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To study the antigenic characteristics and differentiation serotypes of Hantavirus, the produced hybridoma secreting monoclonal antibodies(Mabs) against Hantaan virus, 76/118 strain and Seoul virus, 80/39 strain were produced by fusion of mouse myeloma cell, Sp2/0-Ag14 with spleen cells isolated from Balb/c mice immunized with Hantaan virus or Seoul virus. From the result in this study, the Mabs against Hantaan virus were screened by indirect immunofluorescent antibody techinque(IFA). 587 hybridoma cells were produced out of 768 wells and the fusion frequency was 76%. Seven among 587 hybridoma cells produced Mabs against Hantaan virus continuously. Isotype analysis of Mabs against Hantaan virus revealed that all these Mabs belong to the IgG2a subclass. All 7 Mabs reacted to nucleocapsid (N) proteins (MW.49-50Kd) of Hantaan virus by immunoblot assay. Mabs against Seoul virus were screened, 985 hybridoma cells were produced out of 1152 and the fusion frequency was 86%. Eight arnong 985 hybridoma cells produced Mabs against Seoul virus continuously. Isotype analysis of these Mabs against Seoul virus revealed the IgM and IgG2a subclass. These 8 Mabs reacted to nucleocapsid (N) proteins (MW.49-50Kd) of Seoul virus as demonstrated by immunoblot. Interestingly, these Mabs had neutralizing activity determined by PRNT analysis. Therefore it is suspected that the antigenic sites on N protein may be involved in the virus neutralization. The serological reactivity of 7 Mabs against Hantaan virus with Hantaan virus were strong, although Seoul, Puumala and prospect hill virus were not reacted to these Mabs. 8 Mabs against Seoul virus, reacted to Seoul virus strongly. Hantaan and Puumala virus reacted slightly to these Mabs but Prospect hill virus did not. To comparative study of sensitivity test between IFA and double sandwich ELISA method for human sera using ELISA of which these Mabs coated were tested, the ELISA method was more sensitive than IFA. These Mabs react.ed specifically to Hantaan and Seoul virus. There are antigenic relationships among Hantaviruses could be analysed by using these Mabs.
반상자(Sang Ja Ban),안광수(Kwang Soo Ahn),김주환(Ju Hwan Kim),임종준(Jong Jun Lim),김수연(Soo Yeun Kim),기미경(Mee Kyung Kee),이명숙(Myoung Sook Lee),조수열(Soo Yeul Cho),김영훈(Young Hoon Kim),김지윤(Ji Yoon Kim),이유경(Yoo Kyoung L 한국독성학회 2003 Toxicological Research Vol.19 No.4
Polychlorinated biphenyls (PCBs) has been widely used as plasticizer, insulator, lubricant, paint and ink. The persistence of PCBs in the environment and their bioaccumulation in living<br/> organism make a raise concerns regarding their toxic effects in immune system and subsequent effects on human health. However little has been known about effect of PCB, an endocrine disrupter,<br/> on splenocytes. In this study, for identifying the effect on the organs and immune cell of mice by the concentration and time of commercial PCB mixture (Aroclor 1254), each 3 mice were tested at the concentration of 3, 30, 300, 1,000 mg/kg respectively, and their organ's weight were measured in 4, 7, 14 days, respectively. Also according to concentration and time, PCB was evaluated for the effects on splenocyte viability and lipopolysaccaride (LPS) and concanavaline A (Con A)-induced splenocyte proliferation on mice spleen. In liver and lung, there were significantly defferent by concentration and time of PCB (p<0.0001). In respect of concentration of PCB, no significant effects on mice's liver by Aroclor 1254 concentration below than 300 mg/kg were observed except at the concentration of 1,000 mg/kg doses (p<0.0001). But there was not significant different change in mice spleen by concentration and time of PCB (p=0.2206) and the mode of weight change of spleen was different to of liver and of lung. Viabilities of splenocytes were decreased following treatment with high concentration of PCB. Also, LPS and Con A-induced cell proliferations were decreased by Aroclor 1254 at 1,000 mg/kg. These data suggest that Aroclor 1254 is the immunotoxic compound that may have an effect on mouse immune system.
To estimate the prevalence of Hemorrhagic fever with renal syndrome (HFRS), we tested 1137 people among Korean.100 residents in Kosung-Gun, Kangwon-Do, 100 in Yangyang-Gun, Kang- won-Do, 100 in Buan-Gun, Chunbuk, 100 in Wanju-Gun, Chunlabuk-Do, 295 in Chunlanam-Do, 398 in Cheju-Do and 44 workers from two Laboratories. Vero E-6 cell infected with Hantaan virus used as a antigen and indirect immunofluorescence test (IFT) were performed. The result obtained from this study, 3 out of 100 (3% ) residents in Kosung, 2 out of 100 (2% ) in Yangyang, 1 out of 100 (%) in Buan, 6 out of 100 (6%) in Wanju, 10 out of 295 (3.4%) in Chunnam, 13 out 398(3.3%) and 2 out of 44 (4.5%) from laboratory workers were found to have antibody to Hantaan virus. The titer ranged from 1:32 to 1:256 however no sera were reactive to anti-IgM antibody. The ratio of male and female seropositive was 2.4:1.
To the development of the rapid viral diagnosis for Japanes Encephalitis (JE), we established the dot enzyme immunoassay (DEIA) and RT-PCR to detect an antigen and antibody against JE virus. For detection of JE antibody in swine serum using the DEIA method, we confirmed 100% of the sensitivity and the specificity comparison to the ELISA and HI method. Beside, using three method: SDS-Proteinase K-phenol method, RNA Tack resin Kit (Biotecx) and Guanidine thiocyanate Kit (Promega) for isolation RNA of JE virus. Among these method, RNA Tack resin Kit was detected to JE virus even 10'dilution level which HA titer was 1:320 and this method is the most rapid isolation of RNA. The sensitivity of RT-PCR was found to be at least 10 to 10better, as compared to that of HA test. With the results, DEIA and RT-PCR is rapid di- agnosis method to detection for JE virus and it could be applied to the other viral disease for rapid viral diagnosis.
For easy and rapid screening of hemorrhagic fever with renal syndrome (HFRS) without any laboratory equipment, dot blot enzyme immunoassay was developed and tried to detect anti-hantavirus antibodies. The nucleocapsid protein of Hantaan virus was isolated by affinity chromatography and used for making the dot strip. 28 of 29 Hantaan virus infected sera showed positive signals and 21 of 22 HFRS negative sera showed no positive signals. Anti-Seoul virus monoclonal antibody also exibited positive signal but the intensity of colorization was approximately 5 fold less than that of anti-Hantaan monoclonal antibody. The sensitivity of dot blot assay was equal or superior to indirect immunofluorescent assay (IFA) or ELISA test. Overall, the screening results with dot blot assay showed 92.2 % of concordance with IFA or ELISA test. This results suggests that dot blot assay could be applied a tool for easy and rapid screening of HFRS.
A serological studies on Japanese encephalitis virus among Korean were carried out from April to December in 1991. The total 2,227 human sera (1,532 cases in preepidemic and 695 cases from post-epidemic of Japanese encephalitis (JE) ) were obtained from the 6 provinces in Korea. Haemagglutination inhibition(HI) test and plaque reduction neutralization (PRN) test were employed for antibodies against JE virus. The result obtained from this study, 589 out of 1,532 cases were positive antibody of JE and the rate was 38% in pre-epidemic. 443 out of 695 cases were positive and the antibody rate after epidemic season of JE was 64%. The titer of antibody ranged from 1: 10 to 1: 360. The mean titer in HI test from pre-epidemic season was 1: 17.1 and that from post-epidemic season was 1: 20.1. Obviously, the antibodies of JE increased after epidemic season of JE. The mean neutralizing antibody titer of some subjectives at Chonnam was 1: 10 in preepidemic season and 1: 20 in postepidemic season.
HFRS vaccine inactivated Hantaan virus with formalin prepared from infected suckling rat brain was developed in Korea, the antigenic potency of the vaccine and the Hantaan virus infectivity challenge test was studied using hamster as a model to assay induction of protective immunity by HFRS vaccine. 8-10 weeks old seronegative against Hantaan virus normal hamsters were immunized with 4096 ELISA unit of vaccine by intraperitoneal route two times at a 10days intervals, then were challenged 7 days after booster injection with live Hantaan virus (5 x 10 PFU). The animals were bled by cardiac puncture 3 weeks after virus challenge and various organs were collected for antigen detection using indirect immunofluorescence test (IFT) and Enzyme linked immunosorbent assay (ELISA). Mean antibody titer against Hantaan virus after vaccination was 1:210 by High density particle agglutination (HDPA) test and the neutralizing antibody titer was 1:16 by plaque reduction neutralization test (PRNT). Hantaan virus antigen was detected in the lung of hamster 3 weeks after virus challenge, however the antigen was not detected in the lung of vaccinated hamster. Above result suggested that HFRS inactivated vaccine was effective to production of neutralizing antibody and to protection against Hantaan virus in experimental animal.
The potency of influenza vaccine is determined based on its hemagglutinin (HA) content. In general, single radial immunodiffusion (SRID) assay has been utilized as the standard method to measure HA content. However, preparation of reagents for SRID such as antigen and antibody takes approximately 2~3 months, which causes delays in the development of influenza vaccine. Therefore, quantification of HA content by other alternative methods is required. In this study, we measured HA contents of H1N1 antigen and H1N1 influenza vaccine by reverse phase-high performance liquid chromatography (RP-HPLC) methods. The presence of HA1 and HA2 was investigated by silver staining and Western blot assay. In addition, accuracy and repeatability of HA measurement by RP-HPLC were evaluated. Comparison of HA concentration by SRID and RP-HPLC revealed a precise correlation between the two methods. Our results suggest that RP-HPLC assay can replace SRID in the event of a pandemic flu outbreak for rapid vaccine development.