Double Antibody Sandwich Research Articles

Incidence and molecular characterization of potato leaf roll virus in seed potato production in Serbia

The distribution and frequency of potato leaf roll virus in the four most important potato growing regions in Serbia were studied during the seven years (2012–18). One hundred randomly collected potato tubers were sampled from each seed lot. The young leaves that developed in three weeks were sampled and tested to record infection rate. The presence of potato leaf roll virus was detected by double-antibody sandwich enzyme-linked immunosorbent assay and disease incidence was calculated using standard formula. The obtained result showed that the highest prevalence of potato leaf roll virus was detected from seed potato samples originated from the Raski region during 2018 (20.7%), while in the Moravicki region, only 2.3–11.1% of the potato leaf roll virus was detected every year. The average annual potato leaf roll virus infection was the highest in 2012 (8.4%) and 2018 (8.0%). For further confirmation of potato leaf roll virus infection, reverse transcription (RT)-PCR was performed using specific primers PLRVCPvEcoRI /PLRVCPcNcoI, designed to amplify a 650 bp fragment of the full-length coat protein gene. The PCR products derived from 26 isolates were directly sequenced using the same primer pair as in RT-PCR. The coat protein sequence analysis revealed that the Serbian potato leaf roll virus isolates showed very low nucleotide diversity (95.9–100%). They shared the highest nt identities of 98.08–99.36% with the sequences of potato leaf roll virus isolates deposited in the GenBank from other parts of the world. Phylogenetic analysis and the haplotype network of the coat protein gene sequences showed that the Serbian potato leaf roll virus isolates could be classified in two different groups indicating two possible introductions of the virus to Serbia. The results of this study confirmed the importance of potato leaf roll virus in seed potato production in Serbia. Additionally, this research highlights the need for a continuous monitoring of the potato seeds produced in Serbia as well as imported seeds for the presence of potato leaf roll virus.

Development of a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for the detection of KHV.

Koi herpesvirus disease (KHVD) caused by the koi herpesvirus (KHV) is difficult to diagnose in live fish, presenting a challenge to the koi industry. The enzyme-linked immunosorbent assay (ELISA) method cannot be widely used to detect KHV because few commercial anti-KHV antibody exists. Here, we developed an anti-ORF132 polyclonal antibody and confirmed its reactivity via indirect immunofluorescence assay and Western blotting. A double-antibody sandwich ELISA (DAS-ELISA) was established to detect KHV, monoclonal antibody 1B71B4 against ORF92 was used as the capture antibody, and the detection antibody was the polyclonal antibody against the truncated ORF132. The lowest limit was 1.56ng/ml KHV. Furthermore, the DAS-ELISA reacted with KHV isolates, while no cross-reactions occurred with carp oedema virus, spring viraemia of carp virus, frog virus 3 and grass carp reovirus. Two hundred koi serum samples from Guangdong, China, were used in the DAS-ELISA test, and the positive rate of the koi sera was 13%. The clinical sensitivity and specificity of the DAS-ELISA relative to the traditional PCR method were 66.7% and 97.6%, respectively. Our findings may be useful for diagnosing and preventing KHVD in koi and common carp.

First Report of Garlic virus B infecting Garlic in India.

Garlic (Allium sativum L.) is an economically important spice and vegetable crop grown throughout the world. Garlic viral disease complex caused by multiple virus infections is an important constraint in exploiting the potential yield of garlic. Among these viral pathogens, allexivirus (family Alphaflexiviridae) is the genus of viruses known for their degenerative effect on garlic yield. Their coexistence with other viruses, particularly potyviruses, has an adverse effect on garlic yield and quality (Perotto et al. 2010). During Sept 2018, while screening garlic germplasm accessions for the presence of allexiviruses, symptoms like foliar mosaic and curling were observed on accession G-204, planted at an experimental plot of ICAR-DOGR, Pune, India. A total of five samples comprised of five randomly selected G-204 garlic plants were collected from the experimental plot. Each sample contained leaves from the top, middle, and bottom portion of the individual garlic plants. These samples were subjected to RNA extraction using the RNeasy Plant Mini Kit (Qiagen, Germany) followed by reverse transcription (RT) using the Transcriptor cDNA synthesis kit (Roche Diagnostics, GmbH, Germany). The extracted RNA was then tested for allexiviruses such as garlic virus A (GarV-A), garlic virus B (GarV-B), garlic virus C (GarV-C), garlic virus D (GarV-D), and garlic virus X (GarV-X) by polymerase chain reaction (PCR) (Gawande et al. 2015; Roylawar et al. 2019; Baranwal et al. 2011; Gieck et al. 2009). Leaf samples tested through RT-PCR were found positive for garlic viruses GarV-A, GarV-B, GarV-C, GarV-D, and GarV-X. Allexiviruses other than GarV-B had been previously reported in India and hence further tests were conducted to confirm GarV-B infection. RT-PCR using primers, CF 5'- ATGGGAGACAGGTCGCAA-3' and CR5'- CTAAAATGTAAGCATGAGCGGT-3' designed specific to the coat protein yielded a 735-bp amplicon from all five G-204 plants. The amplified product was purified using QIAquick PCR Purification Kit (Qiagen, Germany) and cloned in pJET1.2 vector (Thermo Scientific, Lithuania). Two clones containing the CP gene were bidirectionally sequenced, and a consensus sequence was submitted to GenBank (MN650206). BLASTn results indicated that this consensus sequence showed 97.96% nucleotide (KP657919.1) and 100% amino acid sequence (AKN19940.1) identity with the CP sequence of GarV-B isolate from Poland. The presence of GarV-B was confirmed by enzyme-linked immunosorbent assay (ELISA) using a double-antibody sandwich ELISA kit (Arsh Biotech, Delhi, India) as per the manufacturer's protocol. An absorbance of reaction was read using a microplate reader at 405 nm. The mean OD values of negative and positive controls were 0.034 and 0.373, respectively. The OD values of five samples tested ranged from 0.210 to 0.296 indicating a positive reaction for GarV-B. To assess the presence of GarV-B in the available genetic stock, we tested 30 garlic germplasm accessions for GarV-B using RT-PCR. Out of these, 17 accessions were found positive for GarV-B. GarV-B has been reported from many countries (Gieck et al. 2009). This is the first report of GarV-B from India. Globally, allexiviruses are known for their adverse impact on garlic production (Oliveira et al. 2014). GarV-B together with other viruses can be a potential threat to garlic production in India. Further, detailed evaluations are needed to study the impact of GarV-B on garlic production in India.

Role of Serum level of IL-1B and MMP-13 in detection of severity and functional impairment in hand Osteoarthritis patients

Hand Osteoarthritis (HOA) is one of the most common chronic degenerative joint diseases. Pain and decreased function are the leading symptom in HOA. Previously HOA was considered a pure degenerative disease, but this concept has changed and current research has demonstrated that inflammation is one of the key factors leading to the cartilage destruction. Cytokines as Interleukin-1β (IL1- β) induces expression and release of proteolytic enzymes such as matrix metalloproteinases (MMPs) and aggrecanases. Chondrocyte-derived MMPs are the main enzymes involved in the breakdown of cartilage collagens and proteoglycans. Research in the area of Hand OA is important to help early diagnosis, stop the progression of the disease, and preventing disability.The aim was to determine role of serum level of IL-1β and MMP-13 in detection of severity and functional impairment in patients with HOA.The study was carried out on 30 patients diagnosed with HOA, fulfilling the ACR 1990 criteria for diagnosis of HOA. Demographic data and anthropometric measurements were recorded for all participants. Clinical characteristics of the disease including duration of disease, pattern of joint involvement, pain and disease severity as well as functional assessment of HOA were assessed. Radiographic severity of HOA was assessed by Kellgren and Lawrence (K-L) score. Serum IL-1β and MMP-13 were assessed using double-antibody sandwich enzyme-linked immunosorbent assay method in both patients and control.The conclusion was that Interleukin-1B and MMP-13 seem to have an insignificant role in detection of severity and functional impairment in mild to moderate HOA.

Higher Levels of Triglyceride, Fatty Acid Translocase, and Toll-Like Receptor 4 and Lower Level of HDL-C in Pregnant Women with GDM and Their Close Correlation with Neonatal Weight

Objectives: In this study, we aimed to compare the levels of maternal blood lipids, placental and venous blood lipid transporters, and inflammatory factor receptors in pregnant women with and without gestational diabetes mellitus (GDM). We also aimed to figure out the relationship between these values and neonatal weight. Methods: Fifty pregnant women with GDM under blood glucose control belong to the case group, and 50 pregnant women with normal glucose tolerance in concurrent delivery belong to the control group. Fasting venous blood of these pregnant women was taken 2 weeks before delivery, and umbilical cord blood was collected after delivery. The levels of triglyceride (TG), serum total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol (HDL-C) in maternal blood and umbilical cord blood were tested in the laboratory department of our hospital. The level of toll-like receptor 4 (TLR4) in serum of umbilical veins was detected by the double-antibody sandwich ELISA. Western blot and RT-PCR were used to detect the protein and mRNA expressions of TLR4, LPL, and FAT/CD36 in the placenta. Results: The level of TG in maternal blood in the case group was remarkably higher than that in the control group, which was opposite to the level of HDL-C. In the umbilical cord blood of women with GDM, the expression of TLR4 increased and was closely correlated with neonatal weight. In the placenta of women with GDM, the expressions of FAT/CD36 and TLR4 increased, and both of them were closely correlated with neonatal weight. Besides, TLR4 in umbilical cord blood increased and was closely correlated with neonatal weight. Although the expression of LPL in the placenta decreased, it had no obvious correlation with neonatal weight. Conclusions: TG in maternal blood, TLR4 in the placenta and umbilical cord blood, and FAT/CD36 in the placenta were positively correlated with neonatal weight. However, HDL-C in maternal blood was negatively correlated with neonatal weight. Although the expression of LPL in the placenta reduced due to GDM, it had no correlation with neonatal weight.

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.

Occurrence and molecular characterization of alfalfa mosaic virus in eggplant in Serbia

In the 2018-19 growing season, a total of 51 leaves of eggplant plants grown under field conditions were collected randomly from nine private gardens at four different localities in the Province of Vojvodina. Eggplants with nearly 40% of plants showing bright yellow to white mosaic or mottling of leaves were found throughout the inspected fields (gardens). The collected samples were analyzed for the presence of alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV) and potato virus Y (PVY) using commercial double-antibody sandwich (DAS)-ELISA kits. Serological analysis of eggplant samples revealed the presence of AMV in 80.39% collected samples. None of the analyzed samples was positive for CMV and PVY. The virus was successfully mechanically transmitted to test plants including Nicotiana benthamiana, Chenopodium quinoa, C. amaranticolor, as well as eggplant seedlings, confirming the infectious nature of the disease. The presence of AMV in eggplants was further verified by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing, using the primers CP AMV1 and CP AMV2 that amplify part of the coat protein (CP) gene. The phylogenetic analysis showed that Serbian AMV isolates grouped into a separate well-supported group together with AMV isolates from Italy, Croatia and previously characterized isolates from Serbia. To our knowledge, this is the first report of AMV infection of eggplant in Serbia.

Rapid, highly sensitive and quantitative detection of interleukin 6 based on SERS magnetic immunoassay.

Sepsis is a systemic inflammatory response syndrome caused by infection, and is a common disease in intensive care units (ICUs), whereby the mortality rate is as high as 30% to 50%. Early diagnosis and treatment can significantly reduce the mortality rate of patients with sepsis. We have developed a method based on SERS for the rapid and quantitative detection of IL-6. Using the principle of double antibody sandwich, the core-shell nanoparticles embedded with a Raman reporter (Au@4MBA@Ag NPs) are coupled to the tracer antibody, while the biotin was coupled to the capture antibody to form an antibody-antigen-antibody sandwich structure with the antigen during detection of the structure. Streptavidin (SA) and biotin had a strong affinity, and the sandwich structure was captured by SA magnetic beads and detected by Raman spectroscopy under the enrichment of an external magnetic field. The results showed a good linear relationship between the Raman signal and the concentration of IL-6 in the concentration range of 0-1000 pg mL-1 (r = 0.9997) with a limit of detection of 1.6 pg mL-1. Also, the recovery rate of standard addition was 93.9-99.1%, and the coefficient of variation intra-assay and inter-assay of the three batches of reagents was less than 15%. Furthermore, it showed excellent specificity with procalcitonin (PCT, 20 ng mL-1) and C-reactive protein (CRP, 100 μg mL-1) and had no cross-reactivity. Except for bilirubin (2 mg mL-1) and hemoglobin (10 mg mL-1), other common interferences in the serum did not interfere, showing good anti-interference performance. Moreover, 57 clinical serum samples were detected via the chemiluminescence method simultaneously, and the detection results showed a good correlation (R2 = 0.9793, P < 0.01). There was no significant difference between the two performances (P > 0.05). The proposed method has numerous advantages such as high sensitivity, wide linear range, short detection time and simple operation, which provide a new technical reference for the clinical detection of sepsis biomarkers.

Rapid detection of Vibrio parahaemolyticus using magnetic nanobead-based immunoseparation and quantum dot-based immunofluorescence.

In recent years, the scale of population exposure and food poisoning caused by Vibrio parahaemolyticus (V. parahaemolyticus) has shown a significant upward trend, becoming one of the primary food-borne pathogens. Herein, we developed a rapid and sensitive detection of V. parahaemolyticus by integrating the technology of magnetic nanobeads (MBs) based immunoseparation (IMS) with quantum dots (QDs) based immunofluorescence. Firstly, specific rabbit polyclone IgG antibodies (IgG) and chicken egg yolk antibodies (IgY) of V. parahaemolyticus were prepared. Then two sizes of MBs (1 μm; 180 nm) were coupled with IgG to form immuno-MB (IMB) capture probes for evaluating the effect of different sizes on the detection efficiency. For QDs, they were conjugated with IgY to form fluorescent reporting probes. In the process of detection, IMB probes were used to separate V. parahaemolyticus and then these complexes were labeled by QD probes on the principle of double antibody sandwich. The fluorescence intensity of the IMB-V. parahaemolyticus-QD complexes was measured by a fluorescence spectrophotometer. The detection method takes 150 min with a detection limit of 102 cfu mL−1 ranging from 102 to 106 cfu mL−1 and it has been shown to work satisfactorily in real food samples.

Maize Seed Contamination and Seed Transmission of Maize Chlorotic Mottle Virus in Kenya

Maize chlorotic mottle virus (MCMV) causes maize lethal necrosis disease in combination with a cereal-infecting potyvirus, leading to high yield losses. There is limited information on seed infection or contamination rate by MCMV and its comparison with transmission rate to maize seedlings. This study was conducted to determine the extent of seed contamination in seed lots from MCMV-infected maize fields in Kenya and the transmission of MCMV from seeds to seedlings. To determine the contamination levels, whole seeds were ground and the extract tested for the presence of MCMV using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Seedling grow-outs were tested for seed transmission of MCMV using DAS-ELISA and real-time reverse transcription polymerase chain reaction (real-time RT-PCR) methods. The seed contamination rates of the four seed lots tested ranged from 4.9 to 15.9%. MCMV transmission frequency for 37,617 seedlings, tested in 820 pools of varying seed amounts by DAS-ELISA, was 0.17%, whereas a transmission frequency of 0.025% was obtained from 8,322 seedlings tested in 242 pools by real-time RT-PCR. Seeds from plants mechanically inoculated with MCMV had an overall seed transmission rate of 0.04% in 7,846 seedlings tested in 197 pools. The study showed that even with substantial contamination of maize seed with MCMV, the transmission of the virus from the seed to seedlings was low. Nevertheless, even low rates of transmission can be significant under field conditions where insect vectors can further spread the disease from infected seedlings, unless diseased plants are detected in time and properly managed. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Biological and molecular characterization of Prunus necrotic ringspot virus isolates from sweet and sour cherry

Biological and molecular properties of six Prunus necrotic ringspot virus (PNRSV) isolates originating from sweet and sour cherry trees were studied. Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) showed PNRSV infection in leaf samples from the respective source trees. While one of the infected source trees was symptomless, the other five showed symptoms, ranging from chlorotic to necrotic spots on the leaves. The reaction of greenhouse-grown seedlings of 22 herbaceous test plant species was investigated. The woody indicators Prunus serrulata cv. Kwanzan, wild cherry Prunus avium, P. cerasus, P. cerasifera and P. tomentosa were chip-budded. Chenopodium quinoa, Cucumis sativus cv. Amelia, C. sativus cv. Levina and P. tomentosa appeared as the most suitable test plants for the bioassay of the studied PNRSV isolates. The nucleotide sequences corresponding to the full-length coat (CP) and movement (MP) proteins were obtained. The nucleotide sequence identity among the studied isolates both in CP and MP genes was from 94% to 100%. The CP and MP amino acid sequence identities were from 95% to 100% and from 94% to 100%, respectively. Phylogenetic analyses performed with MP and CP nucleotide sequences showed that three isolates belonged to PV32-I and the other three to PV96-II phylogroups. Comparative amino acid analyses of MP and CP genes, together with the symptoms on naturally infected trees showed a higher affiliation of the studied isolates with the mild type than with the rugose types of isolates. The molecular variability correlated with the symptomatology on the naturally infected trees.

Determination of plasma β-amyloids by rolling circle amplification chemiluminescent immunoassay for noninvasive diagnosis of Alzheimer's disease.

Arolling circle amplification chemiluminescence immunoassay (RCA-CLIA) was developed for precise quantitation of Aβ in plasma. Capture antibodies conjugated with magnetic beads and detection antibodies with collateral single-stranded DNA (ssDNA) were bound to Aβ42/Aβ40 antigens to form a typical double-antibody sandwich structure. The RCA reaction was triggered by the addition of ssDNA, which generated products with a large number of sites for the binding of acridinium ester (AE)-labeled detection probes, thereby realizing the purpose of the amplification. TheRCA-CLIA method had higher sensitivity than conventional CLIA without loss of specificity. Under optimum conditions, the linear range of Aβ42 and Aβ40 detection was 3.9-140pg/mL and 3.9-180pg/mL, respectively, with corresponding low detection limits of 1.99pg/mL and 3.14pg/mL, respectively. Plasma Aβ42 and Aβ40 were detected in the blood of 21 AD patients and 22 healthy people, wherein this ratio could significantly distinguish AD patients from healthy individuals with a sensitivity of 90.48% and specificity of 63.64% for a cutoff value of 154. The Aβ42/Aβ40 ratio of plasma acts as an accurate indicator for AD diagnosis; therefore, detection of plasma Aβ using the RCA-CLIA exhibits great potential in noninvasive diagnosis and progressive assessment of AD.

Effects of dietary supplementation of iron as sulphates or glycine chelates on the productive performance and concentrations of acute-phase proteins and iron in the serum and liver tissues of broiler chickens

Abstract The aim of the study was to determine the effects of inorganic (ferrous sulfate [FeSO4] and FeSO4 + phytase [FeSO4+F]) and organic forms of iron (ferrous glycine chelate [FeGly], Fe-Gly + phytase [Fe-Gly+F]) on the concentrations of C-reactive protein (CRP), serum amyloid A (SA), alpha- 1-acid glycoprotein (α-AGP), haptoglobin (Hp), transferrin (TRF) and iron in the blood and liver tissue of poultry. Ross 308 roosters were used in this study. The acute-phase protein (AP) levels in the chicken serum and liver tissues were assayed using the double-antibody sandwich ELISA kits for chickens. The iron levels in serum and liver samples were measured using flame atomic absorption spectrophotometry. The results showed that the serum and hepatic iron concentration in the chickens receiving Fe-Gly and Fe-Gly-F were significantly higher than those in the control group and in the chickens receiving iron in the form of sulphates. Further, serum CRP and SA concentrations were lower in the chickens from the supplemented groups than in those from the control. The hepatic CRP concentration was higher in the supplemented groups than in the control group. The serum and hepatic TRF concentration were highest in the chickens receiving FeSO4 and FeSO4-F. The hepatic Hp concentration was higher in the chickens receiving organic forms of iron than in the control chickens, whereas the serum Hp concentrations were similar across the groups. The highest α-1-AGP concentration was found in the livers of the chickens receiving Fe- Gly and Fe-Gly-F. Taken together, these findings suggest that iron had the most beneficial effects for poultry in terms of health, performance and immunity when added to the feed in the form of glycine chelate.

Study of selenomethionine on the selenoproteins expression in hepatocyte L02 and the synergistic effect of serine

To observe the effect of selenomethionine(SeMet)on the selenoproteins expression in hepatocyte L02 and the synergistic effect of serine. The L02 cells were cultured and divided into SeMet group and Serine+SeMet group. SeMet dose was set as 0. 001, 0. 01, 0. 1, 1 and 10 μmol/L. Serine and SeMet were mixed according to 2∶1 molar ratio(Serine∶SeMet=2∶1). The L02 cells were cultured for 48 h after SeMet and Serine added. Finally, the cell culture supernatants and homogenates were collected for the selenoprotein P(SEPP)and glutathione peroxidase 1(GPx1)concentrations detection by a double-antibody sandwich enzyme-linked immuno-sorbent assay(ELISA). The expression of SEPP and GPx1 in cell homogenates was detected by western blot(WB). ELISA and WB results showed that GPx1 and SEPP expressions were dose dependent in a low SeMet concentration range, reached their inflection points when SeMet concentration was 1 μmol/L and 0. 1 μmol/L respectively, and began to decrease when SeMet concentration was further increased to 10 μmol/L. WB result showed that serine had a synergistic effect on GPx1 and SEPP expressions as SeMet concentration was 0. 001 μmol/L to 10 μmol/L. There was an inflection point in selenium protein synthesis when SeMet acted on normal hepatocyte(L02), the specific mechanism needs to be further explored. Secondly, serine had a synergistic effect on GPx1 and SEPP expressions of SeMet in hepatocyte(L02).

EFFECT OF Tobacco etch virus (TEV) ON YIELD AND QUALITY OF RED PEPPER IN TURKEY

The most prevalently grown varieties of red peppers in the Eastern Mediterranean region of Turkey are ‘Sena’ and ‘Dila’ in addition to local red pepper populations. Survey studies conducted on Kahramanmaras pepper growing areas in 2014 and 2015 indicated that Tobacco etch virus (TEV) was the most common virus in collected pepper samples. In this study, the effects of TEV on ‘Sena’ and ‘Dila’ were analyzed. The experiment was designed with 5 replicates and randomized plots in fully controlled greenhouses. The experiment consisted of TEV inoculated and control pepper plots. The pepper plants were mechanically inoculated with TEV at the 4–6 leaf stage and periodical observations were made. Virus transmission was confirmed using the double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) method. The total yield, red pepper flake production, average fruit weight, diameter, length and volume, average fruit wall thickness, fruit color and fresh and dry weights of all green parts of harvested red peppers were evaluated. The quoted data on % reduction in yield and different fruit quality criteria are averaged over two years. According to the results of the study, the highest loss of yield was recorded for ‘Sena’ (58.2%) while the highest red pepper flake loss ratio was in ‘Dila’. In terms of fruit quality criteria, the most reductions in fruit weight (40.1%), fruit diameter (30.9%), fruit length (32.8%) and fruit volume (51.8%) were found in ‘Dila’, the highest losses in fruit wall thickness (27.2%) and average fresh and dry weights of green parts (49.9–43.1%) were in the ‘Sena’. There was a significant effect of TEV inoculation. Overall, virus infected plants were had significantly lower yield and reduced quality compared to control plants.

Associating Insulin Instillation with Negative Pressure Wound Therapy Reduces Insulin Resistance in Diabetic Patients with Infected Foot Wounds

In this work we investigated whether a joint treatment with insulin instillation and negative pressure wound therapy (NPWT) would reduce insulin resistance (IR) in diabetic patients with infected diabetic foot ulcers (DFUs) and improve their healing. We also studied the effects of insulin+NPWT treatment on the inflammatory response coupled with wound healing. Seventy-five diabetic patients with infected DFUs were recruited and randomly divided into equal (n=25) groups treated respectively with NPWT alone, NPWT+insulin, and conventional dressings (controls). Thereafter, the ulcers’ healing progress was assessed. The serum levels of Tumor Necrosis Factor-α (TNF-α), Interleukin-2 (IL-2), and Interleukin-6 (IL-6) were measured at 1 day before and at 7, 14, and 21 days after each treatment using specific double-antibody sandwich enzyme-linked immunosorbent assays (ELISA). Serum glucose was measured via a glucose-oxidase method, and serum insulin via radioimmunoassay. In each patient, IR was appraised via the Homeostasis Model Assessment (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI). Our results showed that at 7, 14 and 21 days post-treatment, with respect to the control group the two NPWT-treated groups had (i) significantly (p &lt; 0.05) lower serum levels of TNF-α, IL-2 and IL-6; (ii) a significantly (p &lt; 0.01) greater reduction in IR; and (iii) significantly (p &lt; 0.05) higher wound healing rates. Moreover, at 14- and 21-days post-treatment, the NPWT+insulin-treated group had a significantly (p &lt; 0.05) higher wound healing rate than the NPWT alone-treated group. In conclusion, the combined NPWT+insulin instillation treatment gave superior results by improving wound healing, decreasing inflammation at the wounds surface, and reducing IR in diabetic patients with infected DFUs.

Combining Negative Pressure Wound Therapy with Insulin Instillation Reduces Insulin Resistance and Accelerates Healing in Patients with Chronically Infected Wounds

This study aimed at evaluating the effects of negative pressure wound therapy (NPWT) combined with insulin instillation on wound healing and insulin resistance (IR) in patients with chronically infected wounds.

Serum Alpha-(1, 3)-Fucosyltransferase IV, Copper, and Zinc Levels in Patients with Colorectal Cancer and Healthy Controls

Background: Colorectal cancer (CRC) has no significant clinical symptoms at the early stages, and the molecular differences in the serum of the patients and healthy subjects could be assessed to identify the biological markers that indicate the detection of this cancer at the levels of the biological system. Objectives: The present study aimed to assess the concentrations of the alpha-(1, 3)-fucosyltransferase IV (FUT4) enzyme and copper (Cu) and zinc (Zn) as biological elements. Methods: This case-control study was conducted on 40 patients with CRC, including 20 men and 20 women. A metal-free sterile tube was used to collect five milliliters of venous blood. The enzyme-linked immunosorbent assay (ELISA) based on the Biotin double-antibody sandwich technology was used to measure the human FUT4 in the sera. In addition, plasma zinc and copper values were determined using Zist Chem Diagnostics kits. Results: The mean FUT4 levels in the CRC patients was slightly higher than the control group (P = 0.17), and the mean serum copper and zinc levels of these patients were lower than the control group with a significance difference in this regard (P &lt; 0.001). In addition, the AUC of FUT4, copper, and zinc was 0.58, 0.80, and 0.77, respectively. Conclusions: According to the results, the altered levels of FUT4, copper, and zinc in the serum of the CRC patients compared to the healthy controls could be an indicator associated with the CRC disease course. Furthermore, the unusual changes in the FUT4, copper, and zinc levels may signify CRC development, which plays a key role in the diagnosis and monitoring of this cancer.

A Chemiluminescent Immunoassay for Osteocalcin in Human Serum and a Solution to the "Hook Effect".

A chemiluminescent immunoassay for human serum osteocalcin, or bone Gla protein, was established using a double-antibody sandwich model. Examination of the hook effect revealed that it was significantly reduced, and no hook effect was observed at an osteocalcin concentration of 4000 ng/mL. The established method showed good analytical performance and thermal stability. The limit of detection was 0.03 ng/mL. The intra-assay coefficient of variation (CV) was 3.22%–5.64%, the interassay CV was 4.42%–7.25%, and the recovery rate was 93.22%–107.99%. Cross-reactivity (CR) was not observed with bovine, rat, mouse, rabbit, or porcine samples. The developed method showed a good correlation with the N-MID product from Roche. In total, 1069 clinical patient samples were measured using the reagent kit developed in this study.

Modulation Effects of Toxoplasma gondii Histone H2A1 on Murine Macrophages and Encapsulation with Polymer as a Vaccine Candidate.

Toxoplasma gondii (T. gondii) is the most common zoonotic protozoa and has infected about one-third of the population worldwide. Recombinant epitopes encapsulated in nanospheres have advantages over traditional T. gondii vaccines. For an efficient delivery system, poly (DL-lactide-co-glycolide) (PLGA) and chitosan are the most frequently used biodegradable polymeric nanospheres with strong safety profiles. In the present study, we first expressed and purified histone H2A1 of T. gondii using the prokaryotic expression system. The effects of recombinant TgH2A1 on the functions of murine macrophages were then studied. Purified recombinant TgH2A1 was then encapsulated in nanospheres with PLGA and chitosan. After subcutaneous vaccination in mice, the immune response was evaluated by double antibody sandwich ELISA kits. The results from this study showed that PLGA and chitosan loaded with rTgH2A1 could trigger a stronger Th1 oriented immune response and prolong the survival time of mice effectively. In conclusion, PLGA and chitosan nanospheres loaded with histone H2A1 are an effective method for the development of vaccines against T. gondii. Further studies should focus on evaluating the regulatory mechanism of TgH2A1, vaccine potency, and cellular response in chronic T. gondii infections.