The ELISA system was optimized and validated using serum samples from 40 healthy individuals and 6 patients with oxalate-related disorders. The cutoff value was determined using the formula (M + 2SD), where (M) is the mean and (SD) is the standard deviation of the healthy sample results. The calculated cutoff value of 0.656750 effectively distinguished between healthy and affected individuals, with a sensitivity of 97.5% and a specificity of 83.3%. These findings provide a valuable tool for the early detection and management of oxalate-related disorders, with significant implications for clinical practice.

Oral Bacterial Lysate OM-85: Advances in Pharmacology and Therapeutics.

Bacterial lysates are known for having immunomodulatory properties and have been used mainly for the prevention and treatment of respiratory tract infections (RTIs). However, rigorous studies are needed to confirm the clinical efficacy of bacterial lysates with various bacterial antigen components, preparation methods, administration routes and course of treatment. OM-85, an oral standardized lysate prepared by alkaline lysis of 21 strains from 8 species of common respiratory tract pathogens, is indicated as immunotherapy for prevention of recurrent RTIs and acute infectious exacerbations of chronic bronchitis. OM-85 acts on multiple innate and adaptive immune targets and can restore type 1helper T (Th1)/Th2 balance. Sporadic studies have shown advances in pharmacology and therapeutics of OM-85, and thus an update review is necessary. Literature was retrieved by searching PubMed, Web of science, Embase, CNKI, and Full Text Database of Chinese Medical Journals. New roles of OM-85 were discovered in prevention and treatment of lung cancer, pulmonary tuberculosis, SARS-CoV-2 infection, allergic rhinitis, pulmonary fibrosis, atopic dermatitis, and nephrotic syndrome. Pharmacoeconomic values of OM-85 were demonstrated in prophylaxis and treatment of RTIs, chronic obstructive pulmonary disease, asthma, chronic bronchitis, rhinosinusitis and allergic rhinitis. Two consecutive courses of OM-85 (6 or 12 months apart) could prevent recurrent RTIs in children. Maternal OM-85 treatment could offer benefits for offspring. Product-specific response was observed. The efficacy of OM-85 may be associated with patient's characteristics (eg, severity of the disease, age, immune response pattern, malignancy risk stratification). OM-85 can improve effectiveness of standard care for some primary diseases, and carry significant pharmacoeconomic implications. The benefits shown by OM-85 in vitro and in vivo, when extrapolated to humans, are exciting but also require caution. Individualized treatment may need to be considered. It is necessary to compare the efficacy and safety of various bacterial lysate preparations.

Comparative evaluation of plasma biomarkers of Schistosoma haematobium infection in endemic populations from Burkina Faso.

Infection with Schistosoma haematobium causes urogenital disease associated with organ disfunction, bleeding, pain, and higher susceptibility to infections and cancer. Timely and accurate diagnosis is crucial for prompt and appropriate treatment as well as surveillance efforts, and the use of plasma biomarkers offers important advantages over parasitological examination of urine, including increased sensitivity and the possibility to use the same specimen for multiple investigations. The present study aims to evaluate the diagnostic performance of different plasma biomarkers in endemic populations from Burkina Faso, West Africa. Schistosoma spp. Circulating Anodic Antigen (CAA), cell free S. haematobium DNA (cfDNA), class M and G antibodies against S. haematobium Soluble Worm Antigen Preparation (SWAP) and Soluble Egg Antigen (SEA) were measured in 406 plasma samples. Results of each biomarker test were compared to those of CAA, a Composite Reference Standard (CRS) and Latent Class Analysis (LCA). An identical proportion of positive samples (29%) was observed as a result of CAA and cfDNA testing, with a substantial agreement (84%, Cohen k = 0.62) between the results of the two tests, and a comparable agreement with the results of CRS and LCA. A higher positivity was observed, as expected, as a result of specific antibody testing (47%-72%), with IgG showing a higher agreement than IgM with the three references. Also, higher IgG levels were observed in current vs past infection, and ROC analysis identified optimal cutoff values for improved testing accuracy. This study provides compelling evidence that can inform the choice of the most appropriate diagnostic plasma biomarker for urogenital schistosomiasis in endemic areas, depending on the purpose, context, and available resources for testing. Either CAA or cfDNA testing can be used for the diagnosis of patients and for epidemiological investigations, even in absence of urine filtration microscopy, whereas anti-SWAP or anti-SEA IgG can be employed for surveillance and integrated monitoring of control interventions against poverty-associated diseases.

Study of the protein composition of antigen preparations from the pathogen Opisthorchis felineus, of various chemical natures.

Study of the protein composition of antigen preparations from the pathogen Opisthorchis felineus, of various chemical natures.

Immunogenic Proteins Characterization in the Larvae of Oestrus ovis by Western Blot Analysis

The present study was undertaken to characterize and identify immunogenic proteins of Oestrus ovis in Telangana. Oestrus ovis is the most predominant nasal bot fly causing nasal myiasis in India. The collected larvae from Jiyaguda slaughter house, Hyderabad are seperated into L2 and L3 larvae were washed with normal saline and PBS pH 7.4 and preserved at -70°C for antigen preparation and some were preserved in 70 % ethanol for identification. The larvae were speciated by light microscopic examination based on morphological features such as ventral spines, anterior hooks and “D” shaped posterior spiracles. In this study, an antigen was prepared from second and third instar larvae of Oestrus ovis using a soluble buffer solution. The resulting Crude Somatic Larval (CSL) antigen had a protein concentration of 27.4 mg per ml. The CSL antigen was resolved in 8% SDS-PAGE, Immunoblotting was performed by electro transferring the separated polypeptides onto a nitrocellulose (NC) membrane. Treatment with specific reagents and antibodies resulted in the identification of 19 immunoreactive polypeptides, with molecular weights matching those observed in SDS-PAGE. Four polypeptides with molecular weights of 58.56, 20.74, 19.09, and 28.91 kDa exhibited the strongest reaction towards the antibodies raised against CSL antigen. These four polypeptides are considered promising candidates for future immunizations and testing their protective efficacy through in vivo assays. These findings have implications for advancing strategies to combat myiasis, a parasitic disease caused by Oestrus ovis larvae.

Evaluation of flow cytometry-based fluorescent antibody to membrane antigen test to measure the humoral immunity against the varicella-zoster virus

The fluorescent antibody to membrane antigen (FAMA) test is the gold standard for measuring the immunity induced by varicella vaccines with high sensitivity and specificity. However, certain aspects of the FAMA test, such as time consumption, non-automation, and subjective interpretation by observers using fluorescence microscopy, are obstacles to handling large amounts of samples. To overcome these hurdles, flow cytometry was adopted to analyze and compare the flow FAMA titer with the classic FAMA titer. In addition, to save time in FAMA antigen preparation and reduce lot-to-lot variation, the stability of the FAMA antigen stored in liquid nitrogen was investigated. The FAMA test was performed on sera from 229 children, and antibody titers were analyzed using fluorescence microscopy (classic FAMA) and flow cytometry (flow FAMA). For comparison, glycoprotein enzyme immunoassay (gpEIA) titer was also measured. A strong correlation was found between the flow and classic FAMA titers, and the flow FAMA and gpEIA titers, with Pearson's r of 0.9316 and 0.8588, respectively. Between the classic FAMA and gpEIA titers, the Pearson's r value was 0.8156. The positive percent agreement, negative percent agreement, and area under the curve of the flow FAMA against classic FAMA were 95.0 %, 86.8 %, and 0.909, respectively. And those of the flow FAMA against gpEIA were 80.0 %, 87.6 %, and 0.838, respectively. The FAMA antigen stored in liquid nitrogen was stable for up to 12 months. Based on the above data, flow FAMA has the potential to be used as an alternative to classic FAMA. Moreover, pre-made FAMA antigen may reduce the preparation time and lot-to-lot variation of FAMA test.

Evaluation of Methodologies in Anti-nephrin Autoantibody Detection.

Recent studies discovered the prominent presence of anti-nephrin autoantibodies in minimal change disease, steroid-sensitive nephrotic syndrome and/or post-transplant recurrent focal segmental glomerulosclerosis (FSGS). However, widely different, and often unconventional autoantibody detection methods were used among these studies, making it challenging to assess the pathogenic role for the antibodies. Here we examined methods of conventional ELISA, magnetic on-beads ELISA, immunoprecipitation-immunoblotting (IP-IB), and cell- and tissue-based antibody assays with 127 plasma samples of kidney and non-kidney diseases. On the antigen side, we compared commercially available recombinant human nephrin extracelluar domain (ECD) produced from human or mouse cell lines, as well as lab-made full length, ECD, and series of ECD truncates for measuring autoantibody reactivity and specificity. Surprisingly, different assay methods and different antigen preparations led to observation of assay-specific false-positive and false-negative results. In general, a set of tests that combines magnetic beads-enhanced ELISA, followed by IP-IB, and epitope mapping showed the most robust results for anti-nephrin autoantibodies, detected in two primary FSGS patients among all cases tested. It is interesting to note that cell/tissue-based results, also supported by antigen truncation studies, clearly suggest steric hindrance of reactive epitopes, as in full length nephrin that forms compact self-associated complexes. In conclusion, anti-nephrin positivity is rare among the tested patients (2/127), including those with FSGS (2/42), and autoantibody results can be affected by the choice of detection methods.

Evaluation of SARS-CoV-2-Specific IgY Antibodies: Production, Reactivity, and Neutralizing Capability against Virus Variants.

The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel. We compared three types of antigens based on ancestral SARS-CoV-2: an inactivated whole-virus preparation, an S1 spike-protein subunit (S1 antigen) and a receptor-binding domain (RBD antigen, amino acids 319-519) coated on lumazine synthase (LS) particles using SpyCather/SpyTag technology. The RBD antigen proved to be the most efficient immunogen, and the resulting chicken IgY antibodies derived from serum or yolk, displayed strong reactivity with ELISA and indirect immunofluorescence and broad neutralizing activity against SARS-CoV-2 variants, including Omicron BA.1 and BA.5. Preliminary in vivo studies using RBD-lumazine synthase yolk preparations in a hamster model showed that local application was well tolerated and not harmful. However, despite the in vitro neutralizing capacity, this antibody preparation did not show protective effect. Further studies on galenic properties seem to be necessary. The RBD-lumazine antigen proved to be suitable for producing SARS-CoV-2 specific antibodies that can be applied to such therapeutic approaches and as reference reagents for SARS-CoV-2 diagnostics, including virus neutralization assays.

Enzyme immunoassay system for serological diagnosis of hemorrhagic fever with renal syndrome based on inactivated purified Puumala virus (Hantaviridae: Orthohantavirus)

Hemorrhagic fever with renal syndrome (HFRS) is the most common zoonotic human viral disease in the Russian Federation. More than 98% of the HFRS cases are caused by Puumala orthohantavirus (PUU). Effective serological tests are required for laboratory diagnosis of HFRS. Construction of an enzyme immunoassay (ELISA) test system for detection of specific antibodies using standard antigen in the form of highly purified inactivated PUU virus as immunosorbent. Preparation of PUU virus antigen, designing the ELISA for detection of specific antibodies, developing parameters of the ELISA system, parallel titration of HFRS patients sera by fluorescent antibody technique (FAT) and the new ELISA. For the first time, ELISA based on purified inactivated PUU virus as standard antigen directly absorbed onto immunoplate was developed. Parallel titration of 50 samples from HFRS patients blood sera using FAT and the developed ELISA showed high sensitivity and specificity of this ELISA, with 100% concordance of testing results and significant level of correlation between the titers of specific antibodies in the two assays. The ELISA based on purified inactivated PUU virus as an immunosorbent can be effectively used for HFRS serological diagnosis and for mass seroepidemiological studies.

Ascaris Mouse Model Protocols: Advancing Research on Larval Ascariasis Biology.

Ascariasis, caused by both Ascaris lumbricoides and Ascaris suum, is the most prevalent parasitic disease worldwide, affecting both human and porcine populations. However, due to the difficulties of assessing the early events of infection in humans, most studies of human ascariasis have been restricted to the chronic intestinal phase. Therefore, the Ascaris mouse model has become a fundamental tool for investigating the immunobiology and pathogenesis of the early infection stage referred to as larval ascariasis because of the model's practicality and ability to replicate the natural processes involved. The Ascaris mouse model has been widely used to explore factors such as infection resistance/susceptibility, liver inflammation, lung immune-mediated pathology, and co-infections and, notably, as a pivotal element in preclinical vaccine trials. Exploring the immunobiology of larval ascariasis may offer new insights into disease development and provide a substantial understanding of key components that trigger a protective immune response. This article focuses on creating a comprehensive guide for conducting Ascaris experimental infections in the laboratory as a foundation for future research efforts. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Acquisition and embryonation of Ascaris suum eggs from adult females Alternate Protocol: Cleaning and purification of Ascaris suum from female A. suum uteri Basic Protocol 2: Preparation of Ascaris suum eggs and murine infection Basic Protocol 3: Measurement of larval burden and Ascaris-larva-induced pathogenesis Basic Protocol 4: In vitro hatching and purification of Ascaris L3 larvae Support Protocol: Preparation of crude antigen from Ascaris infectious stages Basic Protocol 5: Ultrastructure-expansion microscopy (U-ExM) of Ascaris suum larval stages.

Two lateral flow assays for detection of anti-coccidioidal antibodies show similar performance to immunodiffusion in dogs with coccidioidomycosis.

To compare 2 point-of-care lateral flow assays (LFAs) with immunodiffusion (ID) IgG results for anti-coccidioidal antibody detection in dogs with coccidioidomycosis. A further aim was to compare the quantifiable output of 1 of the LFAs to ID antibody titers. Serum banked from 73 client-owned dogs diagnosed with pulmonary or disseminated coccidioidomycosis. ID was used to determine antibody presence and titer against a coccidioidal antigen preparation. All sera were subsequently tested on an LFA based on recombinant chitinase 1 (CTS1) and the commercially available sōna LFA. LFA results were analyzed and compared to ID IgG results and clinical diagnosis. All assays showed similar sensitivities in detecting anti-coccidioidal antibodies (83.6% to 89.0%). When compared with ID IgG, the CTS1 LFA had a positive percent agreement of 100%, while the sōna LFA had a positive percent agreement of 91.4%. Since the CTS1 LFA is semiquantitative, we were able to compare test line densities with ID titers and found a strong correlation between the 2 assays (Spearman ρ = 0.82). This is the first side-by-side evaluation of a commercially available LFA (sōna) and a newer more rapid anti-CTS1 antibody LFA using serum from dogs with coccidioidomycosis. Both LFAs tested have similar sensitivity to ID IgG results. The CTS1 LFA can be read after 10 minutes and is semiquantitative, while the sōna LFA is read after 30 minutes, and the results are subject to interpretation. Accurate and fast detection of anti-coccidioidal antibodies allows clinicians to initiate appropriate treatment without diagnostic delay.

The efficacy of cercarial antigen loaded on nanoparticles as a potential vaccine candidate in Schistosoma mansoni-infected mice.

Schistosomiasis is one of the most common causes of morbidity and mortality from parasitic diseases. Mass treatment has proven to be insufficient because of repeated infection after treatment and the appearance of strains resistant to drug therapy. Hence, immunization is a new approach to control the disease and limit the pathological consequences of schistosomiasis. To evaluate the prophylactic effect of Cercarial antigen (CAP) loaded on chitosan nanoparticles (CSNPs) as a potential vaccine against Schistosoma mansoni-infected mice. 130 mice divided into 2 groups were used: Group I: Control groups (50 mice) subdivided into subgroup Ia (10 mice): Non-infected mice (normal control), subgroup Ib (20 mice): Schistosoma infected mice (infected control) and subgroup Ic (20 mice): Non-infected mice receiving NPs only. Group II: Vaccinated group (80 mice) subdivided equally into subgroup IIa (CAP): Received cercarial antigen and subgroup IIb (CAP + CSNP): Received cercarial antigen loaded on chitosan NPs then both vaccinated groups were infected with S. mansoni 3 weeks following the initial vaccination dose. CAP + CSNP and CAP groups showed significant reduction in adult worms count, hepatic egg count, hepatic granulomas number and size in comparison to the infected control group. Elevation of serum IgG and IgM levels, CD4+ and CD8+ T cell frequencies, IL-4, IL-10 and INF-γ levels was more significant in CAP + CSNP group than CAP group. CAP + CSNP is a promising new preparation of Schistosomal antigens that gave better results than immunization with CAP alone. CSNPs enhanced the immune and protective effect of CAP as validated by parasitological, histopathological and immunohistochemical studies.

Exosome-like Systems: From Therapies to Vaccination for Cancer Treatment and Prevention-Exploring the State of the Art.

Cancer remains one of the main causes of death in the world due to its increasing incidence and treatment difficulties. Although significant progress has been made in this field, innovative approaches are needed to reduce tumor incidence, progression, and spread. In particular, the development of cancer vaccines is currently ongoing as both a preventive and therapeutic strategy. This concept is not new, but few vaccines have been approved in oncology. Antigen-based vaccination emerges as a promising strategy, leveraging specific tumor antigens to activate the immune system response. However, challenges persist in finding suitable delivery systems and antigen preparation methods. Exosomes (EXs) are highly heterogeneous bilayer vesicles that carry several molecule types in the extracellular space. The peculiarity is that they may be released from different cells and may be able to induce direct or indirect stimulation of the immune system. In particular, EX-based vaccines may cause an anti-tumor immune attack or produce memory cells recognizing cancer antigens and inhibiting disease development. This review delves into EX composition, biogenesis, and immune-modulating properties, exploring their role as a tool for prevention and therapy in solid tumors. Finally, we describe future research directions to optimize vaccine efficacy and realize the full potential of EX-based cancer immunotherapy.

An in vitro assay for toxicity testing of Clostridium perfringens type C β-toxin.

Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the β-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the β-toxin. Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for β-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the β-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

Inactivated Aeromonas salmonicida impairs adaptive immunity in lumpfish (Cyclopterus lumpus).

Aeromonas salmonicida, a widely distributed aquatic pathogen causing furunculosis in fish, exhibits varied virulence, posing challenges in infectious disease and immunity studies, notably in vaccine efficacy assessment. Lumpfish (Cyclopterus lumpus) has become a valuable model for marine pathogenesis studies. This study evaluated several antigen preparations against A. salmonicida J223, a hypervirulent strain of teleost fish, including lumpfish. The potential immune protective effect of A. salmonicida bacterins in the presence and absence of the A-layer and extracellular products was tested in lumpfish. Also, we evaluated the impact of A. salmonicida outer membrane proteins (OMPs) and iron-regulated outer membrane proteins (IROMPs) on lumpfish immunity. The immunized lumpfish were intraperitoneally (i.p.) challenged with 104 A. salmonicida cells/dose at 8 weeks-post immunization (wpi). Immunized and non-immunized fish died within 2 weeks post-challenge. Our analyses showed that immunization with A. salmonicida J223 bacterins and antigen preparations did not increase IgM titres. In addition, adaptive immunity biomarker genes (e.g., igm, mhc-ii and cd4) were down-regulated. These findings suggest that A. salmonicida J223 antigen preparations hinder lumpfish immunity. Notably, many fish vaccines are bacterin-based, often lacking efficacy evaluation. This study offers crucial insights for finfish vaccine approval and regulations.

Modulatory actions of Echinococcus granulosus antigen B on macrophage inflammatory activation.

Cestodes use own lipid-binding proteins to capture and transport hydrophobic ligands, including lipids that they cannot synthesise as fatty acids and cholesterol. In E. granulosus s.l., one of these lipoproteins is antigen B (EgAgB), codified by a multigenic and polymorphic family that gives rise to five gene products (EgAgB8/1-5 subunits) assembled as a 230 kDa macromolecule. EgAgB has a diagnostic value for cystic echinococcosis, but its putative role in the immunobiology of this infection is still poorly understood. Accumulating research suggests that EgAgB has immunomodulatory properties, but previous studies employed denatured antigen preparations that might exert different effects than the native form, thereby limiting data interpretation. This work analysed the modulatory actions on macrophages of native EgAgB (nEgAgB) and the recombinant form of EgAg8/1, which is the most abundant subunit in the larva and was expressed in insect S2 cells (rEgAgB8/1). Both EgAgB preparations were purified to homogeneity by immunoaffinity chromatography using a novel nanobody anti-EgAgB8/1. nEgAgB and rEgAgB8/1 exhibited differences in size and lipid composition. The rEgAgB8/1 generates mildly larger lipoproteins with a less diverse lipid composition than nEgAgB. Assays using human and murine macrophages showed that both nEgAgB and rEgAgB8/1 interfered with in vitro LPS-driven macrophage activation, decreasing cytokine (IL-1β, IL-6, IL-12p40, IFN-β) secretion and ·NO generation. Furthermore, nEgAgB and rEgAgB8/1 modulated in vivo LPS-induced cytokine production (IL-6, IL-10) and activation of large (measured as MHC-II level) and small (measured as CD86 and CD40 levels) macrophages in the peritoneum, although rEgAgB8/1 effects were less robust. Overall, this work reinforced the notion that EgAgB is an immunomodulatory component of E. granulosus s.l. Although nEgAgB lipid's effects cannot be ruled out, our data suggest that the EgAgB8/1 subunit contributes to EgAgB´s ability to regulate the inflammatory activation of macrophages.

Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs.

Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance.

In-process quality control in foot-and-mouth disease vaccine production by detection of viral non-structural proteins using chemiluminescence dot blot assay

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-footed animals. Immunization with inactivated virus vaccine is effective to control the disease. Six-monthly vaccination regimen in endemic regions has proven to be effective. To enable the differentiation of infected animals from those vaccinated, non-structural proteins (NSPs) are excluded during vaccine production. While the antibodies to structural proteins (SPs) could be observed both in vaccinated and infected animals, NSP antibodies are detectable only in natural infection. Quality control assays that detect NSPs in vaccine antigen preparations, are thus vital in the FMD vaccine manufacturing process. In this study, we designed a chemiluminescence dot blot assay to detect the 3A and 3B NSPs of FMDV. It is sensitive enough to detect up to 20 ng of the NSP, and exhibited specificity as it does not react with the viral SPs. This cost-effective assay holds promise in quality control assessment in FMD vaccine manufacturing.