Comparative evaluation of lateral flow immunoassays, LAMP, and quantitative PCR for diagnosis of fire blight in apple orchards.

Fire blight remains a serious threat to commercial apple production in the USA and worldwide. Other diseases and spray damage can result in fire blight-like symptoms that can lead to misdiagnosis and affect disease management strategies. Accurate and timely detection of the fire blight pathogen, Erwinia amylovora, is extremely important to deploy appropriate and timely measures to reduce fire blight epidemics in commercial apple orchards. We tested two commercial lateral flow immunoassays (AgriStrip®, and Pocket Diagnostics kit), Loop mediated isothermal amplification (LAMP), and quantitative PCR (qPCR) to diagnose E. amylovora infected samples in lab and field settings. The AgriStrip® and Pocket Diagnostics kits were able to detect actively growing bacteria up to ×106 cfu/ml bacterial concentration. Pocket Diagnostics kit had less specificity and showed positive tests for E. pyrifolia in addition to E. amylovora. The LAMP assay showed high specificity for E. amylovora and was able to detect up to ×103 cfu/ml bacterial concentrations. The qPCR assay was also able to detect bacterial cells up to ×10-3 cfu/ml bacterial concentration with highly specific E. amylovora detection. Grower surveys and comparative cost-benefit analysis indicated that immunoassay kits are less expensive, easier to use, and require less technical expertise for on-site fire blight diagnosis than LAMP and qPCR. However, the choice of a specific diagnostic assay depends on the time, sensitivity, and specificity required for the detection of fire blight and its management.

Role of astaxanthin in the modulation of brain-derived neurotrophic factor and spatial learning behavior in perinatally undernourished Wistar rats.

Objective: Maternal health and nutrition during the perinatal period is the predominant factor influencing the functional development of the brain. Maternal malnutrition during the perinatal period causes retardation of brain development. The current study investigates the role of Astaxanthin (AsX) in spatial learning and memory and BDNF in perinatally undernourished Wistar rats.Methods: The albino wistar rats were perinatally undernourished and administered with different dosages of AsX. The spatial learning and memory performance and BDNF level were assessed. Data were collected and analysed.Results: The % Correct choice during the acquisition phase, performance at the end of the acquisition phase and the mean BDNF level at the Hippocampus, Cerebellum, and Cerebral cortex showed significant decline (P<0.001) in the PUN group and significantly high (P<0.001) in the PUNA2 group compared to the control. However, the mean RME and mean WME during different days of the acquisition phase were significantly high (P<0.001) in the PUN group and insignificant (P>0.05) in PUNA2 compared to the control.Discussion: The results showed that AsX effectively modulated the cognitive deficit that occurred in perinatally undernourished rats. This can be attributed to BDNF upregulation as evidenced by the significant increase of the BDNF level.

Curcumin primed exosomes reverses LPS-induced pro-inflammatory gene expression in buffalo granulosa cells.

Curcumin possesses anti-inflammatory properties and provides a promising treatment for inflammation. The aim of the study is to establish that buffalo granulosa cells when primed with curcumin (20 µM), release improved cellular contents through exosome that can mitigate granulosa cell dysfunction. Recently, we have shown that buffalo granulosa cells exposed to LPS (1 µg/mL) in serum free culture, transiently increased the pro-inflammatory cytokine genes (IL-1ß, TNF-α, IL-6) expression followed by the inhibition of CYP19A1 gene expression and estradiol production. Therefore, LPS-treated granulosa cells were used as a model of inflammation and curcumin primed exosomes were utilized to check their potential for reducing granulosa cell dysfunction. Expression level of pro-inflammatory cytokines and CYP19A1 were detected by real time PCR while estradiol levels were measured by ELISA. Exosomes derived from curcumin-treated cells alleviated LPS mediated inflammation. In conclusion, our study potentiates the use of curcumin primed exosomes in mitigating granulosa cell dysfunction. Results show the therapeutic conservatories of curcumin via primed exosomes.

Detection of undeclared animal by-products in commercial canine canned foods: Comparative analyses by ELISA and PCR-RFLP coupled with slab gel electrophoresis or capillary gel electrophoresis.

BACKGROUND: The potential presence of undeclared animal by-products in pet foods is not subject to routine examination. Previously published methods for species-based identification of animal by-products have not been used routinely owing to inconsistent results. The present study evaluated the utility of several approaches for accurate identification of animal by-products in 11 commercial brands of canine canned foods. RESULTS: Canine canned foods from several countries were analysed by ELISA, PCR-RFLP coupled with slab-gel electrophoresis (SGE) and capillary gel electrophoresis (CGE) to test for evidence of by-products derived from cattle, chicken, sheep or pig. While CGE-based analysis detected all (24) animal-derived by-products that were reported for the 11 test samples, SGE and ELISA detected only 22/24 (92%) and 14/24 (58%) of labelled by-products, respectively. In addition, undeclared animal by-products were found using all three analytical approaches with CGE detecting more positives (19) than SGE (17) or ELISA (5). CONCLUSION: Significant disparities were evident between the labelled contents and the detected content of animal by-products. CGE-based testing for PCR products appears to provide greater sensitivity and accuracy than either SGE or ELISA-based methods. As testing of commercial products becomes more reliable and mainstream, manufacturers will need to develop more thorough and accurate labelling protocols.

WITHDRAWN:Development of Dot Enzyme Linked Immuno Sorbent Assay Using Recombinant Hexon Protein for Detecting Antibodies to Hydropericardium Syndrome Disease Virus.

Ahead of Print article withdrawn by publisher.

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli.

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10Å from the active site, while epitope ß is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a ß epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.

Evaluation of efficacy of various immunochromatographic rapid tests for dengue diagnosis.

OBJECTIVE: The immunochromatographic rapid tests facilitate the early diagnosis of dengue by providing evidence of the presence of virus specific proteins (antigens/ antibody) in human blood. Many products for rapid dengue diagnosis are available in the market; the performance of few selected products was evaluated and compared with enzyme linked immuno sorbent assays (ELISA). METHODS: Sera from a large number of patients (n=184) admitted to National Institute of Blood Diseases & Bone Marrow Transplantation (NIBD) were used to determine the efficiency of non-structural (NS) 1, IgA, IgG and IgM based rapid test devices for dengue diagnosis. RESULTS: The dengue NS1 antigen based device was least efficient while among the antibody based devices the dengue IgA rapid test (RDT) was comparatively better (specificity: 80.95%; sensitivity: 85.21%). This device could detect both primary and secondary dengue infection and was found to be the most sensitive device at all point of sample collection. CONCLUSION: The dengue IgA RDT could be a cost effective and efficient rapid test device for timely dengue diagnosis at all levels of healthcare settings.

Exploratory Research for New Diagnostic Markers of Mucormycosis.

Mucormycosis is a fungal infectious disease caused by Rhizopus oryzae and other members of the order Mucorales, and it is known as one of the most lethal fungal infections. Early diagnosis of mucormycosis improves prognosis because of limited effective treatments and the rapid progression of the disease. On the other hand, the lack of characteristic clinical findings in mucormycosis and the challenge of early definitive diagnosis make early treatment difficult. Our goal was to establish a serodiagnostic method to detect Rhizopus specific antigen (RSA), and we have developed a diagnostic kit by Enzyme-linked immuno-sorbent assay (ELISA) using a monoclonal antibody against this antigen. RSA increased over time in the serum and alveolar lavage fluid of R. oryzae-infected mice. RSA was also detected in serum and alveolar fluid, even at an early stage (Day 1), when the tissue invasion of R. oryzae mycelium was not histopathologically detectable in the lungs of R. oryzae-infected mice. Further evaluation is needed to determine the feasibility of using this assay in clinical practice.

Comparison between radiometry and spectrophotometry for the determination of angiotensin-converting enzyme activity in cerebrospinal fluid.

Determination of angiotensin-converting enzyme (ACE) activity in cerebrospinal fluid (CSF) can help for establishing the diagnosis of neurosarcoidosis. We investigated the performance characteristics of two assays for ACE determination in 57 CSF, radiometry with [glycine-1-14C] benzoyl-L-histidyl-L-leucine and spectrophotometry with furylacryloyl-phenylalanyl-L-glycyl-L-glycine (FAPGG) as substrates. We compared both kinetic assays to an ELISA specific for human ACE. Within run and between run imprecisions were 14-17% for radiometry, 6-19% for spectrophotometry and 5-8% for ELISA. The limit of detection was 0.04 U/L for radiometry, 1.0 U/L for spectrophotometry and 0.156 µg/L for ELISA. The limit of quantification was 0.06 U/L for radiometry, 1.5 U/L for spectrophotometry, but not known for ELISA. The domain for quantification was 0.06-4.0 U/L for radiometry, 1.5-24 U/L for spectrophotometry and 0.156-10 µg/L for ELISA. Deming regression and Bland-Altman plots show good correlations between the three assays, but with high slopes, because both kinetic assays use different substrates and ELISA measures ACE molecule but not activity. Radiometry was more sensitive than spectrophotometry, which has a limit of detection above most pathological levels. ELISA could be an alternative to radiometry but only after complete evaluation, determination of normal values and assessment of its clinical value. We claim for standardization of ACE determination as well as in serum as in other biological fluids, in particular CSF.

Amyloid ß (Aß) ELISA of Human iPSC-Derived Neuronal Cultures.

Amyloid ß (Aß) peptides are the main component of the characteristic insoluble deposits in brain parenchyma and small blood vessels in the patients afflicted with Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). These small peptides are attributed to the pathogenesis of both AD and CAA, suggesting an important index for disease stage and progression. In the brain tissue, Aßs are released mainly from neuronal cells into extracellular space. Here, we describe a step-by-step protocol to measure Aßs secreted from human pluripotent stem cell-derived neuronal cells.

A biotechnological tool for glycoprotein desialylation based on immobilized neuraminidase from Clostridium perfringens.

BACKGROUND: Sialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation. METHODS: Neuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry. RESULTS: Clostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles. CONCLUSIONS: Immobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes. GENERAL SIGNIFICANCE: Immobilized neuraminidase would contribute to understand the role of sialic acid in biological processes.

Standardization of a colorimetric technique for determination of enzymatic activity of diamine oxidase (DAO) and its application in patients with clinical diagnosis of histamine intolerance.

BACKGROUND: Diamine Oxidase (DAO) has an essential role for degradation of exogenous histamine in the intestine; thus, histamine intolerance (HI) mainly has been correlated to a low concentration and/or activity of this enzyme. The objective of the study was to standardize a colorimetric technique to measure the enzymatic activity (function) of hDAO to then apply it to a series of 22 patients with a clinical diagnosis of HI. METHODS: For the standardization variables such as volume and type of sample, incubation time, wavelength of maximum absorption, types of substrates, and concentration of oxidized ascorbate were evaluated. Then the activity and concentration of DAO was determined in 22 patients diagnosed with HI and 22 healthy subjects. RESULTS: The mean of serum DAO concentration in the 22 patients was of 9.268 ± 1.124 U/mL. The mean of serum DAO concentration in the 22 controls was of 20.710 ± 2.509 U/mL, being significantly higher (P value 0.0002) the mean of the samples. The mean of serum DAO activity of the patients was of 1.143 ± 0.085 U/L and the controls was 1.533 ± 0.119 U/L, significantly greater than the patients (P value 0.011). In addition, the sensitivity of both techniques was 0.63. In the measuring of DAO concentration the specificity was 0.9, constituting a good diagnostic test, especially to rule out the true negatives. The determination of DAO activity had a specificity of 0.68. CONCLUSIONS: Although we used a small number of patients and controls and the absorbance values were lower than expected, statistically significant differences were found in the levels of concentration and DAO activity between the patients with histamine intolerance and the controls. Therefore, the measuring of DAO concentration and DAO activity is a good diagnostic strategy for study suspect cases of HI. The simultaneous use of both assays allows to reduce positive and negative false results, for example, patients with normal DAO levels that could present a dysfunction in the activity of this enzyme.

Trends in MERS-CoV, SARS-CoV, and SARS-CoV-2 (COVID-19) Diagnosis Strategies: A Patent Review.

The emergence of a new coronavirus (SARS-CoV-2) outbreak represents a challenge for the diagnostic laboratories responsible for developing test kits to identify those infected with SARS-CoV-2. Methods with rapid and accurate detection are essential to control the sources of infection, to prevent the spread of the disease and to assist decision-making by public health managers. Currently, there is a wide variety of tests available with different detection methodologies, levels of specificity and sensitivity, detection time, and with an extensive range of prices. This review therefore aimed to conduct a patent search in relation to tests for the detection of SARS-CoV, MERS-CoV, and SARS-CoV-2. The greatest number of patents identified in the search were registered between 2003 and 2011, being mainly deposited by China, the Republic of Korea, and the United States. Most of the patents used the existing RT-PCR, ELISA, and isothermal amplification methods to develop simple, sensitive, precise, easy to use, low-cost tests that reduced false-negative or false-positive results. The findings of this patent search show that an increasing number of materials and diagnostic tests for the coronavirus are being produced to identify infected individuals and combat the growth of the current pandemic; however, there is still a question in relation to the reliability of the results of these tests.

Development and evaluation of a sensitive, Diffusive Gradients in Thin-Films (DGT) method for determining microcystin-LR concentrations in freshwater and seawater.

A Diffusive Gradients in Thin-Films (DGT) passive sampling technique was developed for microcystin-LR (MC-LR), one of the most common and toxic microcystins. Three types of resins (HP20, SP700, and XAD18) were evaluated for MC-LR uptake kinetics, capacities, and extraction efficiencies and simple procedures were developed for determining MC-LR concentration in binding disc extracts by Adda-ELISA (U.S. EPA Method 546). The XAD18-DGT/Adda-ELISA method had a 7-d deployment time detection limit of ≈0.05 µg/L and capacity of >250 µg/L of MC-LR in water samples which encompass U.S. EPA and WHO advisory concentrations for drinking and recreational waters. The XAD18-DGT/Adda-ELISA method determined time-averaged MC-LR concentrations in waters with wide ranging pH (4.9-8.3) and ionic strength (0.04-0.8 M) under well-stirred and quiescent conditions with 90-101% accuracy. In addition to high sensitivity and accuracy, the method is simple, inexpensive, and applicable for determining MC-LR and related MCs concentrations in waterbodies with wide ranging chemical characteristics and hydrodynamic conditions.

Sero-prevalence of Helicobacter pylori Infection in Neyshabur, Iran, During 2010-2015.

BACKGROUNDS & OBJECTIVE: The Helicobacter pylori prevalence has continuously decreased during recent years in Iran. The current study aimed at determining H. pylori prevalence in Neyshabur city, Northeast Iran, during 2010-2015. METHODS: The current epidemiologic survey was conducted in Neyshabur from 2010 to 2015 to determine the prevalence of H. pylori infection. A total of 11596 participants (3681 male with the mean age of 31.7±6.2 years and 7915 female with mean age of 68.3±4.7 years) were included. The enzyme-linked immunosorbent assay kits for the detection of H. pylori and Stat Fax 3200® Microplate Reader (USA) with a sensitivity of 95% and specificity of 98% were used. Titers above 12 units were considered positive for IgG, IgA, and IgM (negative <8, equivocal 8 to 12, and positive >12 U). The Chi-square t test and F test were used to analyze data. RESULTS AND CONCLUSION: The overall IgA, IgG, and IgM seropositive samples among the study participants were 852 (7.2%), 9000 (72.8%), and 1256 (5.2%), respectively. The IgA seropositivity was significantly high among the age group above 51 years, compared with the other age groups. Moreover, the IgG and IgM seropositivity were significantly high among the age groups 41 to 50 and 31 to 40 years respectively, compared with the other age groups. There was no significant difference between male and female cases regarding IgA and IgG seropositive samples, but IgM level was significantly higher among females, compared with that of the male cases. Furthermore, there was no significant alteration in IgA, IgG, and IgM seropositivity during 2010-2014 in Neyshabur. The prevalence of H. pylori in Neyshabur was high in the healthy population. Furthermore, the H. pylori prevalence did not change from 2010 to 2014 in the studied city. Effective approaches to improve health, educational, and socioeconomic status should be implemented to minimize and control H. pylori infection.

Quantitative analysis of hemin-induced neutrophil extracellular trap formation and effects of hydrogen peroxide on this phenomenon.

Formation of neutrophil extracellular traps (NETs) can perpetuate sterile inflammation; thus, it is important to clarify their pathophysiological characteristics. Free heme, derived via hemolysis, is a major contributor to organ damage, and reportedly induces neutrophil activation as well as reactive oxygen species (ROS) production and NET formation. For this study, we examined hemin (Fe3+ -protoporphyrin IX)-induced NET formation quantitatively in vitro as well as the effects of oxidative stress. NETs formed in vitro from cultured neutrophils were quantitatively detected by using nuclease treatment and Sytox Green, a nucleic acid stain. Hemin-induced NET production was found to be in a dose-dependent manner, NADPH oxidase-dependent and toll-like receptor (TLR)-4 independent. Additionally, the iron molecule in the porphyrin ring was considered essential for the formation of NETs. In the presence of low concentrations of hydrogen peroxide, low concentrations of hemin-induced NETs were enhanced, unlike those of phorbol myristate acetate (PMA)-induced NETs. Quantitative analysis of NET formation may prove to be a useful tool for investigating NET physiology, and hemin could function as a possible therapeutic target for hemolysis-related events.

Seroprevalence of contagious ecthyma in goats of Assam: An analysis by indirect enzyme-linked immunosorbent assay.

AIM: The objective of this study was to screen the prevalence of contagious ecthyma (CE) among the goat population of Assam owing to its high prevalence rate. MATERIALS AND METHODS: In this study, a total of 231 serum samples were collected from 12 districts of Assam during September 2013 to July 2014. The serum samples were tested for the presence of antibodies against Orf virus (ORFV) by indirect enzyme-linked immunosorbent assay (ELISA). Indirect ELISA was standardized using purified Orf reference virus produced in bulk in primary lamb testes cells. RESULTS: Studies on seroprevalence showed 76.62% of goats were seropositive. The total number of animals were divided into different age groups starting from 0-2 months, 2-4 months, 4-6 months, and above 8 months and accordingly highest prevalence of antibodies against ORFV was recorded in the age-group above 8 months of age. Significantly, lower rates of infection were observed in goats of age group 2-4 months. This study recorded that seropositivity from naturally infected animals and in contact apparently healthy animals to be 53.67% and 46.32%, respectively. CONCLUSION: The results indicated that CE is a prevalent infection in goats of Assam, and the healthy population is at increased risk of infection.

Automated and portable solid phase extraction platform for immuno-detection of 17ß-estradiol in water.

A fully automated and portable system for solid phase extraction (SPE) has been developed for the analysis of the natural hormone 17ß-estradiol (E2) in environmental water by enzyme linked immuno-sorbent assay (ELISA). The system has been validated with de-ionized and artificial sea water as model samples and allowed for pre-concentration of E2 at levels of 1, 10 and 100 ng/L with only 100 ml of sample. Recoveries ranged from 24±3% to 107±6% depending on the concentration and sample matrix. The method successfully allowed us to determine the concentration of two seawater samples. A concentration of 15.1±0.3 ng/L of E2 was measured in a sample obtained from a food production process, and 8.8±0.7 ng/L in a sample from the Adriatic Sea. The system would be suitable for continuous monitoring of water quality as it is user friendly, and as the method is reproducible and totally compatible with the analysis of water sample by simple immunoassays and other detection methods such as biosensors.

Evaluation of a new immunoassay for chromogranin A measurement on the Kryptor system.

BACKGROUND: Chromogranin A (CgA) is a biomarker for neuroendocrine tumors (NETs). The aims of this study were to evaluate differences in measurement between the ThermoFisher Brahms CgA Kryptor assay and the CisBio assay and to investigate the influence of patient covariates. Temperature stability of CgA using both assays was determined. DESIGN AND METHODS: 406 patients were analyzed for serum CgA using both assays. We performed a comparison study to determine whether several patient covariates (gender, use of protein pump inhibitors, impaired kidney function, referral department and tumor location) influenced the results. For the stability study, pooled serum samples were aliquoted and stored at different storage temperatures (room temperature, 4 °C and -20 °C) until assayed. In addition, 15 individual samples were evaluated after storage at 4 °C using the Kryptor assay. RESULTS: Differences in measured concentrations between the assays were statistically significant. Passing & Bablok fit showed ln Y(Kryptor)=1.05 ln X(CisBio) - 0.20 with a bias of 1.0% after logarithmic transformation. Patient covariates were not associated. Patients׳ sera showed variable stability for CgA in the Kryptor assay at room temperature and 4 °C, whereas the recovery in the CisBio assay was stable at both temperatures. CONCLUSION: Differences in measured CgA concentration between the assays could not be explained by the investigated patient covariates. Serum should be stored at -20 °C prior to determination using the Kryptor assay.