Double Sandwich Enzyme-linked Immunosorbent Assay Research Articles

Development of an enzyme-linked immunosorbent assay using a monoclonal antibody to a dominant epitope in non-structural protein 4 of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is one of the most severe swine diseases causing great economic losses for the international swine industry. Non-structural protein 4 (NSP4) is critical to the life cycle of PRRSV and contains dominant B cell epitopes. This study prepared a monoclonal antibody against Nsp4, and 2D11, which contained the sequence 138KQGGGIVTRPSGQFCN153, was confirmed as the epitope. A 2D11-based double antibody sandwich enzyme-linked immunosorbent assay (dasELISA) was next developed with a cut value of 0.1987. A total of 1354 pig serum samples were detected by dasELISA and compared to a commercial ELISA kit (N-coated iELISA), resulting in a positive coincidence rate of 98.8% and negative coincidence rate of 96.9%. A total of 119 sera were positive by dasELISA while negative by iELISA. Higher positive rates by dasELISA were found in pig farms where PRRSV antibody levels varied widely. These results indicated that the dasELISA was a useful tool to detect PRRSV antibody in clinical samples.

Epidemiological assessment of hepatitis E virus infection among 1565 pregnant women in Siem Reap, Cambodia using an in-house double antigen sandwich ELISA.

This study investigated hepatitis E virus (HEV) prevalence among pregnant women in Siem Reap, Cambodia, by developing a cost-effective, user-friendly in-house enzyme-linked immunosorbent assay (ELISA) for detecting total anti-HEV immunoglobulins (Ig). The in-house ELISA was designed for large-scale screening in resource-limited settings. Its performance was benchmarked against two commercial tests: the Anti-HEV IgG EIA (Institute of Immunology, Co. Ltd) and the Anti-HEV IgG RecomLine LIA (Mikrogen). The in-house ELISA demonstrated a sensitivity of 76% and 71.4%, and a specificity of 94.1% and 98.6%, against the two commercial tests, respectively, with overall agreement rates of 92.4% and 94.3%. Among 1565 tested pregnant women, 11.6% were anti-HEV positive. Prevalence increased with age, particularly in women aged 35-40years and over 40years. No significant associations were found with education, number of children, family size, or history of blood transfusion and surgery, except for the occupation of the family head as a public officer. Of the total anti-HEV positive women, 22.7% had anti-HEV IgM, indicating recent or ongoing infection. The study concluded that the in-house ELISA is a viable option for HEV screening in regions with limited resources due to its high accuracy and cost-effectiveness. It is particularly suitable for large-scale studies and public health interventions in areas where HEV is endemic and poses a significant risk to pregnant women.

Influence of genotypes and crop growth stage at time of infection on seed transmission of Maize chlorotic mottle virus

Understanding the effect of seed transmission of Maize chlorotic mottle virus (MCMV) is important in managing the spread of the disease especially secondary transmission caused by vectors. This study was set out to investigate the effect of different genotypes and crop growth stage at the time of infection to rate of MCMV transmission from seeds to seedlings. The experiments were laid out in randomised complete block design with three replications. Detection of MCMV in the grow outs was determined using double antibody sandwich enzyme-linked immunosorbent assay and real time reverse transcription polymerase chain reaction. The results showed low rates of seed transmission from the genotypes in which their was detection of MCMV in their samples (0.08–1%). Similarly, crop growth stage at time of infection also showed low transmission rate of up to 0.1%, with highest transmission rate at the four-leaf stage.

Nanobody-based immunomagnetic separation platform for rapid isolation and detection of Salmonella enteritidis in food samples.

Rapid separation and identification of Salmonella enteritidis (S. enteritidis) in food is of great importance to prevent outbreaks of foodborne diseases. Herein, by using O and H antigens as targets, an epitope-based bio-panning strategy was applied to isolate specific nanobodies towards S. enteritidis. This method constitutes an efficient way to obtain specific antibody fragments and test pairwise nanobodies. On this basis, a double nanobody-based sandwich enzyme-linked immunosorbent assay (ELISA) coupled with immunomagnetic separation (IMS) was developed to rapid enrich and detect S. enteritidis in food. The detection limit of the IMS-ELISA was 3.2×103 CFU/mL. In addition, 1CFU of S. enteritidis in food samples can be detected after 4-h cultivation, which was shortened by 2h after IMS. The IMS-ELISA strategy could avoid matrix interference and shorten the enrichment culture time, which has great potential for application in monitoring bacterial contamination in food.

Investigation of serum surface active protein D and clara cell protein levels in workers exposed to silica dust in ferrous metal foundry

Objective: To investigate the levels of serum surface active protein D (SP-D) and clara cell protein (CCl6) in workers exposed to black silica dust, and analyze its influencing factors. Methods: From July to September 2021, 174 workers in 37 positions exposed to silica dust in 5 ferrous metal foundry were investigated by cross-sectional research method. The exposure concentration of silica dust workers was obtained through occupational health field investigation and detection, and the general situation of the study subjects was obtained through questionnaire survey and peripheral blood was collected. Double antigen sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of SP-D and CC16 in serum of workers. The mean values were compared by one-way ANOVA, and the influencing factors of SP-D and CC16 concentrations in serum were analyzed by ordered multiple logistic regression. Results: The time-weighted average concentration (C-TWA) of 174 workers exposed to silica dust (respirable dust) ranged from 0.09 mg/m(3)~3.58 mg/m(3), and the C-TWA overstandard rate of dust exposed workers was 32.18% (56/174) , with differences among workers in different positions (χ(2)=28.85, P<0.001) . The highest concentration of silica dust was (0.82±0.11) mg/m(3). Using C-TWA<50% OEL occupational exposure limit (OEL) as reference, serum SP-D concentration in workers with ≥50% OEL was increased (OR=4.95, 95%CI: 1.86~13.17, P=0.001) , while CC16 concentration was decreased (OR=0.15, 95%CI: 0.05~0.40, P<0.001) ; Serum CC16 concentration decreased in workers exposed to silica dust C-TWA≥OEL (OR=0.46, 95%CI: 0.28~0.98, P=0.043) . Compared with those with low occupational health literacy, the serum SP-D concentration of workers with high occupational health literacy decreased (OR=0.48, 95%CI: 0.25~0.92, P=0.027) and CC16 concentration increased (OR=2.09, 95%CI: 1.10-3.97, P=0.024) . Conclusion: When no abnormality was found in the physical examination of workers, the serum SP-D and CC16 concentration levels changed, and the change was related to the concentration of workers exposed to silica dust.

First report of Tomato bushy stunt virus naturally infecting sugar beet in the United States.

Sugar beet (Beta vulgaris L.) is an important crop grown for its sucrose content used in sugar production around the world. Tomato bushy stunt virus (TBSV) is an RNA virus that belongs to the Tombusvirus genus of the family Tombusviridae (Hearne et al., 1990). The virus was first isolated from tomato, and it is known to infect a wide range of plants (Smith, 1935; Martelli et al., 1988; Hafez et al., 2010). In 1980, a natural infection of TBSV was reported in sugar beet leaves with chlorotic and necrotic ring spots and line pattern symptoms based on serological affinity to TBSV anti-sera in Czechoslovakia (Novak and Lanzova, 1980). In March 2021, sugarbeet plants showing stunted and bushy growth with yellowing and necrotic leaves were observed in a production field in the Imperial Valley of California. Harvested roots exhibited stunted and abnormal growth compared to roots from healthy plants (sFig. 1A). These symptoms prompted a screen for potential infection by TBSV. Root-tissue harvested from the symptomatic sugar beet was initially screened using a TBSV double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA; Agdia, Inc., Elkhart, IN), which reacted positive for TBSV. To obtain the full-length sequence of TBSV and potentially other viruses in the sample, total RNA isolated using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) from the root-tissue was subjected to high-throughput sequencing (HTS). Libraries were prepared using the TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA) and sequenced using Illumina NovoSeq 6000 paired-end platform (Novogene, Sacramento, CA). A total of 52 million reads were obtained after removing the adapters and reads mapping to the host genome. These high-quality reads were de novo assembled into 75,891 contigs that are larger than 500 base pairs using the SPAdes assembler (Bankevitch et al., 2012; Prjibelski et al., 2020). The resulting contigs were searched for matching sequences to known viruses using the NCBI non-redundant database. A single contig of 4770 nts representing the full-length genome of TBSV was generated (Accession number OP477335), which showed 100% coverage to previously reported TBSV isolates 'statice' (AJ249740.1) and 'nipplefruit' (AY579432.1) with 92.19% and 91.25% nucleotide sequence identities, respectively, and thus confirming the presence of TBSV in sugar beet root-tissue. However, it showed 74% coverage with only 87% nucleotide identity to a previously reported Lettuce necrotic stunt virus (LNSV) from sugar beet, a tombusvirus that was re-classified as Moroccan pepper virus (MPV) due to high degree (>97%) of sequence identity (Obermeier et al., 2001; Wintermantel and Anchieta, 2012; Wintermantel and Hladky, 2013). The coat protein is conserved within species in tombusvirus, and it plays a significant role by providing serological relationships to tombusvirus taxonomy. The coat protein of TBSV-isolate of this study shared 98.45% and 96.91% identities at amino acid level with TBSV 'nipplefruit' (AY579432.1) and TBSV 'statice' (AJ249740.1) isolates, respectively. In contrast, it showed only 61.56% identity with the coat protein of MPV as shown in the phylogenetic tree indicating that the TBSV-isolate reported here is different from MPV (sFig. 2). To confirm the presence of TBSV, reverse-transcription (RT)-PCR was performed using the total RNA isolated from the root-tissue with primers (VR306: 5'-CGCTCACGAGCCCAGCATCCTTGA-3' and VR297: 5'-ACACCGCCACAGGAGCCATGATTG-3') designed based on the HTS data to amplify a portion of the TBSV genome. Sequencing of the RT-PCR product confirmed the presence of TBSV sequence with 99.1% identity to the TBSV-isolate identified in this study. Further, mechanical inoculation of total RNA isolated from the symptomatic sugar beet roots produced local lesions and systemic necrosis symptoms on the leaves of Chenopodium quinoa (sFig. 1B). Sequencing of the amplicon obtained using RT-PCR with primers VR306 and VR297 confirmed the presence of TBSV in C. quinoa. In addition to TBSV, several viral contigs representing Beet necrotic yellow vein virus were identified in the root-tissue indicating mixed infection in the field. To our knowledge, this is the first report that documents the occurrence of TBSV in sugar beet in the United States. Since TBSV is a soil-borne virus, our findings indicate the need for further studies focused on the frequency and coexistence of the TBSV with BNYVV in sugar beet production fields to understand the disease complexity resulting from potential mixed infections.

Toxicokinetics of recombinant human fibroblast growth factor 21 for injection in cynomolgus monkey for 3months.

Introduction: Recombinant human fibroblast growth factor 21 (FGF-21) is a potential therapeutic agent for multiple metabolic diseases. However, little is known about the toxicokinetic characteristics of FGF-21. Methods: In the present study, we investigated the toxicokinetics of FGF-21 delivered via subcutaneous injection in vivo. Twenty cynomolgus monkeys were injected subcutaneously with different doses of FGF-21 for 86days. Serum samples were collected at eight different time points (0, 0.5, 1.5, 3, 5, 8, 12, and 24h) on day 1, 37 and 86 for toxicokinetic analysis. The serum concentrations of FGF-21 were measured using a double sandwich Enzyme-linked immunosorbent assay. Blood samples were collected on day 0, 30, 65, and 87 for blood and blood biochemical tests. Necropsy and pathological analysis were performed on d87 and d116 (after recovery for 29days). Results: The average AUC(0-24h) values of low-dose FGF-21 on d1, d37, and d86 were 5253, 25268, and 60445μgh/L, and the average AUC(0-24h) values of high-dose FGF-21 on d1, d37, and d86 were 19964, 78999, and 1952821μgh/L, respectively. Analysis of the blood and blood biochemical indexes showed that prothrombin time and AST content in the high-dose FGF-21 group increased. However, no significant changes in other blood and blood biochemical indexes were observed. The anatomical and pathological results showed that continuous subcutaneous injection of FGF-21 for 86 days did not affect organ weight, the organ coefficient, and histopathology in cynomolgus monkeys. Discussion: Our results have guiding significance for the preclinical research and clinical use of FGF-21.

First record of potato virus s (PVS) and Detect ordinary strain PVS° in Nineveh Governorate

The results of a field survey of some potato growing areas in Nineveh Governorate showed the presence of Potato virus s PVS, which was detected by the Double Antibody Sandwich Enzyme-Linked ImmunoSorbent Assay DAS-ELISA test. This record is the first in the governorate. Through the absorbance readings using the elisa reader, highest value was recorded in the Sherikhan area samples, which amounted to 1.190 nm, while lowest value was recorded in the Rabia area samples with a value of 0.756 nm, while absorbance value of the negative standard sample was 0.300 nm. The frequency of virus in the Sherikhan area was 53.3%, while frequency of the virus in the Rabia area was 28.8%. As a result of mechanical inoculation with virus isolates diagnosed by DAS-ELISA test, spread of the common strain PVSO through symptoms on Chenopodium quinoa, which was in the form of small chlorotic spots on the inoculated leaves, and new leaves were devoid of any disease symptoms.

Impact of Toll-like Receptor 4 Expression on Inflammatory Responses Related to Premature Membrane Rupture Induced by Lipopolysaccharide.

This study aimed to determine the mechanism through which the expression of Toll-like receptor 4 (TLR4) influences the lipopolysaccharide (LPS)-induced inflammatory response, a condition that is associated with premature rupture of membranes (PROM). Human myeloid leukemia mononuclear cells (THP-1) were employed as the experimental model. These cells were treated with LPS and the TLR4 inhibitor CLI-095 and subsequently divided into three groups. A range of assays were utilized, including methyl thiazolyl tetrazole (MTT) assay, real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) for measuring TLR4 and tumor necrosis factor α (TNF-α) mRNA levels, double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for assessing monocyte chemoattractant protein 1 (MCP-1) and matrix metalloproteinase 9 (MMP-9), as well as secretion levels of interleukin (IL)-6 and IL-1β. And western blotting was used to detect the expression of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) p65, which are components of the TLR4 downstream signaling pathway. The LPS-induced proliferation of THP-1 cells was significantly inhibited (p < 0.05) when compared with normal THP-1 cells. Moreover, LPS also promoted TLR4 mRNA and protein expression levels, TNF-α mRNA expression, secretion of inflammatory factors, and phosphorylation of ERK and NF-κB p65 proteins (p < 0.05). On the other hand, administration of the TLR4 inhibitor CLI-095 significantly inhibited the expression of TLR4 mRNA and protein. It also effectively increased the proliferative activity of THP-1 cells and inhibited the secretion of TNF-α and inflammatory factors, as well as the phosphorylation of ERK and NF-κB p65 proteins (p < 0.05). In summary, suppressing TLR4 expression can mitigate inflammatory responses, thereby reducing the likelihood of premature rupture of membranes during pregnancy, which is often triggered by such inflammation.

Development and evaluation of recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (ELISA) and double antigen sandwich ELISA for detection of antibodies to Chikungunya virus

Chikungunya virus (CHIKV) reemerged and caused millions of human infections since 2004. The disease could be established, when the virus has been introduced to areas where the appropriate vectors are endemic. The differential diagnosis of CHIKV infection varies based on place of residence, travel history, and exposures. Serological tests are commonly used to diagnose CHIKV infection, but their availability and assessments of the performance of the diagnostics have been limited. To develop and evaluate antibodies detection methods for chikungunya diagnosis and serological investigation. Recombinant E2 protein based IgM capture enzyme-linked immunosorbent assay (Mac-ELISA) and double antigen sandwich ELISA (Das-ELISA) for detection of antibodies to Chikungunya virus were developed and evaluated. The repeatability was evaluated by testing of three reference sera at single dilutions in triplicated for 5 times. The sensitivity, specificity, accuracy, and agreement of the MAC-ELISA and Das-ELISA were obtained by comparing the detection results of 225 serum samples (45 positive; 180 negative) with a real-time RT-PCR assay and an IFA commercial tests manufactured by Euroimmun. The established ELISA assays were standardized by determining the optimal concentrations of the key reagents. The coefficient values of repeat testing were within 10% and 20% for intraassay and interassay precision, respectively. A sensitivity of 60.0% and 52.5%, a specificity of 96.2% and 96.8%, and an accuracy of 89.8% and 88.9% were obtained for the Mac-ELISA and Das-ELISA, respectively, when compared to a CHIKV qRT-PCR method. And a sensitivity of 100%, a specificity of 97.5% and 99.5%, and an accuracy of 97.8% and 99.6% were yielded respectively when using the IIFT as a reference method, which showed a highly consistence to the commercial IIFT assay with a Kappa value greater than 0.90. The Mac-ELISA and Das-ELISA based on recombinant E2 protein of CHIKV were developed and standardized, which could detect IgM or total antibodies against CHIKV in 2-3 hours with acceptable sensitivities and specificities. These assays can be used for laboratory diagnosis and serological investigation of CHIKV infections to evaluate the risk of CHIKV transmission.

Optimization of culture conditions for stable expression of recombinant fc-fused human extracellular CD99 in HEK293T cells

CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T-cell activation, cell adhesion, and cell migration. We have also demonstrated that CD99 and its ligands regulate proinflammatory cytokines in NK cells, monocytes and activated T cells. These data suggest CD99 as a potential therapeutic target in cancer. However, the molecular mechanisms by which CD99 and CD99 counter receptors participate in such processes are unclear. High-quality CD99 recombinant proteins produced in large amounts are essential for biological studies and clinical research. In this study, we optimized the various culture conditions for increasing amounts of recombinant protein production with good biological activity. Intracellular immunofluorescence staining was performed to identify the highly expressing CD99HIgG cells. We further investigated the culture conditions for recombinant protein production. A double antibody sandwich enzyme-linked immunosorbent assay was employed to determine the level of secreted CD99HIgG proteins in the culture supernatant of various culture conditions. Later, affinity chromatography using protein G was used to purify CD99HIgG proteins from the culture supernatant of three proper culture conditions. According to our previous report, which utilized Western blotting, the purified CD99HIgG obtained from all tested culture conditions is composed of the CD99 extracellular part fused with the human IgG Fc part in dimer form. For biological activity, the obtained CD99HIgG proteins showed the ability to ligate with the CD99 counter receptor, resulting in the induction of cytokine production.

Low VGF is associated with executive dysfunction in patients with major depressive disorder

ObjectivesExecutive dysfunction is considered to be one of the cognitive impairment dimensions that are easily observed in depression, but its underlying molecular mechanism is still unclear. Study have shown that the neuropeptide VGF (non-acronymic) plays an important role in the regulation of hippocampal neurogenesis and neuroplasticity. Previous studies have shown that VGF may be related to the psychopathology of depression and cognitive impairment. However, the correlation between VGF and executive dysfunction in MDD has not been investigated. MethodsA total of 35 MDD patients and 31 healthy control patients were enrolled in this study. The 17-item Hamilton Depression Rating Scale (HDRS) was used to measure the severity of depression, and the Wisconsin Card Sorting Test (WCST) was used to assess executive dysfunction. Double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to determine serum VGF in peripheral blood. ResultsThe level of serum VGF in MDD patients was significantly lower compared to that in the healthy control group (p < 0.001). Moreover, Response Administered (RA) scores, Response preservative errors (RPE), and Non-response preservative errors (NRPE) were all higher in the MDD group (all p < 0.05). In contrast, Categories Completed (CC) and Response Correct (RC) scores were lower (all p < 0.05). Further results showed a significant correlation between serum VGF with RA (r = −0.372, p = 0.028) and RPE scores (r = 0.507, p = 0.002) in patients with depression, while serum VGF showed no correlation with the severity of depression in either group. ConclusionsVGF may play an important role in executive function dysfunction in MDD patients, and VGF levels may become a new marker for predicting executive function dysfunction in depression.

Inhibitory Effect of Chitosan and Phosphate Cross-linked Chitosan against Cucumber Mosaic Virus and Pepper Mild Mottle Virus.

Cucumber mosaic virus (CMV) and Pepper mild mottle virus (PMMoV) causes severe economic loss in crop productivity of both agriculture and horticulture crops in Korea. The previous surveys showed that naturally available biopolymer material – chitosan (CS), which is from shrimp cells, reduced CMV accumulation on pepper. To improve the antiviral activity of CS, it was synthesized to form phosphate cross-linked chitosan (PCS) and compared with the original CS. Initially, the activity of CS and PCS (0.01%, 0.05%, and 0.1% concentration) compound against PMMoV infection and replication was tested using a half-leaf assay on Nicotiana glutinosa leaves. The total number of local lesions represented on a leaf of N. glutinosa were counted and analyzed with phosphate buffer treated leaves as a negative control. The leaves treated with a 0.1% concentration of CS or PCS compounds exhibited an inhibition effect by 40–75% compared with the control leaves. The same treatment significantly reduced about 40% CMV accumulation measured by double antibody sandwich enzyme-linked immunosorbent assay and increased the relative expression levels of the NPR1, PR-1, cysteine protease inhibitor gene, LOX, PAL, SRC2, CRF3 and ERF4 genes analyzed by quantitative reverse transcriptase-polymerase chain reaction, in chili pepper plants.

Occurrence and Distribution of Viruses Associated with Okra and Their Alternative Hosts in Kaduna and Zamfara States, Nigeria.

One of the major constraints to production of okra (Abelmoschus esculentus L.) in Nigeria and in particular in Kaduna and Zamfara States, is the problem of okra mosaic virus and okra leaf curl virus. This study was carried out to provide information on the occurrence and distribution of okra mosaic and okra leaf curl viruses on okra, in Kaduna and Zamfara states, Nigeria. A survey of okra-producing farms was carried out during dry and wet seasons of 2017 cropping season in Kaduna (Zaria, Lere, and Igabi Local Government Areas) and Zamfara (Gusau, Bungudu, and Zurmi LGAs) states. Leaf samples (15) of symptomatic okra plants were collected from each farm in the study area. The total number of plants and the number of symptomatic plants within each subplot were recorded, and the disease incidence was determined. Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS- ELISA) kit was used in the detection of Okra Mosaic Virus while Polymerase Chain Reaction (PCR) was employed for the detection Okra Leaf Curl Virus. The results showed that all the okra leaf samples tested for OLCV were negative in this study while OkMV was tested positve in all the samples with a recorded incidence of 20 % and 14 % in Kaduna and Zamfara states respectively, however, only 8 out of total weed samples were also tested positive for OKV, but all were tested negative to OLCV.

Preparation of Let-7b Nanocomposite and Its Effect on the Level of Inflammatory Factors in Cerebrospinal Fluid of Purulent Meningitis

This study was to explore the effect and mechanism of Let-7b nanocomposite on the expression of inflammatory factors in the cerebrospinal fluid (CSF) of purulent meningitis. 45 patients with purulent meningitis (PM) were selected as observation group (group A), and 38 patients with normal CSF without central nervous system diseases were selected as the control group (group B). The CSF of the two groups were collected to detect the inflammatory factors interleukin-8 (IL-8), macrophage inflammatory protein-1α (MIP-1α), matrix metalloproteinase 9 (MMP9), interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), and Let-7b level with the double antibody sandwich enzyme-linked immunosorbent assay (ELISA). The Let-7b nanocomposite was prepared, and its morphology, particle size, and Zeta potential were analyzed. In addition, the degradation kinetics, cytotoxicity, and phagocytic efficiency (PE) of Let-7b nanocapsules were detected. 36 healthy adult New Zealand (NZL) rabbits were randomly grouped into a control group (group C) (0.9% normal saline (NS)), a model group (Escherichia coli (E. coli) modeling, group D), and a test group (E. coli modeling + Let-7b nanocapsules, group E), with 12 rabbits in each group. The changes of inflammatory factors in CSF of the three groups were detected and compared. It was found that the expression levels of IL-8 and IL-1 β in the group A were much higher than those in the group B (P &lt; 0.01), and the MMP9 and TNF-α levels in the group B were much lower in contrast to the group A (P &lt; 0.001). The expression of Let-7b in the group A was lower obviously in contrast to the group B (P &lt; 0.001). Let-7b nanocapsules were irregularly spherical, with an average particle size (APS) of 23.1 nm, a polydispersity index (PDI) of 0.232, and the Zeta potential of around +15 mV. Let-7b nanocapsules showed obvious polymer shell absorption peaks at 1,015 cm-1, 1,228 cm-1, and 1,547 cm-1. The IL-8 and IL-1β levels of the group D were greatly different from those in the other two groups (P &lt; 0.01). The levels of TNF-α and MMP9 in the group D were greatly different in contrast to the group C (P &lt; 0.001) and the group E (P &lt; 0.01). It indicated that Let-7b nanocomposite could lower the expression levels of IL-8, IL-1β, MMP9, and TNF-α in the CSF of patients with purulent meningitis dramatically, which provides a reliable basis for immunotherapy of purulent meningitis with Let-7b.

Development and Validation of an ELISA for the Detection of Bluetongue Virus Serotype 4-Specific Antibodies.

In this article, we describe the development and evaluation of a double antigen sandwich enzyme-linked immunosorbent assay (ELISA) able to detect serotype 4-specific antibodies from BTV-4 infected or vaccinated animals using a recombinant BTV-4 VP2 protein. The coding sequence of VP2 was inserted into a pVote plasmid by recombination in the Gateway® cloning system. Vaccinia virus (VacV) was used as a vector for the expression of the recombinant VP2. After production in BSR cells, recombinant VP2 was purified by immunoprecipitation using a FLAG tag and then used both as the coated ELISA antigen and as the HRP-tagged conjugate. The performance of the ELISA was evaluated with 1186 samples collected from BTV negative, infected or vaccinated animals. The specificity and sensitivity of the BTV-4 ELISA were above the expected standards for the detection of anti-BTV-4 VP2 antibodies in animals reared in Europe or in the Mediterranean basin. Cross-reactions were observed with reference sera for serotypes 10 and 20, and to a lesser extent with serotypes 12, 17 and 24, due to their genetic proximity to serotype 4. Nevertheless, these serotypes have never been detected in Europe and the Mediterranean area. This ELISA, which requires only the production of a recombinant protein, can be used to detect BTV serotype 4-specific antibodies and is therefore an attractive alternative diagnostic method to serum neutralization.

Effects of β-Glucans from Aureobasidium pullulans on Cucumber Mosaic Virus Infection in Chili Pepper

Cucumber mosaic virus (CMV), the most prevalent virus in chili pepper (Capsicum annuum L.), negatively affects chili pepper production in South Korea. In this study, foliar spraying with β-glucans obtained from the mycelial walls of the yeast-like fungus Aureobasidium pullulans inhibited CMV infection of chili pepper if applied before virus inoculation. At three concentrations, β-glucans from A. pullulans significantly ameliorated CMV symptoms in treated chili pepper; the effect was greater in plants treated with 0.01% β-glucans than 0.005% or 0.001% β-glucans. Double antibody sandwich enzyme-linked immunosorbent assay showed that these β-glucans treatments resulted in 1.7- to 10-fold reductions in CMV accumulation in the treated chili pepper. The glucans did not act directly on the virus and did not interfere with virus disassembly or replication. Foliar spraying with 0.01% β-glucans from A. pullulans at 24 hr intervals for 3 days significantly increased plant height, the total number of fruit, and the fresh weight of chili pepper fruit. However, the stem diameter of chili pepper treated with β-glucans did not increase significantly. These results indicate that foliar spraying with β-glucans from A. pullulans acts an antiviral agent against CMV infection and stimulates chili pepper growth.

Assessment of heterogeneity of two cultivars of Olea europaea based on the study of their Ole e 1 protein content.

Olive pollen is one of the main causes of allergic disease in the Mediterranean area. Ten different proteins with allergenic activity have been described in olive pollen, with major allergen Ole e 1. Olea europaea L. may cause allergenic effects of different severity depending on the Ole e 1 content of cultivars. In this paper, we aimed to assess the heterogeneity of two olive cultivars concerning concentrations of the major allergen Ole e 1 during a period of 2 years. Pollens from two most common olive cultivars, known as “Gemlik” and “Celebi,” were analyzed on regular basis. Ole e 1 amounts were measured by double-sandwich enzyme-linked immunosorbent assay (ELISA). The results were expressed as μg of Ole e 1 per μg of total freeze-dried extract. Comparisons of Ole e 1 levels were made both between individual trees and between cultivars. It was analyzed the influence of some meteorological parameters on pollen counts/allergenic content on a local scale, for 2 years. Pollen sampling was carried out continuously for 2 years, using a Hirst-type volumetric trap. “Gemlik” had the higher value (mean ± standard deviation) of Ole e 1 content (2.44 ±0.70 and 1.87 ±1.03 μg/μg, respectively) when compared to “Celebi” (2.16 ±0.86 and 0.20 ±0.30 μg/μg, respectively) in the years 2013 and 2015. In our research, daily variations were observed in pollen samples of two olive cultivars and even different trees of the same cultivar. Furthermore, during certain sampling days, discrepancies between airborne pollen counts and Ole e 1 concentrations were detected for both cultivars. It was found that meteorological changes, especially temperature and precipitation fluctuations, could affect airborne pollen and Ole e 1 allergen levels in the atmosphere. Therefore, pollen samples of different O. europaea cultivars demonstrated great differences in Ole e 1 content. We believe that these findings were a result of alternate bearing behavior modulated by meteorological factors.

Virus Quantification, Flowering, Yield, and Fruit Quality in Tropical Pumpkin (Cucurbita moschata Duchesne) Genotypes Susceptible or Resistant to Two Potyviruses

Infecting cucurbits around the world, Papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus (ZYMV) are members of the genus Potyvirus and family Potyviridae. Tropical pumpkin is grown globally in the lowland humid tropics. In Puerto Rico, tropical pumpkin is the second most important vegetable crop in economic value. In trials in Puerto Rico in 2016 and 2017, susceptible genotypes ‘Waltham’, Mos166, ‘Taína Dorada’ (2016 only), ‘Soler’ with moderate resistance to ZYMV, and resistant ‘Menina’ and ‘Nigerian Local’ were inoculated with PRSV and ZYMV and evaluated in the greenhouse and field. Mock-inoculated (buffer) controls were included. Puerto Rico strains of PRSV and ZYMV were originally collected from plants of Cucurbita moschata in Puerto Rico. Presence of virus was determined by Double Antibody Sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) and symptom severity was evaluated on a 0 to 5 scale in both trials. Days to anthesis of first staminate and pistillate flower were recorded for each plot. Number of fruits, fruit weight per plant, average fruit weight, fruit and mesocarp diameter, mesocarp color, °Brix, and percentage dry matter were measured in 2017. ‘Waltham’, Mos166, ‘Taína Dorada’, and ‘Soler’ tested positive for PRSV when inoculated with PRSV and positive for ZYMV when inoculated with ZYMV. For both PRSV and ZYMV, symptom severity was less (severity = 0) in resistant genotypes ‘Menina’ and ‘Nigerian Local’ than in all other genotypes. ‘Soler’ inoculated with ZYMV exhibited less symptom severity than that of susceptible genotypes. The degree of symptom severity of ‘Soler’ inoculated with PRSV was similar to susceptible genotypes. Symptom severity in plants inoculated with ZYMV was generally greater than when inoculated with PRSV. Compared with controls, yield per plant was unaffected by inoculation with potyvirus in resistant cultivar ‘Menina’. Unexpectedly, yield in resistant ‘Nigerian Local’ was reduced an average of 45% over control plots. Yield loss was 100% in inoculated plots of susceptible ‘Waltham’. Yield reduction ranged from 35% to 80% for susceptible Mos166 and moderately resistant ‘Soler’. There was little evidence that days to anthesis, average fruit weight, fruit diameter, and fruit quality (mesocarp thickness, chroma, hue angle, °Brix and dry matter) of plants inoculated with virus were different from that of uninoculated control plants. The exception was moderately resistant ‘Soler’ where plants inoculated with ZYMV produced fruits with a 32% reduction in average weight, as well as reductions in diameter, mesocarp thickness, and color saturation (chroma) compared with controls. This was unexpected given that ‘Soler’ has some resistance to ZYMV. Greenhouse evaluations by ELISA or symptom severity were generally useful in predicting field resistance to PRSV and ZYMV. In summary, yield reductions of up to 100% can be expected in C. moschata genotypes susceptible to PRSV or ZYMV, but fruit quality traits are usually unaffected. Moderate resistance to ZYMV in ‘Soler’ was observed to reduced symptom severity but not negative effects on yield and other traits. ‘Soler’ was not resistant to PRSV. ‘Menina’ rather than ‘Nigerian Local’ appears to be the best source of resistance because yield of the former was not impacted by inoculation with either potyvirus.

Utility of procalcitonin as an early diagnostic marker of bacteremia in individuals with periodontitis Stage II and III.

The aim of the present study is to assess the ability of procalcitonin (PCT) to differentiate between periodontal health and Stage II and III periodontitis. We further assessed, if PCT can reflect early bacteremia induced by non-surgical periodontal treatment (NSPT). Sixty-four systemically healthy individuals were divided into Group I, periodontally healthy, and Group II, Stage II and III periodontitis. NSPT was done for both the groups. Standardized serum and salivary samples were obtained and analyzed for PCT levels using highly sensitive double antibody sandwich enzyme-linked immunosorbent assay at baseline and 2 weeks. In addition, the serum levels of PCT were recorded at immediate and 1-hour post-NSPT. Mean PCT levels (saliva=0.03ng/mL and serum=0.05ng/mL) in periodontally healthy group were considerably lower than that in the periodontitis group (saliva=0.22ng/mL and serum=1.85ng/mL) with significant intergroup comparison at P< 0.001. Post NSPT the mean serum PCT values increased from 1.854ng/mL to 1.871ng/mL at the immediate interval and remained at 0.879ng/mL after 2 weeks at P< 0.001. Spearman correlation showed highly significant positive correlation between serum and salivary PCT values to clinical attachment level (CAL) at P< 0.001 and rho=0.78 and 0.75, respectively. Linear regression model showed serum PCT to be a significant predictor for CAL. Screening for serum PCT levels in patients with periodontitis could act not only as a guide to assess the bacterial load and use of antibiotics but also as a predictor for CAL loss in patients with periodontitis.