Enzyme-linked Immunosorbent Assay For Detection Research Articles

Microfluidic Time-Delay Valve Mechanism on Paper-Based Devices for Automated Competitive ELISA.

Paper-based technologies have been drawing increasing attentions in the biosensor field due to their economical, ecofriendly, and easy-to-fabricate features. In this paper, we present a time-delay valve mechanism to automate a series of procedures for conducting competitive enzyme-linked immunosorbent assay (ELISA) on a paper-based device. The mechanism employs a controllable time-delay valve, which has surfactants to dissolve the hydrophobic barriers, in a fluid pathway. The valves can regulate the liquid and sequentially deliver the sample flow for automating ELISA procedures in microchannels. Competitive ELISA is achieved in a single step once the sample, or small molecule pesticide (e.g., Imidacloprid), is applied onto the paper-based device with a comparable sensitivity to plate-based competitive ELISA. The results further demonstrate the appositeness of using paper-based devices with the valve designs for on-the-go ELISA detection in agriculture and biomedical applications.

Development and Validation of a S1 Protein-Based ELISA for the Specific Detection of Antibodies against Equine Coronavirus.

Equine coronavirus (ECoV) is considered to be involved in enteric diseases in foals. Recently, several outbreaks of ECoV infection have also been reported in adult horses from the USA, France and Japan. Epidemiological studies of ECoV infection are still limited, and the seroprevalence of ECoV infection in Europe is unknown. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) method utilizing ECoV spike S1 protein was developed in two formats, and further validated by analyzing 27 paired serum samples (acute and convalescent sera) from horses involved in an ECoV outbreak and 1084 sera of horses with unknown ECoV exposure. Both formats showed high diagnostic accuracy compared to virus neutralization (VN) assay. Receiver-operating characteristic (ROC) analyses were performed to determine the best cut-off values for both ELISA formats, assuming a test specificity of 99%. Employing the developed ELISA method, we detected seroconversion in 70.4% of horses from an ECoV outbreak. Among the 1084 horse sera, seropositivity varied from 25.9% (young horses) to 82.8% (adult horses) in Dutch horse populations. Further, sera of Icelandic horses were included in this study and a significant number of sera (62%) were found to be positive. Overall, the results demonstrated that the ECoV S1-based ELISA has reliable diagnostic performance compared to the VN assay and is a useful assay to support seroconversion in horses involved with ECoV outbreaks and to estimate ECoV seroprevalence in populations of horses.

Development of indirect enzyme-linked immunosorbent assay for detection of porcine epidemic diarrhea virus specific antibodies (IgG) in serum of naturally infected pigs

BackgroundPorcine epidemic diarrhea virus (PEDV) infection is a highly contagious infectious disease causing watery diarrhea, vomiting, dehydration and high mortality rate in newborn piglets. PEDV infection can cause high economic losses in pig industry. In Japan, a PEDV outbreak occurred with high mortality from 2013 to 2015. Even though until now, PEDV infection occurs sporadically. For the control and monitoring of PEDV infection, not only symptomatic pigs, but also asymptomatic pigs should be identified. The objective of this study is to develop and optimize novel indirect ELISA as a simple, rapid, sensitive and specific method for the detection of anti-PEDV antibodies and evaluate the efficacy of the assay as a diagnostic method for PED.ResultsOne hundred sixty-two serum samples, consisting of 81 neutralization test (NT) positive and 81 NT negative sera, were applied to the assay. Indirect ELISA test based on whole virus antigen (NK94P6 strain) derived from Vero cell culture was evaluated by receiver operating characteristic (ROC) analysis with neutralization test (NT) as a reference method, and cut-off value was determined as 0.320 with sensitivity and specificity of 92.6 and 90.1%, respectively. The area under curve (AUC) was 0.949, indicating excellent accuracy of indirect ELISA test. There was significant positive correlation between indirect ELISA and neutralization test (R = 0.815, P < 0.05). Furthermore, the kappa statics showed the excellent agreement between these two tests (kappa value = 0.815). In addition, the sensitivity and specificity of preserved plates with different periods (1 day, 2 weeks, 1, 2, 3, 4, 5 and 6 months) after drying antigen coated plates were 100% and 80–100%, respectively.ConclusionsThe developed indirect ELISA test in our study would be useful as a reliable test for serological survey and disease control of PEDV infection, and our pre-antigen coated ELISA plates can be preserved at 4 °C until at least 6 months.

Development and Evaluation of Epitope-Blocking ELISA for Detection of Antibodies against Contagious Caprine Pleuropneumonia in Goat Sera.

Enzyme linked immunosorbent assays (ELISAs) have been developed for the detection of antibodies against contagious caprine pleuropneumonia (CCPP), the causative agent of which is Mycoplasma capricolum subsp. Capripneumoniae (Mccp). The currently available commercial CCPP competitive ELISA (CCPP cELISA) kit produced and supplied by IDEXX Company (Westbrook, Maine, United States) is relatively expensive for most African laboratories. To address this issue and provide a variety of choices, a sensitive and specific blocking-ELISA (b-ELISA) test to detect antibodies against CCPP was developed. We describe the newly developed CCPP blocking-ELISA based on the blocking of an epitope of a monoclonal antibody (Mccp-25) by a positive serum sample against the Mccp protein coated on a plate. The Percentage Inhibition (PI) cut-off value for the CCPP b-ELISA was set at 50 using 466 CCPP negative and 84 CCPP positive small ruminant sera. Of the negative sera, 307 were obtained from the Botswana National Veterinary Laboratory (BNVL) and 159 from the Friedrich-Loeffler-Institute (FLI) Germany. The 84 positive sera samples came from experimentally vaccinated goats at the AU-PANVAC facility in Debre-Zeit, Ethiopia. The relative diagnostic sensitivity and specificity of the CCPP b-ELISA was 93% and 88%, respectively. This test result indicated good correlation with that of the commercial CCPP cELISA by IDEXX Company (Westbrook, Maine, United States) with a Cohen’s κ agreement of κ agreement of 0.85. The newly developed CCPP b-ELISA will be useful in the detection of antibodies for the diagnosis CCPP and for sero-surveillance during vaccination campaigns.

Reproducibility of antigen-immobilized enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA for quantitative detection of NNV particles

Nervous necrosis virus (NNV) is a fish virus belonging to family Nodaviridae. In this study, we prepared partially aggregated and monometric NNV particles to determine reproducibility of two different enzyme-linked immunosorbent assays (ELISAs): antigen-immobilized ELISA and sandwich ELISA. Passing ratios of purified NNV particles through ultrafilters with molecular weight cut off (MWCO) of 105, 3 × 105 and 106 were 0%, 35.2% and 80.3%, respectively, suggesting that purified NNV particles were partially aggregated whereas those in filtrates with MWCO of 3 × 105 could be monometric. Both NNV particles were subjected to ELISAs. Reduction ratios of ELISA values by 2-fold dilution of antigens were 50% in sandwich ELISA regardless of aggregation state of NNV particles. In contrast, those in antigen-immobilized ELISA were 42% (partially aggregated NNV) to 43% (monometric NNV), which were lower than the theoretical value (50%). This could be due to changes in aggregation state of NNV particles during dry-immobilization. Sandwich ELISA has excellent reproducibility from five times of experiments, in comparison with antigen-immobilized ELISA. Furthermore, available range of regression lines (R2 > 0.99) in sandwich ELISA was wider than that in antigen-immobilized ELISA. These results revealed that sandwich ELISA had better quantitativeness, reproducibility and available range of ELISA values than antigen-immobilized ELISA.

6-Bromoindirubin-3'-Oxime Suppresses LPS-Induced Inflammation via Inhibition of the TLR4/NF-κB and TLR4/MAPK Signaling Pathways.

We investigated the efficacy of the traditional herbal extract 6-bromoindirubin-3'-oxime (BIO) against lipopolysaccharide (LPS)-induced mastitis in mice and inflammatory signaling in mouse mammary epithelial cells (MMECs). In vivo, breast inflammation scores and enzyme-linked immunosorbent assay (ELISA) detection of pro-inflammatory factor expression were used to assess the effect of BIO against mastitis. In vitro, the effects of BIO on LPS-induced changes in the expression levels of pro-inflammatory factors, anti-inflammatory cytokines, and signaling factors of the toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) and TLR4/mitogen-activated protein kinase (MAPK) pathways were examined by qRT-PCR and ELISA. In LPS-injected mice, BIO pretreatment downregulated the expression of the pro-inflammatory factors interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and myeloperoxidase (MPO) in mammary glands and reduced inflammatory lesions in breast tissue. In MMECs, BIO pretreatment downregulated the LPS-induced expression of IL-1β, IL-6, and TNF-α. Further, BIO inhibited both the expression and phosphorylation of TLR4/NF-κB and TLR4/MAPK signaling factors. Thus, BIO downregulates IL-6, IL-1β, TNF-α, and MPO expression, upregulates IL-10 expression, and suppresses LPS-induced inflammation by inhibiting the TLR4/NF-κB and TLR4/MAPK pathways. BIO may be a potential treatment agent for mastitis and other inflammatory diseases.

Authentication approach using enzyme-linked immunosorbent assay for detection of porcine substances

Food manufacturers across the world commonly add animal substances in their food products. Some food products may contain porcine substances including pork, gelatine, blood and pepsin. These substances significantly affect the texture, colour or taste of the end products. Aside from enhancing sensory qualities, additional ingredients also contribute to preservation, bulk and nutrition. However, the inclusion of porcine substances might not be suitable among certain communities. One primary concern is fraud labelling which includes hiding the addition of porcine substances in food. Therefore, analytical techniques such as enzyme-linked immunosorbent assay (ELISA) have been developed to detect the porcine proteins in food. The ELISA delivers specificity and sensitivity in detecting the targeted animal species in food. This review provides an overview of the ELISA technique which has been developed for potential detection of porcine substances in laboratory-prepared food samples and commercial food products.

Development and application of a fiber2 protein-based indirect ELISA for detection of duck adenovirus 3

Duck adenovirus 3 (DAdV-3) is a newly identified duck adenovirus that has recently emerged in China. The incidence of duck infection caused by this virus is very high, with very large economic losses to the poultry industry. Thus, there is an urgent need for a serological assay for the specific detection of DAdV-3. To this end, prokaryotic expression of the fiber2 protein of DAdV-3 was used as a coating antigen to establish an indirect enzyme linked immunosorbent assay (ELISA) method for the specific detection of antibodies against DAdV-3. The method was found to be specific, repeatable and more sensitive than the agarose gel precipitation test (AGP). This indirect ELISA method based on the recombinant fiber2 protein may be used for the clinical detection of DAdV-3 infection and for monitoring antibody levels after vaccine immunization and is of great significance for the effective prevention and control of the disease.

Ultrasimple Single-Cell Detection of Multiple Cytokines by a Nanowell Chip Integrated with Encoded Microarrays.

Cytokine production is often regarded as the marker of immune cells' activation status. The spectrum and temporal secretion of cytokines are dramatically varied between cell phenotypes and even within the same phenotype. Multiparameter analysis of individual immune cell's cytokine secretion has always been a challenging and complicated process that needs special facilities in a laboratory setting. Herein, we present an ultrasimple method with high sensitivity and high robustness to quantify cytokine expression at the single-cell resolution. A microchip is developed based on poly(dimethylsiloxane) nanowells on sticky tape, while each nanowell is integrated with a DNA-antibody convertible microarray. Only pipetting is needed for the whole single-cell analysis process. The sensitivity of the assay is evaluated by measuring various concentrations of six recombinant cytokine proteins, which was found comparable to conventional methods. Once single cells are loaded to nanowells and incubated there, a Fluorinert FC-40 is used to isolate nanowells; so, cytokines from those cells are captured by separate microarrays. The rest of the sandwich enzyme-linked immunosorbent assay detection process is also executed simply by pipetting of various reagents. This method is validated by measuring cytokine production from hundreds of single cells. It has simplified a typically sophisticated multiplex single-cell assay into an instrument-free, point-of-detection technology, and thus it may find a broad utility in clinical diagnostics.

Development of an enzyme-linked immunosorbent assay for detection of Chlorpyrifos-ethyl and its metabolites 3,5,6-Trichloro-2-Pyridinol and Diethylthiophosphate

ABSTRACT A competitive enzyme-linked immunosorbent assay (ELISA) test using polyclonal rabbit antibodies for detection of Chlorpyrifos-ethyl (CPE) and its two major metabolites of degradation [3,5,6-Trichlororpyridin-2-ol (TCP) and O,O’-diethylthiophosphoric acid (DETP)], has been developed. We have first synthetized haptens of these three molecules, with 4 and 5 carbons spacer arms having carboxyl function at their ends. Haptens with 5 carbons’ spacer arm have been coupled to Bovine Serum Albumin (BSA) then injected to rabbits for polyclonal antibodies production. Haptens with 4 carbons spacer arm have been coupled to Human Albumin (HAS) to be in well plates coating to perform the ELISA tests. After adjusting the ELISA parameters, we have plotted the B/B0 (%) values against the logarithm of analyst concentrations. The IC50 values and detection limits were obtained through fitting the curves. The IC50 value of CPE and TCP were 651 and 5.61 ppb, with detection limits about 2.3410−2 and 2.18 10−3 ppb, respectively. We report for the first time, ELISA system developed for DETP with IC50 of 76.5 and a detection limit of 8.81 10−3 ppb. To evaluate the potential interferences that may be encountered in environmental water samples, pH, ionic strength, humic acid and some heavy metals were tested upon IC50 of the developed ELISA systems. In addition, several pesticides as well as CPE metabolites were tested for cross reactivity study. In all cases, the interference of the CPE assay was negligible. TCP assay represented a high cross reactivity to CPE but no interference with the others compounds. For DETP, no cross reactivity with the TCP was detected, but it was higher with CPE and the others pesticides. The developed ELISA systems have been compared with HPLC technique. Both techniques gave values of the same order of CPE and TCP prepared concentrations with a better sensitivity for ELISA system.

Strongyloides stercoralis in Patients on Corticosteroids Therapy Using Enzyme-Linked Immunosorbent Assay and Gelatin Particles Indirect Agglutination Tests: A Diagnostic Approach

Patients on corticosteroids therapy (POCT) are more likely to flare up concealed Strongyloides infection and develop Strongyloides hyperinfection syndrome and/or dissemination. Such critical complications can lead to high mortality rates. Rapid detection methods are, therefore, necessary to detect Strongyloides infection in POCT with the advantage of being applicable in a developing country. Two hundred POCT have been enrolled in this study to determine the rate of infection with Strongyloides. Three different groups of POCT (Strongyloides infected, non-infected, infected with other parasites) were used to evaluate the antibodies detection capability of two serological techniques (enzyme-linked immunosorbent assay (ELISA) and gelatin particles indirect agglutination (GPIAT) against the results of the gold standard agar plate culture (APC). With APC, the infection rate of Strongyloides stercoralis in POCT was 9.5% (19/200). POCT with Strongyloides infection displayed related risk factors (job, rural settlements, and soil contact) for infection combined with the subtropical nature of Ismailia Governorate. With regard to serology, ELISA detection results were poor compared to APC with sensitivity and specificity of 42.1% and 82.6%, respectively, and positive and negative predictive values of 72% and 30%. GPIAT appeared to be closely related to APC with sensitivity and specificity of 89.4% and 81.8%, respectively, and positive and negative predictive values of 80.9% and 96.7%. Statistical moderate correlation was detected between GPIAT and ELISA. The GPIAT technique is more convenient, easier, cheaper and faster to rule out the infection of Strongyloides in POCT. It might be the test of choice for routine immunodiagnosis of human strongyloidiasis.

Sero-prevalence of anti- CMV- IgM and anti -CMV- IgG in Iraqi Aborted Women Infected with Human Cytomegalovirus in Nasiriyah city

Many viruses infections are associated with abortion as cytomegalovirus infection. CMV is almost asymptomatic there for, diagnosis depends primarily on serological tests namely Enzyme linked immunosorbent assay (ELISA) to detect immunoglobulin M and G (IgM, IgM) in serum of aborted women. This study was carried out in Bent- Al Huda hospitals for the period (April 2015 to June 2015). A number of 50 aborted women serum collected during 2015. Serum samples were collected and then tested by ELISA for detection of anti-CMV IgM and IgG antibodies. The number of abortion more incidences in second group aged (20 to30 years). CMV specific IgM antibody was detected in 3pataint (6%) of the 50 aborted women ,while the specific IgG antibody detected in high percentage 46% in 23 samples. these result was statistically significant (P<0.05) .Seropositivity results for cytomegalovirus (CMV) is highly occurrences in women with first trimester of gestation.

A commercial ELISA for detection of interferon gamma in white rhinoceros.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is endemic in Kruger National Park, South Africa, home to the largest population of white rhinoceros (Ceratotherium simum) in the world. In 2016, the first cases of naturally occurring bTB were reported in white rhinoceros; however, there is a lack of understanding of infection and disease process in this species. Prevention and control of transmission depends on the availability of accurate tools to detect M. bovis infection. Interferon gamma (IFN-γ) assays are a reliable detection method for TB in other animal species, and studies have indicated that these tests can be used in white rhinoceros. We sought to screen and optimize a commercial IFN-γ enzyme-linked immunosorbent assay (ELISA) to detect endogenous white rhinoceros IFN-γ in mitogen-stimulated whole blood as a basis for developing a test for M. bovis infection. Optimizations included identifying ELISA antibodies and determining the effect of sample matrix, ELISA plate incubation temperature, ELISA linearity, assay reproducibility, and the assay's limit of quantification. The optimized assay employed an equine IFN-γ antibody pair that was used to create a commercial ELISA kit. This ELISA had a linear response to recombinant equine and endogenous rhinoceros IFN-γ (range: 7.8-125 pg/mL). When incubated at 37°C, the ELISA was highly reproducible, with an optimal recovery and a low limit of quantification, indicating that the Mabtech equine IFN-γ ELISAPRO kit is a robust assay for measuring white rhinoceros IFN-γ.

IMM-H004 Protects against Cerebral Ischemia Injury and Cardiopulmonary Complications via CKLF1 Mediated Inflammation Pathway in Adult and Aged Rats.

(1) Background: Chemokine-like factor 1 (CKLF1) is a chemokine with potential to be a target for stroke therapy. Compound IMM-H004 is a novel coumarin derivative screened from a CKLF1/C-C chemokine receptor type 4 (CCR4) system and has been reported to improve cerebral ischemia/reperfusion injury. This study aims to investigate the protective effects of IMM-H004 on cerebral ischemia injury and its infectious cardiopulmonary complications in adult and aged rats from the CKLF1 perspective. (2) Methods: The effects of IMM-H004 on the protection was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining, behavior tests, magnetic resonance imaging (MRI) scans, enzyme-linked immunosorbent assay (ELISA), Nissl staining, histo-pathological examination, and cardiopulmonary function detection. Immunohistological staining, immunofluorescence staining, quantitative real-time PCR (qPCR), and western blotting were used to elucidate the underlying mechanisms. (3) Results: IMM-H004 protects against cerebral ischemia induced brain injury and its cardiopulmonary complications, inhibiting injury, and inflammation through CKLF1-dependent anti-inflammation pathway in adult and aged rats. IMM-H004 downregulates the amount of CKLF1, suppressing the followed inflammatory response, and further protects the damaged organs from ischemic injury. (4) Conclusions: The present study suggested that the protective mechanism of IMM-H004 is dependent on CKLF1, which will lead to excessive inflammatory response in cerebral ischemia. IMM-H004 could also be a therapeutic agent in therapy for ischemic stroke and cardiopulmonary complications in the aged population.

Identification of Low-Abundance Urinary Biomarkers in Lupus Nephritis Using Electrochemiluminescence Immunoassays.

To investigate the utility of a sensitive platform using electrochemiluminescence (ECL) for the identification of low-abundance urinary protein biomarkers in lupus nephritis (LN). Forty-eight urine samples were obtained from subjects in 2 independent cohorts, each consisting of 3 groups (matched for age, sex, and race) of 8 patients with active LN (renal Systemic Lupus Erythematosus Disease Activity Index [SLEDAI] >0), 8 patients with inactive SLE (renal SLEDAI 0), and 8 healthy controls. Samples were tested using a preexisting 40-plex ECL panel. A custom 5-plex ECL panel was then developed for further validation studies and used to test 140 urine samples (from 44 patients with active LN, 41 patients with inactive SLE, 28 healthy controls, and 27 patients with other kidney diseases). Levels of 17 urinary proteins were elevated (P < 0.05 by 2-tailed Mann-Whitney U test) in samples from patients with active LN compared to samples from patients with inactive SLE and healthy controls in cohort 1, while 9 were similarly elevated in cohort 2. Of these, interleukin-7 (IL-7), IL-12p40, IL-15, interferon-γ-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC) were chosen for further validation. These 5 proteins were undetectable by enzyme-linked immunosorbent assay (ELISA). Hence, a custom 5-plex ECL panel was developed and used to validate the results from the initial 40-plex screening panel. Urinary IL-7, IL-12p40, IL-15, IP-10, and TARC levels were again significantly elevated in patients with active LN compared to those with inactive SLE and healthy controls, and correlated well with the renal SLEDAI and physician's global assessment of disease activity (R > 0.67, P < 0.05). All 5 urinary proteins were more frequently elevated in LN compared to controls with other chronic kidney diseases, although overall group differences attained significance only for urinary IL-7 and IL-15. Urinary levels of IL-7, IL-12p40, IL-15, IP-10, and TARC are potentially useful diagnostic tools in LN. The use of ECL assays may allow detection of urinary biomarkers that are below ELISA detection limits.

Plasma Hepcidin Levels in Healthy Children: Review of Current Literature Highlights Limited Studies.

There are few studies that highlight pediatric hepcidin reference ranges especially from Asian subcontinent. In current study, plasma from 131 children (72 boys and 59 girls; 1 to 12 y) was analyzed for hepcidin-25 by enzyme-linked immunosorbent assay. The median (interquartile range) plasma hepcidin in boys was 21.89 ng/mL (16.50 to 51.70 ng/mL) and girls was 21.95 ng/mL (19.20 to 47.70 ng/mL). No statistically significant difference (P=0.937) of plasma hepcidin levels in sex was noted. However, multiple regression analysis revealed a significant correlation between plasma hepcidin levels and ferritin (P=0.000). Our study results highlight relatively lower median hepcidin values in children 1 to 12 years of age as compared with western data. This may be attributed to either lack of a harmonized and standard enzyme-linked immunosorbent assay detection methodology or to presence of clinically significant polymorphisms in hepcidin gene in our population.

Upregulation of miR-216a exerts neuroprotective effects against ischemic injury through negatively regulating JAK2/STAT3-involved apoptosis and inflammatory pathways.

Ischemic stroke remains a significant cause of death and disability in industrialized nations. Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) of the JAK2/STAT3 pathway play important roles in the downstream signal pathway regulation of ischemic stroke-related inflammatory neuronal damage. Recently, microRNAs (miRNAs) have emerged as major regulators in cerebral ischemic injury; therefore, the authors aimed to investigate the underlying molecular mechanism between miRNAs and ischemic stroke, which may provide potential therapeutic targets for ischemic stroke. The JAK2- and JAK3-related miRNA (miR-135, miR-216a, and miR-433) expression levels were detected by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis in both oxygen-glucose deprivation (OGD)-treated primary cultured neuronal cells and mouse brain with middle cerebral artery occlusion (MCAO)-induced ischemic stroke. The miR-135, miR-216a, and miR-433 were determined by bioinformatics analysis that may target JAK2, and miR-216a was further confirmed by 3' untranslated region (3'UTR) dual-luciferase assay. The study further detected cell apoptosis, the level of lactate dehydrogenase, and inflammatory mediators (inducible nitric oxide synthase [iNOS], matrix metalloproteinase-9 [MMP-9], tumor necrosis factor-α [TNF-α], and interleukin-1β [IL-1β]) after cells were transfected with miR-NC (miRNA negative control) or miR-216a mimics and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) damage with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V-FITC/PI, Western blots, and enzyme-linked immunosorbent assay detection. Furthermore, neurological deficit detection and neurological behavior grading were performed to determine the infarction area and neurological deficits. JAK2 showed its highest level while miR-216a showed its lowest level at day 1 after ischemic reperfusion. However, miR-135 and miR-433 had no obvious change during the process. The luciferase assay data further confirmed that miR-216a can directly target the 3'UTR of JAK2, and overexpression of miR-216a repressed JAK2 protein levels in OGD/R-treated neuronal cells as well as in the MCAO model ischemic region. In addition, overexpression of miR-216a mitigated cell apoptosis both in vitro and in vivo, which was consistent with the effect of knockdown of JAK2. Furthermore, the study found that miR-216a obviously inhibited the inflammatory mediators after OGD/R, including inflammatory enzymes (iNOS and MMP-9) and cytokines (TNF-α and IL-1β). Upregulating miR-216a levels reduced ischemic infarction and improved neurological deficit. These findings suggest that upregulation of miR-216a, which targets JAK2, could induce neuroprotection against ischemic injury in vitro and in vivo, which provides a potential therapeutic target for ischemic stroke.

Development of Chemically Signal Amplified Nano-Biosensor Mediated by Poly-L-Lysine.

Amyloid β (Aβ) is considered to be one of a potential biomarker to monitor Alzheimer's Disease (AD) not only for diagnostic purposes but for early detection. Here we describe a novel nano-biosensor for Aβ mediated by poly-L-lysine (PLL) which was used for the amplification of detection signal for Aβ. The indirect enzyme-linked immunosorbent assay (ELISA) method was modified using PLL for the amplification of the Aβ detection signal. A commercially available ELISA plate was modified by PLL using chemical agent and the amplified amino groups were activated by a chemical agent for the detection of Aβ. The detection was carried out by the traditional immunochemistry using primary antibody and fluorescence molecules conjugated secondary antibody. In the result, the fluorescence intensity was increased by the increasing treated Aβ amount, and the sensitivity was approximately 2 times higher in the concentration of 2 ng/mL Aβ treatment, and approximately 4 times higher in the concentration of 200 ng/mL Aβ treatment compare with that of indirect ELISA detection method. We suggest our novel signal amplification method for the Aβ early detection.

Survey of Viral Hemorrhagic Septicemia Virus (VHSV) in Olive Flounder, Paralichthys olivaceus Hatchery in Korea

Viral hemorrhagic septicemia (VHS) generally occurs after juvenile olive flounders (Paralichthys olivaceus) are moved from the hatchery to on-growing system in Korea during spring. However, it remains unclear whether fish are infected by VHS virus (VHSV) in the hatchery or the on-growing system. In the present study, a survey was conducted to investigate VHSV infection in 39 olive flounder hatcheries from 2014 to 2017. Fish were tested for the presence of VHSV by inoculating sample to fathead minnow (FHM) and chinook salmon embryo (CHSE-214) cells to observe cytopathic effect, reverse transcription-polymerase chain reaction (RT-PCR), and antibody detection enzyme-linked immunosorbent assay (ELISA). VHSV was not detected in any of the 2,430 fish (from 461 pooled and 156 unpooled samples), although 34 (20.3%) of 167 samples was found to be positive for marine birnavirus (MABV) by cell culture and RT-PCR. Antibody detection ELISA results showed that all 212 fish sera have optical density (OD) values below of 0.1, suggesting that these fish had no VHSV-specific antibodies. Moreover, VHSV was not detected in any of 40 pooled samples (172 fish) collected after shifting rearing water temperature from 17-21°C to 10-15°C. In conclusion, the 39 olive flounder hatcheries surveyed in Korea was not infected by VHSV.

Evaluation of a Rapid Immunochromatographic Assay and Two Enzyme-Linked Immunosorbent Assays for Detection of IgM-Class Antibodies to Zika Virus.

There are currently five serologic assays available for detection of anti-Zika virus (ZIKV) IgM-class antibodies with U.S. Food and Drug Administration emergency use authorization. Among these are the Chembio DPP Zika IgM system (DPP Zika ICA; Chembio, Medford, NY), a rapid immunochromatographic assay (ICA), and the InBios ZIKV Detect 2.0 IgM antibody capture enzyme-linked immunosorbent assay (ZIKV 2.0 MAC-ELISA; InBios international, Inc., Seattle, WA), which has replaced the original InBios ZIKV Detect MAC-ELISA. We evaluated performance of these three serologic assays using 72 specimens characterized by plaque reduction neutralization testing (PRNT) for the presence or absence of neutralizing antibodies (NAbs) to ZIKV, dengue virus (DENV), and West Nile virus (WNV). The InBios ZIKV 2.0 MAC-ELISA was "presumptive Zika positive" in all 15 PRNT-confirmed ZIKV samples, while the Chembio DPP Zika ICA was nonreactive in three (20%) and the InBios ZIKV MAC-ELISA was negative in four (27%). The Chembio DPP Zika ICA and InBios ZIKV 2.0 MAC-ELISA showed >95% specificity in 22 ZIKV/DENV-seronegative specimens and in 13 samples positive for NAbs to non-ZIKV flaviviruses. Comparatively, the InBios ZIKV MAC-ELISA was "presumptive" or "possible Zika positive" in 8 of 12 WNV or DENV PRNT-positive samples and in 12 of 22 PRNT-seronegative sera. Our findings suggest that replacement of the InBios ZIKV MAC-ELISA with the InBios ZIKV 2.0 MAC-ELISA will lead to fewer samples requiring PRNT, minimizing unnecessary anxiety among patients ultimately determined to be seronegative for ZIKV and DENV by PRNT and alleviating some of the testing burden on laboratories performing PRNT.