ABSTRACT
Biosensors are rapid and portable detection devices with great potential for the instant screening of infectious diseases. Receptors are the critical element of biosensors. They determine the specificity, sensitivity and stability. However, current receptors are mainly limited to antibodies and aptamers. Herein, we developed a glycosylated extracellular vesicle-like receptor (GlycoEVLR) for the rapid detection of virus antigens, specifically using SARS-CoV-2 as a model. The human angiotensin-converting enzyme 2 (ACE2)-overexpressed and heparin-functionalized HEK-293T cell membrane-cloaked Fe3O4 nanoparticles (NPs) were prepared as functionalizing GlycoEVLR. They were characterized as spherical core–shell structures with a diameter of around 100 nm, which were perfectly comparable to natural extracellular vesicles. Binding affinities between GlycoEVLR and spike1 (S1) antigen were demonstrated using surface plasmon resonance (SPR). The GlycoEVLR was fixed on magnetic electrodes to construct electrochemical biosensors. Using electrochemical impedance spectroscopy (EIS) as a measurement technique, the S1 antigen was detected down to 1 pg/mL within 20 min and showed a good linearity range from 1 pg/mL to 1 ng/mL. Also, the GlycoEVLR-based electrochemical biosensors showed excellent antifouling performance and stability. Overall, our work provides a useful methodology for developing extracellular vesicle-like receptors for biosensors. Combining the inherit natural receptor proteins and antifouling lipids from the host cells with engineered glycan motifs to target and sense viral antigens will open a newavenue for biosensors.
ABSTRACT
Against a pandemic of emerged infectious disease, COVID-19, new generation vaccines based on nucleic acids or recombinant viruses, which had not been used as vaccines in humans, have been inoculated and shown to be successful. They are, however, heat-labile and need a cold-chain including deep-freezers for storage and transportation. Vaccinia virus (VAC) vector vaccine (VACV) is a pioneer of new generation of vaccines constructed by using molecular biological technology. VACV, which has contributed to eradication of smallpox, has excellent characteristics of vaccinia virus such as a high heat-stability and long-lasting immunological effects. It is possible to distinguish the immunological responses of vaccination from those of natural infections. We started our developmental researches 35 years ago, using attenuated VAC strains established in Japan. In this article, we first describe the early researches of VACVs;development of two VACVs for Bovine leukemia virus and Rinderpest morbillivirus antigens and their protective immunity in large mammals, sheep and cows. Second, application of VACV is described;Rabies-VACV, which has already been licensed, used in the field in Europe and USA, and resulted in a prominent decrease of rabies. Then, current status of VACV research is described;non-replicating VACVs in mammalian cells have been developed as new-generation and ultimately-safe vaccines. We discuss the possibility of future application of VACV for wildlife.
ABSTRACT
Feline coronavirus (FCoV) belongs among pathogens with common occurrence in the cats population in the whole world. FCoV is ubiquitous in environments with a higher concentration of cats, e.g. in shelters, multicat households and kennels. FCoV primarily attacks the digestive feline tract, replicates in its cells and is excreted in the feces to surroundings of permanently or transiently infected cats. The aim of the study was the detection of FCoV in the feces of newly admitted cats to the shelter by the qPCR method and by means of commercial rapid immunochromatographic (antigen) tests from three different producers. For each of the antigen tests, sensitivity and specifity were determined by comparison with the qPCR analysis result. Out of 70 examined fecal samples, viral RNA was by the qPCR analysis identified in 44 samples (62.9%). Neither the age nor the gender of cats played a significant role in the viral excretion. Found sensitivity of the antigen tests was at a low (< 35%;tests A and C) to a satisfactory level (> 50%, test B). The number of viral particles in the samples determined by the qPCR method did not correlate significantly with the result of the antigen tests. The results of this study suggest that the use of rapid antigen tests for routine screening of FCoV shedding in feline shelters is limited due to the high rate of false-negative results.
ABSTRACT
Domestic and wild dogs of all ages and breeds are susceptible to Canine Coronavirus(CCoV) infections and be seen in T..rkiyeand amongst world. CCoV has recently been declared a zoonotic disease agent and the eighth pathogenic human coronavirus. This study was conducted on 143 naturally infected dogs with gastroenteritis which were not vaccinated against CCoV in T..rkiye in 2015-2020. The data of dogs were analyzed seroepidemiologically, clinicopathologically and statistically. CCOV antibodies in serum and CCOV antigens in stool were detected by ELISA and lateral immunochromatography. The rising CCoV IgG antibody titers were detected at all dogs and were as follows;<10 ng/L in 3 (2%), 10-20 ng/L in 18 (13%), 20-30 ng/L in 16 (11%), 30-40 ng/L in 14 (%10), 40-64 ng/L in 11 (8%) and >64 ng/L in 81 (81%) dogs. CCOV and Canine Parvovirus (CPV) antigen were detected together in the stool of the 41 (28.7%) dogs. As a result, it was concluded that the CCOV agent is in circulation among dogs living in T..rkiye. CCOV and CPV can cause co-infections and increased mortality. Although infection can be seen in dogs of all ages, it can be seen more frequently in dogs younger than 1 year of age, and especially in dogs younger than 6 months, and can cause enteritis, low hemoglobin, erythropenia, lymphopenia, leukopenia, thrombocytopenia, and hypoproteinemia.
ABSTRACT
Since its emergence in late 2019, the coronavirus disease 2019 (COVID-19) pandemic has caused severe disruption to key aspects of human life globally and highlighted the need for timely, adaptive, and accessible pandemic response strategies. Here, we introduce the cell-free dot blot (CFDB) method, a practical and ultra-low-cost immune diagnostic platform capable of rapid response and mass immunity screening for the current and future pandemics. Similar in mechanism to the widely used enzyme-linked immunosorbent assays (ELISAs), our method is novel and advantageous in that (i) it uses linear DNA to produce the target viral antigen fused to a SpyTag peptide in a cell-free expression system without the need for traditional cloning and antigen purification, (ii) it uses SpyCatcher2-Apex2, an Escherichia coli-produced peroxidase conjugate as a universal secondary detection reagent, obviating the need for commercial or sophisticated enzyme conjugates, and (iii) sera are spotted directly on a nitrocellulose membrane, enabling a simple "dipping" mechanism for downstream incubation and washing steps, as opposed to individual processing of wells in a multiwell plate. To demonstrate the utility of our method, we performed CFDB to detect anti-severe acute respiratory syndrome coronavirus 2 nucleocapsid protein antibodies in precharacterized human sera (23 negative and 36 positive for COVID-19) and hamster sera (16 negative and 36 positive for COVID-19), including independent testing at a collaborating laboratory, and we show assay performance comparable to that of conventional ELISAs. At a similar capacity to 96-well plate ELISA kits, one CFDB assay costs only ~$3 USD. We believe that CFDB can become a valuable pandemic response tool for adaptive and accessible sero-surveillance in human and animal populations. IMPORTANCE The recent COVID-19 pandemic has highlighted the need for diagnostic platforms that are rapidly adaptable, affordable, and accessible globally, especially for low-resource settings. To address this need, we describe the development and functional validation of a novel immunoassay technique termed the cell-free dot blot (CFDB) method. Based on the principles of cell-free synthetic biology and alternative dot blotting procedures, our CFDB immunoassay is designed to provide for timely, practical, and low-cost responses to existing and emerging public health threats, such as the COVID-19 pandemic, at a similar throughput and comparable performance as conventional ELISAs. Notably, the molecular detection reagents used in CFDB can be produced rapidly in-house, using established protocols and basic laboratory infrastructure, minimizing reliance on strained commercial reagents. In addition, the materials and imaging instruments required for CFDB are the same as those used for common Western blotting experiments, further expanding the reach of CFDB in decentralized facilities.
ABSTRACT
The aim of this study is to establish an indirect ELISA technique for detecting the SIgA antibody against porcine epidemic diarrhea virus (PEDV) to evaluate its mucosal immunity. Firstly, the S1D gene (534-789 aa) of PEDV was cloned into the pET-28a(+) vector, and induced in Escherichia coli BL21 (DE3) by IPTG, the product of which was in the form of inclusion bodies. According to Western-blot, the target protein S1D with antigenic activity was 32 ku in molecular weight and could be well detected. Then, the S1D protein was denatured by 8 mol/L urea, purified and gradient as the coating antigen to establish an indirect ELISA for detecting the PEDV specific SIgA antibody in nasal or oral mucus by optimizing conditions. And the optimal antigen coating concentration of ELISA was 2 micro g mL, the working concentrations of nasal mucus was 1:1 and the optimal blocking solution was 50 g/L skimmed milk, while the working concentrations and optimal blocking solution were 1:2 and 30 g/L BSA in oral mucus, the working concentrations of the enzyme-labeled antibody was 1:2 000 in nasal and oral mucus. Finally, 84 samples of oral and nasal mucus from immunized pigs were detected by S1D of ELISA, and the coincidence rate could reach 95.2% compared with purified PEDV of ELISA. In conclusion, the indirect ELISA established in this study provided a quick, simple, sensitive, and specific method to detect PEDV specific SigA for evaluating the level of PEDV mucosal immunity.
ABSTRACT
This study aims to investigate the protective effect of infected piglets which were immunized with different dose of inactivated porcine epidemic diarrhea virus (PEDV) vaccines. The number of infective virus particles and total virus particles of PEDV with different concentrations were determined, and the mice were immunized with different concentration vaccine prepared as antigen, respectively. The humoral and cellular immune production were determined by ELISA antibody detection method, neutralization test and ELISPOT method. Vaccine with appropriate antigen content was selected to immunize piglets, then the antibody was determined. The relationship between concentrated vaccine and protective effect was studied by challenge experiment. The results showed that, when the antigen dose was equal or greater than 8x106 pfu.mL-1, the inactive vaccine could effectively stimulate mice to produce humoral and cellular immunity. The piglets immunized with 2 mL inactivated PEDV vaccine containing 8x106 pfu.mL-1 antigen could resist diarrhea and continuous viral shedding caused by PEDV challenge. Compared with the total number of virus particles, the number of infectious virus particles was significantly correlated with antibody production (r=0.998 1), and neutralization titer was significantly correlated with piglet protection (r=0.974 7). PEDV inactivated vaccine can provide good immune protection, in which the number of infectious virus particles is the key factor to improve the antibody level. Antibody titer, as an index of humoral immunity, is an important reference for judging immune protection.
ABSTRACT
This bulletin highlights the effects of COVID-19 in the dairy cattle sector with emphasis on milk prices and milk yield.
ABSTRACT
The purpose of this study is to establish a blocking ELISA antibodies detection method for porcine epidemic diarrhea virus (PEDV). The purified N protein was used as the coating antigen, and the ELISA reaction conditions were optimized by the chess rboard titration. A blocking ELISA method for detecting PEDV antibodies was established, and its specificity, sensitivity and repeatability tests were carried out. One hundred and forty clinical serum samples were tested, and the results were compared with commercially IDvet PEDV indirect ELISA antibodies detection kit. The results showed that the best antigen coating concentration was 625 ng.mL-1, and the best dilution ratio of serum was 1:1;The best dilution of the HRP-conjugated antibody working solution was 1:5 000;There was no cross-reaction with healthy pig serum and the positive sera of common pig disease pathogens, such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and transmissible gastroenteritis virus (TGEV). The sensitivity of PEDV positive serum was 1:16, which was equivalent to that of IDvet ELISA kit (titer 1:32). The coefficient of variation of within-run and between-run repeatability test is less than 10%, so it showed that the blocking ELISA established in this study had good repeatability and stability;the kappa value of detected 140 clinical porcine serum using this method was 0.87 when compared with IDvet ELISA. The above results indicated that the established blocking ELISA method for detecting PEDV antibodies in this study could be applied to the prevention and control of PEDV, epidemiological investigation and the monitoring of antibody levels after vaccine immunization.
ABSTRACT
Bovine coronavirus (BCoV) infections are widespread in newborn calf diarrhea, which is one of the critical problems in cattle breeding. This study aims to investigate BCoV infection in calves with diarrhea in Sanliurfa province. In this study, 94 calves with diarrhea (3 months) following clinical examination were sampled. Enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of BCoV antigen. A total of 5 stool samples were found to be BCoV positive (5.32%). This result showed that BCoV was low in calves with diarrhea in Sanliurfa province. However, considering the rapid spread of the infection in cattle populations, it is thought that it may cause significant economic losses due to treatment costs and calf deaths.
ABSTRACT
The development of versatile and sensitive biotools to quantify specific SARS-CoV-2 immunoglobulins in SARS-CoV-2 infected and non-infected individuals, built on the surface of magnetic microbeads functionalized with nucleocapsid (N) and in-house expressed recombinant spike (S) proteins is reported. Amperometric interrogation of captured N- and S-specific circulating total or individual immunoglobulin (Ig) isotypes (IgG, IgM, and IgA), subsequently labelled with HRP-conjugated secondary antibodies, was performed at disposable single or multiplexed (8×) screen-printed electrodes using the HQ/HRP/H2 O2 system. The obtained results using N and in-house expressed S ectodomains of five SARS-CoV-2 variants of concern (including the latest Delta and Omicron) allow identification of vulnerable populations from those with natural or acquired immunity, monitoring of infection, evaluation of vaccine efficiency, and even identification of the variant responsible for the infection.
Subject(s)
Biosensing Techniques , COVID-19 , Antibodies, Viral , COVID-19/diagnosis , COVID-19 Testing , Humans , Immunity , Immunoglobulin G , SARS-CoV-2 , Spike Glycoprotein, CoronavirusABSTRACT
The development of new, low-cost vaccines and effective gene therapies requires accurate delivery and high-level expression of candidate genes. We developed a plasmid vector, pIDV-II, that allows for both easy manipulation and high expression of exogenous genes in mammalian cells. This plasmid is based upon the pVax1 plasmid and shares a common structure with typical mammalian transcription units. It is composed of a chicken ß-actin promoter (CAG), followed by an intron and flanked by two restriction sites, and also includes a post-transcriptional regulatory element, followed by a transcriptional termination signal. While the modification of pVax1 elements either decreased eGFP expression levels or had no effect at all, replacement of the promoter, the poly-A signal, deletion of the T7 and AmpR promoters, and inversion of the ORI-Neo/Kan cassette, significantly increased in vitro eGFP expression with the modified plasmid called pIDV-II. To further evaluate our vector, expression levels of three viral antigens were compared in cell lines transfected either with pVax1 or pCAGGS backbones as controls. Higher transgene expression was consistently observed with pIDV-II. The humoral and cellular responses generated in mice immunized with pIDV-II vs pVax1 expressing each viral antigen individually were superior by 2-fold or more as measured by ELISA and ELISPOT assays. Overall these results indicate that pIDV-II induces robust transgene expression, with concomitant improved cellular and humoral immune responses against the transgene of interest over pVax1. The new vector, pIDV-II, offers an additional alternative for DNA based vaccination and gene therapy for animal and human use.
Subject(s)
Vaccines, DNA , Animals , DNA , Immunity, Humoral , Mice , Mice, Inbred BALB C , Transgenes , Vaccines, DNA/geneticsABSTRACT
Fifty years ago, the first landmark structures of antibodies heralded the dawn of structural immunology. Momentum then started to build toward understanding how antibodies could recognize the vast universe of potential antigens and how antibody-combining sites could be tailored to engage antigens with high specificity and affinity through recombination of germline genes (V, D, J) and somatic mutation. Equivalent groundbreaking structures in the cellular immune system appeared some 15 to 20 years later and illustrated how processed protein antigens in the form of peptides are presented by MHC molecules to T cell receptors. Structures of antigen receptors in the innate immune system then explained their inherent specificity for particular microbial antigens including lipids, carbohydrates, nucleic acids, small molecules, and specific proteins. These two sides of the immune system act immediately (innate) to particular microbial antigens or evolve (adaptive) to attain high specificity and affinity to a much wider range of antigens. We also include examples of other key receptors in the immune system (cytokine receptors) that regulate immunity and inflammation. Furthermore, these antigen receptors use a limited set of protein folds to accomplish their various immunological roles. The other main players are the antigens themselves. We focus on surface glycoproteins in enveloped viruses including SARS-CoV-2 that enable entry and egress into host cells and are targets for the antibody response. This review covers what we have learned over the past half century about the structural basis of the immune response to microbial pathogens and how that information can be utilized to design vaccines and therapeutics.