• 대한수의학회지
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• 제31권4호
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• pp.491-500
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• 1991

This study was conducted to detect the serum antibodies in the experimentally toxoplasma infected dogs and street dogs by use the of an enzyme-linked immunosorbent assay (ELISA). And this test was performed on the polystylene microplate by coating with the tachyzoites soluble antigen of T gondii (RH strain), incubated with sera diluted then, added with HPO-conjugated rabbit anti-dog IgG and o-phenylenediamine used as a substrate. Tachyzoites of T gondii harvested from mouse peritoneal cavity were purified by 30, 40 and 50% Percoll density gradient centrifugation and used as the source of antigen. The results obtained were summarized as follows; 1. The highest ratio of positive to negative (P/N ratio) was obtained at the level of $l{\mu}g/ml$ protein concentration of antigen with the 1/4000 dilution of the conjugate measured by checker-board titration. It was regarded as the optimum concentration of the antigen and conjugate. 2. Cut-off value in this IgG ELISA was 0.375 that was determined by mean absorbance (at 492nm) of IFA negative serum added with the dauble value of the standard deviation $(mean{\pm}2S.D.)$. 3. Serum ELISA IgG antibodies to T gondii in the exyerimentally infected dogs were detected firstly at the Week 3 after inoculation and the highest titer was recognized at the Week 4, 5 and 6 after inoculation. 4. Stability of the antigen absorbed in the microplates that were preserved at $4^{\circ}C$ and $-25^{\circ}C$ separately were prolonged up to 3 weeks and 10 weeks at $4^{\circ}C$ and $-25^{\circ}C$, respectively. However the reproducibility was not reliable after the preservation of 4 weeks and longer. 5. Positive rate of the specific antibodies in 312 test sera was 28.5% and there was no significant differences between the male (27.8%) and female (29.5%), respectively. 6. The IgG ELISA was proved to be a specific procedure for the detection of antibodies to canine toxoplasma infection and also evaluated as a screening test for the large scale of test samples in laboratory.

• 한국식품위생안전성학회지
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• 제9권3호
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• pp.123-131
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• 1994

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of residual gentamicin(GM) in edible animal products. The immunogen(GM-KLH conjugate) and coating antigen(GM-BSA conjugate) were prepared by coupling GM sulfate to keyhole limpet hemocyanin(KLH) and bovine serum albumin(BSA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, respectively. Polyclonal antibody to GM was produced in rabbits(New Zealand White, female) by using the immunogen and the antibody titer was measured by indirect ELISA. A competitive ELISA was developed using GM-bovine serum albumin conjugate as a coating antigen, GM(as standards or sample), polyclonal antibody to GM, secondary antibody conjugated with horseradish peroxidase as an enzyme, and H2O2 and o-phenylenediamine dihydrochloride as a substrate and a chromophore, respectively. The detection limit of GM was 10 ng/ml and the standard curve of GM(n=26) was linear up to 10 $\mu\textrm{g}$/ml in this competitive ELISA system. There were no cross-reactivities of the partially purified antibody between GM and the various antibiotice such as amikacin, benzyl-penicillin, chloramphenicol, erythromycin, furazlidone, kanamycin, neomycin, oleandomycin, streptomycin, sulfathiazole and thiamphenicol(CR50<0.05%)

• BMB Reports
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• 제38권6호
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• pp.683-689
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• 2005

Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying ahexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5% of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100% with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.

• 한국어병학회지
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• 제9권2호
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• pp.169-176
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• 1996

연어과 어류의 이상유영 원인 바이러스 RVS의 ELISA법에 의한 신속 진단 방법을 개발하였다. 주화세포를 이용한 바이러스 배양액 및 감염 무지개송어의 뇌조직 마쇄액을 사용하여 실험하였다. 바이러스 배양액을 이용한 ELISA법의 검출 감도 조사에서 최소 바이러스 감염가 검출 한계치는 $10^{2.6}$ $TCID_{50}/100{\mu}l$ 이었다. 또한, 인공감염어의 뇌조직 마쇄액 내의 RVS 항원도 검출 되었다. 본 방법은 현장에서의 RVS 감염어 조사에 효과적으로 사용되어질 수 있는 방법으로 생각 되어진다.

• Parasites, Hosts and Diseases
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• 제23권1호
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• pp.95-101
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• 1985

Fascioliasis in cattle is one of the most common and very serious trematode diseases in Korea. In the present study, the enzyme linked immunosorbent assay (ELISA) was applied in the diagnosis of fascioliasis using antigen of Fasciela hepatica, perokidase of conjugate anti-cattle Is G and orthophenylenediamine as a substrate by micro-method technique of Volley et at. (1976b) and MacLaren (1978) with a slight modification. Results obtained from the present study are as follows. 1. In assay for optimal dilution of stock antigen, the antigen (protein contents; 0. Bmgymz) was diluted from 1150 to 1/600 with carbonate buffer (pH 9.6), and then absorbance values were measured with 1/100 diluted sera. The regression equations between the OD values of ELISA and dilution of antigen were log Y: -0.181-0.00127X in infected sera, and log Y: -0.578-0. 000879X in normal sera. The significantly higher (p<0.05) OD value was observed in the former. 2. In assay for optimal dilution of sera, the sera were diluted from 1125 to 1/400 with in PBSJ Tween 20 (pH 7.4), and absorbance values were measured with 1/200 diluted antigen. The regression equation between the OD values of ELISA and dilution of sera were log Y: -0.1540-0.0007238X in infected sera and log Y: -0.4834-0.00116X in normal sera. The former was higher than the latter (p<0.05). 3. In the 27 cases of negative intradermal test, OD values of the ELISA are $0.447{\pm}0.144$, the 95% confidence interval (Mean+2 H SD) of the values was 0.735, and there was no case over the values. Therefore, the sensitivity of the antigen to diagnose fascioliasis was 100% in the negative case. The OD value 0.7 which is designed as a criterion (detection level of positive one) is useful for the performance of the ELISA in fascioliasis. 4. According to the OD value of criterion in the regression equations, the optimal dilutions of stock antigen and serum were 1/250 and 1/100, respectively. 5. In the 58 cases of fascioliasis from which the adult could be found in the bile ducts, the OD value was $0.846{\pm}0.224$. The 75% (44 cattle) among them had higher value with compared to the criterion, and the 60% (20 cattle) of the cases of proliferative cholangitis of 33 cattle which had been infected previousely with Fasciola sp. is higher than the criterion. 6. Prevalence of fascioliasis was 43.4% in the application of the ELISA to 272 cattle which were reared in Jeonbug district.

• Bulletin of the Korean Chemical Society
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• 제23권4호
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• pp.605-609
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• 2002

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of organophosphorus insecticide cyanophos. An analogue (hapten) of cyanophos was synthesized and was coupled to BSA to produce polyclonal antibodi es from rabbits. The antisera were screened against another hapten coupled to ovalbumin (OVA). Using the sera of highest specificity, an antigen-coated ELISA was developed, which showed an I50 of 310 ng/mL with the detection limit of 20 ng/mL. The antibodies showed negligible cross-reactivities with other organophosphorus pesticides except for parathion-methyl, which makes the assay suitable for the selective detection of cyanophos.

• 한국임상수의학회지
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• 제24권2호
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• pp.169-171
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• 2007

The aim of this study was to detect bovine viral diarrhea virus (BVDV) from calves in Chonbuk province. Blood samples were taken from ninety-two dairy calves. Capture enzyme-linked immunosorbent assay (ELISA) was used to detect BVDV. BVDV were detected in eight out of ninety-two (8.6%) dairy calves. BVDV were detected in one of twenty five of female calves and one of twenty three of male calves of 4 months old, whereas in the 5 months age group, BVDV were detected in low of twenty three of female calves and two of twenty one of male calves. There were no significant differences (p>0.05) in the detection rate of BVDV on the basis of sex. On the other hand, ages of calves had significant differences (p<0.05) on the prevalence of BVDV.

• 대한수의학회지
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• 제38권4호
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• pp.793-802
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• 1998

Salmonellosis caused by a number of serotypes of Salmonella is an infectious, acute or chronic, zoonotic disease and characterized by enteritis and diarrhea, septicemia in animal. In these studies we investigated the prevalent serotypes of Salmonella causing animal salmonellosis in Korea and the 71 strains of Salmonella spp. were isolated from materials such as mesenteric lymph nodes, fecal samples from slaughtered animal. With the identification test results, the most prevalent serotypes were, in order, S stanley 31 strains (43.7%), S typhimurium 19 strains (26.8%) and S montevideo 11 strains (15.5%), respectively. And we could establish the method for detection of antibodies to broad variety of Salmonella serotypes. Lipopolysaccharide(LPS) antigen extracted from Salmonella was more sensitive and specific than outer membrane protein antigen from that for detection of Salmonella antibody by using an indirect ELISA. The optimal concentration of antigen was 100ng/ml of LPS, the dilutions of conjugate and serum were 1 : 1,000~2,000 and 1 : 200~400, respectively. The mix LPS-ELISA which was used by mixing LPS from S typhimurium (group B), S choleraesuis (group C) and S enteritidis (group D) were more rapid and effective than that used LPS from individual strain for detection of Salmonella serogroup O4, O7 and O9 antibody at the same time. We could obtain the high values of optical density ($0.73{\pm}0.32$) by mix LPS-ELISA on the farm which had occurred salmonellosis, but very low values of $0.17{\pm}0.06$ on the negative farm of salmonellosis. So, the mix LPS-ELISA may be used to monitor the serological surveillance for the presence of infection with a number of serotypes of Salmonella and would be useful for prevention and control of salmonellosis in animal.

• Parasites, Hosts and Diseases
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• 제59권6호
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• pp.565-572
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• 2021

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.

• 대한수의학회지
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• 제33권1호
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• pp.131-135
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• 1993

Enzyme-linked Immuno sorbent Assay (ELISA) for the serological diagnosis of Brucella abortus was developed and compared with plate agglutination test. Cell wall antigen was extracted from Brucella abortus 1119-3 by sonication and with a sodium deoxychlate solution. Optimum protein concentration of coating antigen were $0.4{\mu}g/100{\mu}{\ell}$ protein on each microtiter plate well. Horse radish peroxidase (HRP) labeled protein-G was used as a tracer of reacted antibodies. ELISA confirmed the agreeable results of 40 cases out of 43 cases by plate aggulutination test. ELISA diagnosed positive cases(10 out of 12) and negative cases (1 out of 12) with dubious sera by plate agglutination test. From this results ELISA could be used for the early diagnostic tools of Brucellosis in cattle.