Double-antibody Sandwich Enzyme-linked Immunosorbent Assay Research Articles

Molecular characterization of wheat dwarf virus isolates from Serbia based on complete genome sequences.

Wheat dwarf virus (WDV), the species Mastrevirus hordei of the genus Mastrevirus in the family Geminiviridae, is a cereal virus commonly detected in several European, African and Asian countries that causes economic losses. In the spring of 2019, a severe outbreak of wheat dwarfing and yellowing was observed in many winter wheat crops across Serbia. A total of 161 samples were tested for the presence of WDV and other common wheat viruses using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). To obtain the complete genome of 23 selected isolates, several overlapping segments of the WDV genome were amplified and sequenced. A phylogenetic tree was constructed using the whole genome sequences of the WDV isolates identified in this study and 40 selected sequences from GenBank. The results of DAS-ELISA indicated the presence of WDV in all samples collected from 21 sites in all nine districts surveyed. Further molecular characterization based on complete genome sequencing of 23 selected isolates showed that the Serbian WDV isolates had low nucleotide diversity and were closely related to wheat-infecting isolates from Europe, suggesting the presence of wheat-adapted forms of WDV in Serbia. The constructed phylogenetic tree revealed that Serbian isolates grouped in clade E within the wheat-adapted forms. This study provided the first insight into the genetic structure of WDV in Serbia based on its whole genome sequence. Further studies on the vector biology and population dynamics are needed to better understand the factors influencing the emergence and spread of WDV under local agroecological conditions.

Development of a double-antibody sandwich ELISA for detection of SARS-CoV-2 variants based on nucleocapsid protein-specific antibodies.

The COVID-19 pandemic, driven by the SARS-CoV-2 virus, has posed a severe threat to global public health. Rapid, reliable, and easy-to-use detection methods for SARS-CoV-2 variants are critical for effective epidemic prevention and control. The N protein of SARS-CoV-2 serves as an ideal target for antigen detection. In this study, we achieved soluble expression of the recombinant SARS-CoV-2 N protein using an Escherichia coli expression system and generated specific monoclonal antibodies by immunizing BALB/c mice. We successfully developed 10 monoclonal antibodies against the N protein, designated 5B7, 5F2-C11, 5E2-E8, 6C3-D8, 7C8, 9F2-E9, 12H5-D11, 13G2-C10, 14E9-F6, and 15H3-E10. Using these antibodies, we established a sandwich ELISA with 6C3-D8 as the capture antibody and 5F2-C11 as the detection antibody. The assay demonstrated a sensitivity of 0.78 ng/mL and showed no cross-reactivity with MERS-CoV, HCoV-OC43, HCoV-NL63, and HCoV-229E. Furthermore, this method successfully detected both wild-type SARS-CoV-2 and its variants, including Alpha, Beta, Delta, and Omicron. These findings indicate that our sandwich ELISA exhibits excellent sensitivity, specificity, and broad-spectrum applicability, providing a robust tool for detecting SARS-CoV-2 variants.

Screening of potato germplasm for resistance to potato virus y and potato leafroll virus using DAS-ELISA

Potato (Solanum tuberosum L.) ranks as the fourth most important staple food globally, after rice, wheat, and maize. In Pakistan, Potato virus Y (PVY) and Potato leafroll virus (PLRV) are among the most critical pathogens affecting potato crops, causing yield losses of up to 90%. Fifteen advanced potato lines from the Potato Program of the Horticultural Research Institute (HRI) were screened for resistance to these economically significant viruses. The experiment was conducted in the glasshouse of the Crop Diseases Research Institute, NARC, Islamabad, during 2023-24, following a completely randomized design with three replications per clone. For screening, small tubers of fifteen potato clones, including one check variety, were planted in pots within the glasshouse. PVY was mechanically transmitted, while a chip graft inoculation assay was used for screening against PLRV. Symptoms began to appear two weeks after inoculation, and data were recorded four weeks post-inoculation. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was performed on all plants to confirm the presence of both viruses serologically. The findings revealed that the line HRI-P1 remained asymptomatic and was categorized as highly resistant to both viruses, based on symptom observation and ELISA testing. Six clones exhibited varying levels of resistance, three showed moderate susceptibility, one was susceptible, and three were highly susceptible to PVY. The study highlights the availability of valuable sources of resistance within the Potato Program. HRI, NARC, which could be utilized in breeding potential future varieties. The disease-resistant genotypes identified will be instrumental in future potato breeding programs.

Development of a double-antibody sandwich ELISA for quantification of mutated EPSPS gene expression in rice

Glyphosate resistance is a critically important trait for genetically modified (GM) crops. Mutation of the rice EPSPS gene results in a high level of glyphosate resistance, presenting significant potential for the development of glyphosate-tolerant crops. The resistance of rice to glyphosate is correlated with the expression levels of resistance genes. Therefore, developing a convenient, stable, and sensitive method for quantifying the OsmEPSPS protein is crucial for the development of glyphosate-resistant crops. We developed a double-antibody sandwich quantitative ELISA (DAS-ELISA) using a specific monoclonal antibody (mAb) for OsmEPSPS capture and an HRP-conjugated anti-OsmEPSPS rabbit polyclonal antibody (pAb). The method could be used to detect OsmEPSPS within a linear range of 16–256 ng/mL with robust intra- and inter-batch duplicability (%CV values: 0.17 %–7.24 %). OsmEPSPS expression in the transgenic rice lines (54.44–445.80 μg/g) was quantified using the DAS-ELISA. Furthermore, the expression of the OsmEPSPS gene was validated through Western blotting. This study demonstrated the reliability and stability of the DAS-ELISA for OsmEPSPS detection in GM rice.

Association between DEFB1 rs11362 and Caries Susceptibility in Permanent Dentition: A Cross Sectional Study.

Dental caries is a multifactorial chronic bacterial infectious disease. Variations in the predisposition of the general population to dental cavities suggest that genetic and immunological factors play significant roles in its pathogenesis. This study aims to explore the impact of the Beta-Defensin 1 (DEFB1) rs11362 polymorphism on caries susceptibility in permanent dentition among the Bai Kuyao and Zhuang ethnic groups in China. A sample of 754 adolescents aged 12-15 was randomly selected from primary and junior high schools in Nandan County, Guangxi, China. All adolescents underwent clinical examinations, and DNA samples were collected. The genotype of DEFB1 rs11362 was determined using single nucleotide polymorphism (SNP) typing. The concentration of human β Defensin 1 (hBD-1) protein in saliva was measured using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The distribution of the DEFB1 rs11362 T allele was lower in the Bai Kuyao group compared to the Zhuang group. The disparity in the rs11362 genotype was statistically significant in the superficial dentin caries subgroup of the Bai Kuyao population (p = 0.017). Following adjustment for all potential confounding variables, the analysis revealed a heightened risk of superficial dental caries among CT genotype carriers in the Bai Kuyao population under a co-dominant model (odds ratios (OR) = 2.70; 95% confidence intervals (CI) [1.35-5.44]; p = 0.005), and an increased risk among CC genotype carriers in the Bai Kuyao population under a dominant model (OR = 2.35; 95% CI [1.18-4.67]; p = 0.015). A significant difference (p < 0.05) was noted in the distribution of rs11362 genotypes and salivary hBD-1 levels among the Bai Kuyao group. Salivary hBD-1 levels were notably higher in the CC genotype group (4.12 ± 2.07 ng/mL) compared to both the CT (2.77 ± 1.62 ng/mL) and TT genotype groups (2.32 ± 0.98 ng/mL). The DEFB1 rs11362 polymorphism showed an association with caries susceptibility in permanent teeth and influenced hBD-1 protein expression in saliva. Consequently, the DEFB1 polymorphism likely represents a concealed risk factor for caries.

In situ growth of double-hybrid nanoflowers on paper for the visual detection of carcinoembryonic antigen

In this study, the hemoglobin, egg white, copper phosphate organic–inorganic hybrid nanoflowers (H-E-Cu-NFs), and filter paper-based-egg white-Ca3(PO4)2 HNFs (FP-E-Ca-NFs) were synthesized using a mild biomineralization method. Antibodies (Ab) were labeled on the surface of nanoflowers through highly specific interactions between avidin and biotin. The FP-E-Ca-NFs@Biotin N-hydroxysuccinimide ester–Anti-carcinoembryonic antigen primary antibodies (Biotin-NHS-Ab1)/carcinoembryonic antigen (CEA)/H-E-Cu-NFs@Biotin-NHS-Ab2 immune-biosensing system was constructed by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to achieve the visual detection of CEA. Among them, H-E-Cu-NFs have abundant enzyme-like catalytic sites of Fe3+/Cu2+, which can effectively improve the efficiency of the colorimetric reaction. In contrast, E-Ca-NFs have no catalytic sites, and the directional design of catalytic performance realizes the specific identification and signal amplification of CEA detection. The as-developed immune-biosensing system showed a detection range of 0.5 ∼ 100 ng/mL, with the lowest detection limit of 0.5 ng/mL and the CEA recovery between 89.5 % ∼ 108.1 % in human serum samples. Compared to traditional ELISA methods, this immune-biosensing system has the advantages of easy operation, low cost, rapid detection, and on-site detection.

Alfalfa Mosaic Virus and White Clover Mosaic Virus Combined Infection Leads to Chloroplast Destruction and Alterations in Photosynthetic Characteristics of Nicotiana benthamiana.

Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses; it often exhibits combined infection with white clover mosaic virus (WCMV). Even so, little is known about the effects of co-infection with AMV and WCMV on plants. To determine whether there is a synergistic effect of AMV and WCMV co-infection, virus co-infection was studied by electron microscopy, the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and real-time fluorescence quantitative PCR (RT-qPCR) of AMV and WCMV co-infection in Nicotiana benthamiana. Meanwhile, measurements were carried out on the photosynthetic pigments, photosynthetic gas exchange parameters, and chlorophyll fluorescence parameters. The results showed that the most severe disease development was induced by AMV and WCMV co-infection, and the disease grade was scale 7. N. benthamiana leaves induced mottled yellow-green alternating patterns, leaf wrinkling, and chlorosis, and chloroplasts were observed to be on the verge of disintegration. The relative accumulation of AMV CP and WCMV CP was significantly increased by 15.44-fold and 10.04-fold upon co-infection compared to that with AMV and WCMV single infection at 21 dpi. In addition, chlorophyll a, chlorophyll b, total chlorophyll, the net photosynthetic rate, the water use efficiency, the apparent electron transport rate, the PSII maximum photochemical efficiency, the actual photochemical quantum yield, and photochemical quenching were significantly reduced in leaves co-infected with AMV and WCMV compared to AMV- or WCMV-infected leaves and CK. On the contrary, the carotenoid content, transpiration rate, stomatal conductance, intercellular CO2 concentration, minimal fluorescence value, and non-photochemical quenching were significantly increased. These findings suggest that there was a synergistic effect between AMV and WCMV, and AMV and WCMV co-infection severely impacted the normal function of photosynthesis in N. benthamiana.

Rational design and application of broad-spectrum antibodies for Bt Cry toxins determination

Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12–9.86 μg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66–20.46 μg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36–36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.

Human tau N-terminal domain-specific monoclonal antibodies: screening and application in blood detection

The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21-37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50-70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8-500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.

Prognostic Value of B-Type Natriuretic Peptide in Hospitalized Patients with Pneumonia

Background: Pneumonia is a major medical problem and is foremost infectious cause of death in developed countries. A rapid and reliable predictor might aid physicians in identifying hospitalization, intensive care unit admission, and treatment changes. B-type natriuretic peptide (BNP) has consistently been observed to improve the diagnosis and management of hospitalized patients with pneumonia. This investigation aimed to study the importance of BNP in the assessment of severity of pneumonia in a tertiary care center in South India. Subjects and Methods: A hospital-based observational research was carried out on patients admitted during the period of September 2016 to July 2018 with sample size of 108. All patients satisfying the inclusion criteria were enrolled. Detailed clinical history and physical examination findings were noted. Diagnostic criteria symptoms were recorded following the standard protocol. BNP was measured using the double-antibody sandwich enzyme-linked immunosorbent assay. The statistical analyses were done using the statistical software R version 3.6.3 and MS Excel. Results: Out of 108 patients, 48 (44.44%) were female and 60 (55.56%) were male. Of these, 45 (41.67%) patients identified with community-acquired pneumonia, 27 (25%) with health-care-associated pneumonia, 26 (24.07%) with aspiration pneumonia, and 10 (9.26%) with pneumonia with comorbid conditions. BNP levels among survivors and nonsurvivors were 193.52 ± 124.93 and 413.08 ± 149.38 pg/ml, respectively. The total mortality was 13/108 (12.04%). Out of 13 patients, 12 (92.31%) nonsurvivors had BNP level ≥200 pg/ml and 1 (7.69%) had 100–200 pg/ml. Conclusion: BNP levels could be a useful single prognostic marker for pneumonia. BNP levels correlated with the clinical severity of pneumonia.

Development a high-sensitivity sandwich ELISA for determining antigen content of porcine circovirus type 2 vaccines

Porcine circovirus type 2 (PCV2) is intensely prevalent in global pig farms. The PCV2 vaccine is an important means of preventing and controlling PCV2. The quality control of PCV2 vaccines is predominantly based on detection techniques such as animal testing and neutralizing antibody titration. Measuring the content of effective proteins in vaccines to measure vaccine efficacy is an excellent alternative to traditional methods, which can greatly accelerate the development speed and testing time of vaccines. In this study, we screened a monoclonal antibody (mAb) that can effectively recognize not only the exogenous expression of PCV2 Cap protein but also PCV2 virus. The double antibody sandwich ELISA (DAS-ELISA) was developed using this mAb that specifically recognize PCV2 Cap. The minimum protein content detected by this method is 3.5 ng/mL. This method can be used for the quality control of PCV2 inactivated vaccine and subunit vaccine, and the detection results are consistent with the results of mice animal experiments. This method has the advantages of simple operation, good sensitivity, high specificity and wide application. It can detect the effective antigen Cap protein content of various types of PCV2 vaccines, which not only shorten the vaccine inspection time but also save costs.

Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis

BackgroundRicin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin. MethodsRicin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin–biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked. ResultsThe LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90–62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples. ConclusionThe developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.

Development of a double antibody sandwich ELISA method for the quantitative detection of serum C-reactive protein based on nanobody

In this study, we successfully developed a nanobody-based double antibody sandwich ELISA kit for the detection of clinical serum C-reactive protein (CRP) by using two novel CRP specific nanobodies.The developed method exhibited a linear detection range of approximately 6–200 ng/mL, with a detection limit of 1 ng/mL. Furthermore, the method demonstrated excellent specificity, as there was no cross-reactivity with interfering substances such as total bilirubin and hemoglobin and so on. To assess reproducibility, independent measurements of the samples were conducted under experimental conditions, resulting in intra- and inter-batch coefficients of variation below 10% and a recovery rate of 93%–102%. These results indicate robust reproducibility of the method.To evaluate the performance of the developed kit, we collected 90 clinical samples for correlation analysis with commercial kits. The results showed a high correlation coefficient value (R2) of 0.98, indicating accurate concordance between the developed and commercial kits.In conclusion, our study successfully developed a nanobody-based double antibody sandwich ELISA kit to detect clinical serum CRP. The utilization of nanobodies represents a significant advancement in the field of CRP immunoassay development. The developed kit demonstrates excellent performance characteristics and holds promise for clinical applications.

Development of a Double-Antibody Sandwich ELISA Based on a Monoclonal Antibody against the Viral NS1 Protein for the Detection of Chicken Parvovirus.

Chicken parvovirus (ChPV) infection can cause runting-stunting syndrome (RSS) in chickens. There is currently no commercially available vaccine for controlling ChPV, and ChPV infection in chickens is widespread globally. The rapid detection of ChPV is crucial for promptly capturing epidemiological data on ChPV. Two monoclonal antibodies (mAbs), 1B12 and 2B2, against the ChPV NS1 protein were generated. A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for detecting ChPV based on the mAb 1B12 and an anti-chicken polyclonal antibody against the ChPV NS1 protein. The detection limit for the ChPV recombinant pET32a-NS1 protein was approximately 31.2 ng/mL. A total of 192 throat and cloaca swab samples were analyzed for ChPV by the established DAS-ELISA and nested PCR methods. The concordance rate between the DAS-ELISA and the nested PCR method was 89.1%. The DAS-ELISA can detect the ChPV antigen without any cross-reaction with FAdV-4, FAdV-1, NDV, AIV, MS, CIAV, aMPV, EDSV, IBV, or AGV2. The method also has high repeatability, with a coefficient of variation (CV) of less than 5%. These findings indicate that the DAS-ELISA exhibits high accuracy, good sensitivity, and specificity, making it suitable for viral detection, field surveillance, and epidemiological studies.

Establishment of enzyme-linked immunosorbent assay for beef and lamb contents in cooked meat

In this study, an enzyme-linked immunosorbent assay (ELISA) was established to detect beef and lamb components, and its performance was tested. Double-antibody sandwich ELISA was adopted and determined a coating concentration of capture antibody 3G5 of 1:4 000, a working concentration of enzyme-labeled antibody 2E7-horseradish peroxidase (HRP) of 1:1 000, a sample incubation time of 60 min and a detection antibody reaction time of 60 min. The specificity, sensitivity, repeatability and stability of this assay were determined. The limit of detection for beef and lamb skeletal muscle troponin I was 45 mg/kg, the inter-assay and intra-assay recovery rates ranged from 80.4% to 115.7%, the coefficients of variation were below 13.6%, and the cross reaction rates of the tissue components of chicken, duck and fish were below 13.4%. The sandwich ELISA method established in this study is stable and has high accuracy. The test results were consistent with the polymerase chain reaction (PCR) method at 50 and 100 g/kg. Therefore, this ELISA method can be used to quantitatively detect beef and lamb components in meat products.

STUDY THE RELATIONS BETWEEN ANGIOPOIETIN-LIKE PROTEIN 4 (ANGPTL4) AND LIPOPROTEIN LIPASE AS PREDICTORS FOR ISCHEMIC HEART DISEASE IN PATIENTS WITH T2DM

Betatrophin production and blood lipid profile have been proven in previous research to have a correlation, both in the laboratory and in clinical settings. Investigations on the connection and contact behavior of lipoprotein lipase, betatrophin, and angiogenin- likeprotein3 (ANGPTL3) continue to vary from one another (LPL). This study aimed to evaluating the relationship between betatrophin, ANGPTL4, and LPL in addition to the severity of IHD in individuals with diabetes. There were sixty people with diabetes in the case group (30 with IHD and 30 without IHD). The control group included thirty people in total. While the serum concentration of ANGPTL4 was assessed using a double antibody sandwich ELISA, the serum concentrations of betatrophin, LPL, and ANGPTL4 were assessed using the enzyme-linked immunosorbent assay (ELISA). The levels of total cholesterol, fasting blood glucose, and triglycerides in each group were determined using an automated biochemical analyzer. The clinical baseline data for each patient group was incorporated into the analysis within the same time frame. Consequently, compared to diabetic patients without IHD and those in the control group, individuals with IHD had significantly higher levels of betatrophin, ANGPTL4 and LPL (p 0.05). With diabetic patients with IHD, the Gensini rating significantly and favorably correlated with TC, TG, LDL-C, betatrophin, LPL, and BMI (r = 0.201, 0.501, 0.045, 0.562, 0.611, and 0.431 and P = 0.041, 0.008, 0.038, 0.009, 0.002, and 0.039 respectively) In all groups of gensini score with (P&lt;0.05), there is a statistically significant difference found in the levels of betatrophin, ANGPTL4, and LPL. In patients with diabetes, the degree of IHD was associated with the betatrophin, ANGPTL4, and LPL parameters.

Detection of six potato viruses using double antibody sandwich ELISA from in vitro, screen house and field grown potato crops in Ethiopia

Virus infection in seed potato reduces yield, and the problem is exacerbated when an early-generation seed is affected. The prevalence of six key potato viruses, PVY, PVX, PLRV, PVA, PVS, and PVM, was assessed among decentralized seed multipliers such as individuals, farmer seed group cooperatives, private companies, and agricultural research centers that produce early generation seed in six major potato growing districts in Ethiopia. A total of 262 leaf samples were randomly collected from potato plants and analyzed using a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for six major potato viruses. Potato virus prevalence was calculated as the proportion of samples that tested positive for the viruses against the total number of samples tested. The prevalence of infection with at least one of the six viruses was 98.2%. Among the samples analyzed, 17.2% had a single viral infection with one of the six viruses while the majority had multiple infections. The ELISA tests confirmed presence of latent virus infection in early generation seeds from the three EGS producers and in different seed classes. This result indicates that virus infection is widespread in the country, limiting potato production. To address this issue, it is critical to develop a robust system that prevents viral infection build-up and spread in the seed system through regular seed quality assurance and certification, particularly for early generation seed.

Biological Exploration and Physicochemical Characteristics of Tomato Brown Rugose Fruit Virus in Several Host Crops

Tomato brown rugose fruit virus (ToBRFV) is considered an emerging disease and a viral pandemic for tomato consumers. The objectives of this research were to analyze the biological and physicochemical characteristics of ToBRFV in tomato and tobacco plants, as well as to evaluate its natural host range. Inoculant seeds were recovered from ToBRFV-infected tomato samples in Coahuila, Mexico, and confirmed by RT-PCR. In the first greenhouse experiment, tomato plants of the F1 hybrid variety 172–300 (Yüksel), infected with ToBRFV, were used to evaluate viral inclusions (VI), dilution endpoint (DEP), the incubation period (IP), and latency period (LP). In a greenhouse experiment, Nicotiana longiflora plants were inoculated with ToBRFV to determine the in vitro longevity (IVL) and thermal inactivation (TI) of the virus in sap. Finally, the inoculation of tomato plants grown in open fields was carried out to evaluate transmission to natural hosts. The plants tested for possible ToBRFV reservoirs near the inoculum source were inspected and confirmed by a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The results indicate that the VIs on tomato leaves manifested as X-bodies and rounded, stacked plaques within epidermal cells. The DEP required to induce the infection in plants was from a ToBRFV concentration of 1 × 10−5.5, the IP of ToBRFV occurred 9 to 12 days post-inoculation, and LP could be detected one day after inoculation. The TI of ToBRFV in N. longiflora plants occurred at 85 °C for 30 min. Ipomoea purpurea, Mirabilis jalapa, Clematis drummondii, and Solanum tuberosum were newly identified hosts of ToBRFV. The results found contribute to a better understanding of the impact of ToBRFV, managing and preventing the spread of ToBRFV in diverse environments.

Development and Standardization of Sandwich ELISA for the Detection of Canine Parvovirus Infection

Canine parvovirus (CPV) is a highly contagious viral pathogen affecting dogs aged 6-21 weeks primarily targeting rapidly dividing cells of GI tract, which leads to haemorrhagic gastroenteritis and symptoms such as fever, vomiting, in appetence and abdominal pain. Several variants include CPV-2, CPV-2b and CPV-2c. CPV can be prevented by timely and complete vaccination and can be treated effectively if timely diagnosis is done. It can be diagnosed by effective methods such as PCR, fecal ELISA, HA and HI, the most effective being ELISA and PCR. Therefore, the present research was aimed at developing and standardizing a Double Antibody Sandwich ELISA for the detection of CPV by using rabbit hyperimmune sera as primary antibody and mice hyperimmune sera as secondary antibody.

The Detection of Circulating Antigen Glutathione S-Transferase in Sheep Infected with Fasciola hepatica with Double-Antibody Sandwich Signal Amplification Enzyme-Linked Immunosorbent Assay

Fasciolosis is a global zoonotic parasitic disease caused by F. hepatica infection that is particularly harmful to cattle and sheep. A biotin–streptavidin signal amplification ELISA (streptavidin-ELISA/SA-ELISA) based on circulating antigens can allow for the early detection of F. hepatica-infected animals and is suitable for batch detection. It is considered to be a better means of detecting F. hepatica infection than traditional detection methods. In this study, using the serum of sheep artificially infected with F. hepatica, the cDNA expression library of F. hepatica was screened, 17 immunodominant antigen genes of F. hepatica were obtained, and glutathione s-transferase (GST) was selected as the candidate detection antigen. Firstly, the GST cDNA sequence was amplified from F. hepatica, followed by the preparation of recombinant protein GST (rFhGST). Then, monoclonal and polyclonal antibodies against rFhGST were prepared using the GST protein. Afterward, the immunolocalization of the target protein in the worm was observed via confocal microscopy, and it was found that the GST protein was localized in the uterus, intestinal tract, and body surface of F. hepatica. Finally, a double-antibody sandwich SA-ELISA based on the detection of circulating antigens was established. There was no cross-reaction with positive sera infected with Dicrocoelium lanceatum (D. lanceatum), Haemonchus contortus (H. contortus), Neospora caninum (N. caninum), or Schistosoma japonicum (S. japonicum). Forty serum and fecal samples from the same batch of sheep in Nong’an County, Changchun City, Jilin Province, China were analyzed using the established detection method and fecal detection method. The positive rate of the SA-ELISA was 17.5%, and the positive rate of the fecal detection method was 15%. The detection results of this method were 100% consistent with commercial ELISA kits. A total of 152 sheep serum samples were tested in Nong’an County, Changchun City, Jilin Province, and the positive rate was 5.92%. This study laid the foundation for the development of serological detection preparations for F. hepatica infection based on the detection of circulating antigens.