• Korean Journal of Poultry Science
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• v.37 no.2
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• pp.167-172
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• 2010

An immunochromatograhy (IC) based infectious bursal disease virus (IBDV) detection kit, which employed two anti-IBDV VP2 monoclonal antibodies, was evaluated for rapid diagnosis of infectious bursal disease virus (IBD). The detection limit of the IC kit for IBDV was $10^{3.1}$ to $10^{3.9}$ $EID_{50}$/mL, indicating that the IC kit detected IBDV sensitively as same as double antigen capture ELISA but less than a RT-PCR assay. The IC kit did not detect other viral pathogens such as Newcastle disease virus, infectious bronchitis, avian influenza virus, and infectious larynotracheitis virus. When applied to tissue samples of experimental chickens died 3 or 4 days post infection after very virulent IBDV (strain Kr/D62) infection, the IC kit detected IBDV in all samples of the bursa of Fabricius, spleen, kidney, cecal tonsil and in 87.5%, 37.5% and 0% of liver, thymus and proventriculus samples. In particular, BF tissue samples showed stronger signal bands than other tissues. Positive signal was observed. All except for one thymus sample of samples having negative results by the IC kit showed the same result with DAS-ELISA but RT-PCR assay detected IBDV in some of IC kit negative samples of thymus and proventriculus. When swab samples from the bursa of Fabricius of dead chickens (n=231) on field farms were tested, the sensitivity and specificity of the IC assay relative to RT-PCR was 100% (109/109) and 97.5% (119/122), respectively and kappa value between both assay was 0.97. The kit can provide a useful aid for rapid detection of IBDV in chickens under field circumstances.

• KSBB Journal
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• v.21 no.5
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• pp.325-330
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• 2006

This study presents the characterization of an integrated portable microfluidic electrical detection system for fast and low volume immunoassay using polystyrene microbead, which are used as immobilization surfaces. In our chip, a filtration method using the microbead was adopted for sample immobilization and immunogold silver staining(IGSS) was used to increase the electrical signal. The chip is composed of an inexpensive and biocompatible Polydimethylsiloxane(PDMS) layer and Pyrex glass substrate. Platinum microelectrodes for electric signal detection were fabricated on the substrate and microchannel and pillar-type microfilters were formed in the PDMS layer. With a fabricated chip, we reacted antigen and antibody according to the procedures. Then, silver enhancer was injected to increase the size of nanogold particles tagged with the second antibody. As a result, microbeads were connected to each other and formed an electrical bridge between microelectrodes. Resistance measured through the electrodes showed a difference of two orders of magnitude between specific and nonspecific immuno-reactions. The detection limit was 10 ng/ml. The developed immunoassay chip reduced the total analysis time from 3 hours to 50 min. Fast and low-volume biochemical analysis has been successfully achieved with the developed microfilter and immuno-sensor chip, which is integrated to the microfluidic system.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.3
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• pp.1813-1817
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• 2013

Background: Multiple etiologic factors are suspected to cause gastric cancer, the most important of which is infection with virulent types of Helicobacter pylori. Materials and Methods: We have compared 102 gastric cancer patients with 122 non-ulcer, non-cancer dyspeptic patients. Gastric specimens were evaluated for H. pylori infection by tissue-based detection methods. Patient sera underwent antigen-specific ELISA and western blotting using a Helicoblot 2.1 kit and antibody responses to various H. pylori antigens were assessed. Results: The absolute majority (97-100%) of both groups were H. pylori seropositive. Multivariate regression analysis demonstrated serum antibodies to the low molecular weight 35kDa protein to be protective and reduce the risk of gastric cancer by 60% (OR:0.4; 95%CI:0.1-0.9). Conversely, seroreactivity to the 89kDa (VacA) protein was significantly higher in gastric cancer patients (OR:2.7; 95%CI:1.0-7.1). There was a highly significant association (p<0.001) between seroreactivity to the 116kDa (CagA) and 89kDa (VacA) proteins, and double positive subjects were found at nearly five fold (OR:4.9; 95%CI:1.0-24.4) enhanced risk of gastric cancer as compared to double negative subjects. Conclusions: Seroreactivity to H. pylori low (35kDa) and high (116kDa/89kDa) molecular weight antigens were respectively revealed as protective and risk indicators for gastric cancer.

• IMMUNE NETWORK
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• v.11 no.6
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• pp.383-389
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• 2011

Background: EY-6 is one of the newly synthesized indoledione derivatives to induce tumor cell-specific cell death. In this study, we investigated the mechanism of immunological death induced by EY-6 at mouse colon cancer cell as well as at the normal immune cell represented by dendritic cell. Methods: C57BL/6 mouse syngeneic colon cancer cell MC38 was treated with EY-6, and analyzed by MTT for viability test, flow cytometry for confirming surface expressing molecules and ELISA for detection of cytokine secretion. Normal myeloid-dendritic cell (DC) was ex vivo cultured from bone marrow hematopoietic stem cells of C57BL/6 mice with GM-CSF and IL-4 to analyze the DC uptake of dead tumor cells and to observe the effect of EY-6 on the normal DC. Results: EY-6 killed the MC38 tumor cells in a dose dependent manner (25, 50 and $100{\mu}M$) with carleticulin induction. And EY-6 induced the secretion of IFN-${\gamma}$ but not of TNF-${\alpha}$ from the MC38 tumor cells. EY-6 did not kill the ex-vivo cultured DCs at the dose killing tumor cells and did slightly but not significantly induced the DC maturation. The OVA-specific cross-presentation ability of DC was not induced by chemical treatment (both MHC II and MHC I-restricted antigen presentation). Conclusion: Data indicate that the EY-6 induced tumor cell specific and immunological cell death by modulation of tumor cell phenotype and cytokine secretion favoring induction of specific immunity eliminating tumor cells.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.4
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• pp.420-426
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• 1999

We examined the immune response in flounder, Paralichthys olivaceus, with immunization of formalin killed Edwardsiella tarda as an antigen. The ELISPOT-assay (enzyme-linked immunospot assay) was optimized technically and applied to count the number of total and specific antibody secreting cells (TASC and SASC) in lymphocytes of different lymphatic organs. Incubation of lymphocytes on 96 well plate for more than 2.5hrs came out enough time in ELISPOT-assay for counting the antibody secreting cells in the anterior kidney and spleen. However, too much of plate-coated antigen or rabbit anti-flounder immunoglobulin for SASC or TASC counting, respectively, was appeared to decrease the sensitivity of the assay system. Specificity of the system was also confirmed by the absence of TASC in lymphocytes treated with cycloheximide to prevent protein synthesis. The peak numbers of SASC appeared at wk 3 post immunization after that there was a sharp decrease and reached to almost zero at wk 7. In the spleen and kidney, the timing and numbers of SASC on peak response were concurrent without preferential organ distribution. The specific antibody level in the sera increased rapidly between wk 2 and 3 after immunization, i.e. like the specific cellular response found with ELISPOT-assay on that period, However, the remained high level of specific serum antibody from wk 5 after immunization until the end of experiment was clearly distinguishable from the kinetics of SASC response decreased sharply.

• Journal of Veterinary Clinics
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• v.26 no.2
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• pp.130-133
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• 2009

The use of screening tests as part of a diagnostic work-up is common in domestic canine practice, but understanding of the diagnostic test characteristics and factors affecting diagnostic accuracy is not clear among clinicians. This article was aimed to provide clinicians with a better understanding on the selection of test kits and with a proper interpretation of test results using an example from heartworm(Dirofilaria immitis) studies. From the literatures, diagnostic accuracy varied depending on the kits: percent average sensitivity and specificity of ELISA antigen-detecting kits were DiroChek(Synbiotics, USA) 78.1 and 95.2, SNAP(IDEXX, USA) 66.3 and 98.1, and Solo Step(Heska, Switzerland) 69.5 and 97.5, respectively, while the values for three hematological methods(Modified Knott's, direct smear and capillary tube) ranged from 38.4 to 81.8% and from 96.9 to 100%, respectively. Furthermore, it was also reported that the prevalence of heartworm disease in domestic dog populations varied depending on the regions studied: 2.5-22.8% for microfilarial test and 2.2-66.3% by ELISA. The values of predictive values for positive(PPV) and negative(NPV) provide useful information to clinicians on the probability of heartworm infection, but the PPV and NPV are greatly dependent on the heartworm prevalence. This suggests that PPV or NPV values of a test should be interpreted carefully in different clinical settings. Practical methods on the interpretation taking into account heartworm prevalence were discussed.

• Journal of Life Science
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• v.24 no.9
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• pp.995-1000
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• 2014

The studies of marine viruses in terms of viral isolation and detection have been limited due to the high mutation rate and genetic diversity of marine viruses. Of the modern methods currently used to detect marine viruses, serological methods based on enzyme-linked immunosorbent assay (ELISA) are the most common. They depend largely on the quality of the antibodies and on highly purified suitable antigens. Recently, a new experimental system for using viral capsid protein as an antigen has been developed using the yeast surface display (YSD) technique. In the present study, the capsid protein gene of the red-spotted grouper nervous necrosis virus (RGNNV) was expressed and purified via YSD and HA-tagging systems, respectively. Two regions of the RGNNV capsid protein gene, RGNNV1 and RGNNV2, were individually synthesized and subcloned into a yeast expression vector, pCTCON. The expressions of each RGNNV capsid protein in the Saccharomyces cerevisiae strain EBY100 were indirectly detected by flow cytometry with fluorescently labeled antibodies, while recognizing the C-terminal c-myc tags encoded by the display vector. The expressed RGNNV capsid proteins were isolated from the yeast surface through the cleavage of the disulfide bond between the Aga1 and Aga2 proteins after ${\beta}$-mercaptoethanol treatment, and they were directly detected by Western blot using anti-HA antibody. These results indicated that YSD and HA-tagging systems could be applicable to the expressions and purification of recombinant RGNNV capsid proteins.

• Journal of Preventive Medicine and Public Health
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• v.28 no.2 s.50
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• pp.526-541
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• 1995

This study was conducted to estimate the validity of reverse transcriptase-polymerase chain reaction(RT-PCR) compared to enzyme immunoassay(EIA) for the detection of hepatitis C virus (HCV) infection. EIA for antibody to HCV(anti-HCV) and RT-PCR for HCV was executed on the subjects from Pusan and Kyungnam area with questionnaire survey to collect some relating factors of HCV infection. As the result from 617 cases, the prevalence of HCV infection was 1.5% by EIA and 3.7% by RT-PCR(p<0.05), and the age standardized rate was 1.7% and 3.4% by EIA and RT-PCR, respectively. The prevalence of hepatitis B surface antigen(HBsAg) was 6.8% by enzyme linked immunosorbent assay(ELISA) and the age standardized rate was 7.7%. It was the higher in male group comparing to female group(p<0.01). Both of the prevalence of HCV and HBsAg were higher in elevated asparate aminotransferase(AST) and alanine aminotransferase (ALT) group than in normal AST and ALT group(p<0.01). There was no specific risk factor of HCV infection. Though the degree of agreement of EIA and RT-PCR by gamma statistics was 97.2%, it showed a significant difference between the two methods(p<0.01). For the detection of HCV infection, positive predictive value of EIA was 66.7% and negative predictive value of EIA was 97.2%. This study suggests that negative result to anti-HCV by EIA didn't mean the free state of HCV infection, therefore it would be helpful that further monitoring for HCV infection by RT-PCR in the case of elevated AST and ALT and/or clinically suspected.

• Journal of Veterinary Clinics
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• v.24 no.2
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• pp.77-81
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• 2007

Diagnostic performance of polymerase chain reaction (PCR) for detecting Dirofilaria immitis in dogs was evaluated when no gold standard test was employed. An enzyme-linked immunosorbent assay test kit (SnapTM, IDEXX, USA) with unknown parameters was also employed. The sensitivity and specificity of the PCR from two-population model were estimated by using both maximum likelihood using expectation-maximization (EM) algorithm and Bayesian method, assuming conditional independence between the two tests. A total of 266 samples, 133 samples in each trial, were randomly retrieved from the heartworm database records during the year 2002-2004 in a university animal hospital. These data originated from the test results of military dogs which were brought for routine medical check-up or testing for heartworm infection. When combined 2 trials, sensitivity and specificity of the PCR was 96.4-96.7% and 97.6-98.8% in EM and 94.4-94.8% and 97.1-98% in Bayesian. There were no statistical differences between estimates. This finding indicates that the PCR assay could be useful screening tool for detecting heartworm antigen in dogs. This study was provided further evidences that Bayesian approach is an alternative approach to draw better inference about the performance of a new diagnostic test in case when either gold test is not available.

• Journal of Korean Medicine Rehabilitation
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• v.34 no.2
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• pp.15-28
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• 2024

Objectives We used the D-galactose (D-gal) induced C2C12 myoblast senescence model to investigate whether ethanol extract of Perilla. fructescens leaves (EEPF) could delay cellular senescence and regulate related mechanisms. Methods C2C12 myogenic cells were cultured in an incubator under 37 ℃ and 5% CO₂ conditions. EEPF, dried perilla leaves were pulverized and extracted at 1:10 (v/v) at 50 ℃ for 4 hours. Cell counting kit-8 and western blot analysis was performed. Annexin V-FITC apoptosis detection kit and DAPI staining was applied. Catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde analysis kits were used. To measure the level of reactive oxygen species generation, staining and flow cytometry was used. To analyze the mitochondrial activity, membrane potential changes were measured using JC-1. 𝛽-gal activity was analyzed using SA-𝛽-gal staining solution, and DNA damage was analyzed by using 𝛾-H2AX. Quantikine ELISA kit was used to analyze inflammatory cytokine production. Results According to the results of this study, EEPF significantly alleviated the decrease in cell viability in C2C12 cells treated with D-gal and suppressed the decrease in the expression of proliferating cell nuclear antigen. EEPF also markedly blocked D-gal-induced C2C12 cell apoptosis and restored reduced activity of CAT, GSH-Px, T-AOC, SOD. In addition, EEPF suppressed the decrease in 𝛽-galactosidase activity, the induction of DNA damage and the increase in expression of senescence-associated secretory phenotype proteins such as p16, p53 and p21 in D-gal-treated C2C12 cells. Furthermore, EEPF significantly attenuated D-gal-induced production and expression of inflammatory cytokines such as interleukin (IL)-6 and IL-18. Conclusions The results of this study indicate that EEPF can be used as a potential candidate for the prevention and treatment of muscle aging.