• Korean Journal of Food Science and Technology
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• v.28 no.6
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• pp.1184-1187
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• 1996

A simple and rapid detection system for $aflatoxin\;M_1\;(AFM_1)$ in cow's milk by an enzyme-linked immunosorbent assay (ELISA) was developed. Specific antibodies against $AFM_1$, conjugated to bovine serum albumin $(AFM_1-BSA)$ were raised in rabbits and purified. The cross-reactivities of the antibodies against aflatoxin analogs were less than 29.9%. When a competitive direct ELISA (cdELISA) for $AFM_1$, established by use of the antibodies was applied to the spike test of $AFM_1$ onto uncontaminated cow's milk, the assay recovery was unstable unless cow's milk was diluted to 40% (2:3) with phosphate buffered saline (PBS). In that condition of sample dilution, the mean ELISA recovery of $AFM_1$, from the cow's milk was 113% (coefficient of variation (CV) of each recovery percentage, 8.2%) in the range of $0.3{\sim}3.0\;ppb$. These results showed that the ELISA system could be a convenient tool to monitor the contamination of AFM1 more than 0.5 ppb in cow's milk (FDA allowance limit) easily.

• Parasites, Hosts and Diseases
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• v.29 no.4
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• pp.397-402
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• 1991

Paragonimus westermani is a common fluke in Korea. The present study aimed to determine serum total IgE and specific IgG levels in experimental paragonimiasis of rats. Each Wistar rat was inoculated orally with 20~25 metacercariae of P. westermani from Cambaroides similis. Before and after infection(1,2,3,4,6,8 weeks) of P. westermani, the blood was collected from the retro- orbital venous plexus of rats and kept serum at $-70^{\circ}C$. Serum total IgE and specific IgG levels were determined by the capture and conventional enzyme-linked immunosorbent assay, respectively. The results were as follows; 1. Serum IgE values were increased to 0. 18${\pm}$0.042 at 2 weeks, $0.28{\pm}0.151$ at 4 weeks and 0.43${\pm}0.055$ at 8 weeks after infection. The absorbances of non-infected rats ranged $0.07{\pm}0.021~0.12{\pm}0.025$. 2. Specific IgG values were slightly increased at 3 weeks ($0.20{\pm}$0.032) and gradually increased up to 8 weeks($0.31{\pm}0.067$) after infection. The absorbances of non-infected rats ranged $0.11{\pm}0.035~0.18{\pm}0.019$. The present results suggested that p. westermani could elevate serum IgE and specific IgG antibodies in Wistar rats which were not a good definitive host.

• Toxicological Research
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• v.27 no.2
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• pp.125-131
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• 2011

Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1-carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with ${\lambda}$-type light chains. The $IC_{50}s$ of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml ($R^2$ > 0.99) and from 1 to 100 ng/ml ($R^2$ > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.

• Korean Journal of Veterinary Research
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• v.31 no.4
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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.

• Korean Journal of Veterinary Service
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• v.21 no.3
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• pp.255-260
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• 1998

Since bovine leukosis caused considerable economic loss to the dairy industry, seroepidemiologi-cal survey on bovine leukosis was carried out for the dairy herds in Kyunggi province. 1. When compared the results of immunodifussion test with those of enzyme-linked immunosorbent assay(ELISA) for 94 dairy herds sera, the relationship between the immunodifussion test and ELISA were showen high corresponding rate with sensitivity(97.5%) and specificity(92.6%). 2. In immunodiffusion test for bovine leukosis virus (BLV) antibody in 570 dairy cattle from 30 herds, mean positive rate for BLV antibody was 28.2%. The positive rate by districts were 16.5% in central, 35.4% in east, 17.3% in west, 29.1% in south, 31.6% in north, 43.7% in northeast. 3. When the results of serological studies was analyzed by age groups, the number of positive was increased gradually with the advanced in age of herds. The highest positive rate was found in the age over 6 years. 4. Of 30 dairy herds examined, 5 herds(16.7%) have no reactions against BLV antigen while 15 herds (50%) showed the range of 1∼5 positive cattle and 5 herds(16.7%), the rang of over 11 positive cattle.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.711-717
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• 1993

This study was performed to examine the charges of serum cortisol concentrations on the normal conditions in Korean native goats by enzyme-linked immunosorbent assay(ELISA). The Korean native goats were 4 to 30 months old and weighted 6.0 to 28kg. The goats were allowed ad libitum access to water and food. To minimize the stress with handing and blood sampling, animals were adapted for 2 weeks before the experiment. After adaptation, intravenouse catheter was inserted left intact a jugular vein of 6 goats and blood samples were done 2~3 days later. Experimental animals were divided into 2 groups which was non-catheterized and catheterized group in order to examine the effect for changes of serum concentration and circadian rhythm of cortisol. The results were obtained as follows ; The sensitivity of serum cortisol concentration was $20pg/m{\ell}$. The intra-assay and inter-assay coefficient of variation were below 2.5% and 5%, respectively. Serum concentration of cortisol was more higher in noo-catheterized group$(32.84{\pm}16.78ng/m{\ell})$ than catheterized group$(23.20{\pm}10.29ng/m{\ell})$. The difference according to months old in serum concentration of cortisol was the more higber on 4~6 than 7~12 and lowest over 12 months old. A circadian rhythm in the serum concentration of cortisol in Korean native goats was not found significantly with 2 hours sampling intervals.

• Parasites, Hosts and Diseases
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• v.26 no.2
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• pp.73-86
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• 1988

The sensitivity and specificity of crude and affinity-purified antigens of Clcnorchis sinensis obtained from the infected rabbits were studied. Stage-specific antigenic proteins from the eggs, metacercariae and adult worms were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent astray (ELISA). The results were as follows: 1. The antibody.binding antigen (ABA) purified from whole worm crude antigen (IVWA) by CNBr-activated Sepharose 4B affinity chromatography made :l specific bands against rabbit antisera on Ouchterlony gel diffusion plate, while WWA made 7 bands. Major WWA protein bands by SDS-PAGE were found at 16, 300~18, 500 and 28, 000~29, 000 daltons, while major ABA protein bands were at 18, 000~21, 000 and 29, 000~31, 000 daltons. The reactivity of ABA with rabbit anti-sera in ELISA was remarkably less sensitive than that of WWA. 2. Molecular weights of egg antigen (EGA), metacercarial antigen (MEA) and adult worm antigen (WWA) of C. sinensis ranged from 15, 000-200, 000 daltons, 15, 000-100, 000 daltons and 11, 000~80, 000 daltons, respectively. Major WWA proteins consisted mainly of polypeptide bands of low molecular weight, less than 31, 000 daltons, while those of EGA and MEA consisted of higher molecular T.eights than 30, 000 daltons. 3. The ELISA reactivities of WWA to rabbit anti.sera were remarkably greater than those of MEA. EGA showed negative reaction throughout the experiments. WWA showed higher optical density (O.D.) than 1.0, when reacted with rabbit anti-sera obtained at 4~6 weeks after the infection. In the rabbit anti-sera later than 12 weeks after the infection, the O.D. reacting witll WWA showed a plateau without variation. MEA shoT.ed relatively low O.D. values (<0.6), when reacted with anti-sera from lightly in(ected groups throughout the experiments, althougll there were some wealth positive cases (O.D.>0.6) ill heavily infected groups. MEA reacted with rabbit anti-sera showed negative results on Ouchterlony gel diffusion plates. Summarizing the above results, it is suggested that the whole worm antigen prepared from the adult worms of C. sinensis is most highly antigenic. However, this antigen might reveal cross reactions with other trematodes such as Paragonimus westermani, therefore, purification of antigenic proteins from the crude antigen is essential 18 increase the sensitivity and specificity for the immuncdiagnosis of clonorchiasis.

• Tuberculosis and Respiratory Diseases
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• v.45 no.2
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• pp.333-340
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• 1998

Background: Lung cancer is the leading cause of cancer over the world. P53 alteration is by far the most common genetic defect in lung cancer. The mutation of p53 protein involves the loss of inhibitory function of p53 related tumor suppressor gene and resultant oncogenesis. The analysis of p53 alterations consists of immunohistochemical stain, PCR based assay, or serologic ELISA (enzyme-linked immunosorbent assay). Methods : Serum levels of p53 mutant protein were measured in 69 cases of lung cancer (adenocarcinoma n=29, epidermoid n=16, small cell n=13, large cell n=1, undifferentiated n=1, undetermined n=9) and 42 controls of respiratory disorders using ELISA. Immunohistochemical stain in tissue was performed using monoclonal antibody of p53 in lung cancer subjects. Results: Both serum p53s in nonsmall cell cancer ($0.28{\pm}0.44ng/ml$) and in small cell cancer ($0.20{\pm}0.14ng/ml$) were not different from controls ($0.34{\pm}0.20ng/ml$). Also there was no significant difference in serum p53 according to tumor stages. P53 immunohistochemical stain showed 50% positivity overall in lung cancer. There were no close correlation between serologic level and positivity of immunohistochemical stain. Conclusion: The serologic determination of p53 mutant protein is thought to have no diagnostic role in lung cancer. Immunohistochemical stain in lung cancer specimen shows 50% positivity.

• Journal of Veterinary Clinics
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• v.28 no.2
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• pp.204-210
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• 2011

The presence of the tick-borne pathogens Ehrlichia and Borrelia in German Shepherd dogs in Korea was determined by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). A total of 291 dogs were randomly selected from five Korean provinces from October 2005 through September 2006. The seroprevalence of antibodies to canine Ehrlichia and Borrelia agents detected by ELISA (Snap$^{(R)}$ 3Dx$^{(R)}$ Test, IDEXX Laboratories) was 7.56% (22 dogs) and 1.72% (5 dogs) respectively, throughout the country. Positive antibodies against both pathogens were detected in two dogs (0.69%). The provincial distribution of seroprevalence against Ehrlichia was 1.28% (1 of 78) in Gyeonggi-do, 12.64% (11 of 87) in Gangwon-do, 9.76% (4 of 41) in Chungcheong-do, 8.93% (5 of 56) in Gyeongsang-do, and 3.45% (1 of 29) in Jeolla-do. According to PCR analysis, Ehrlichia chaffeensis target DNA was amplified in 3.09% (9 of 291 dogs) of blood samples, 2.41% (7 of 291) from Gangwon-do and 0.69% (2 of 291) from Chungcheong-do. The oligonucleotide sequences (SNU-EC3 and SNU-EC5) from the PCR fragment examined in Korea were closely related to E. chaffeensis isolated from the tick Haemaphysalis longicornis, in China and the state of Arkansas in the US. Based on these results, the presence of E. chaffeensis infection was identified in German Shepherds being bred in Korea. These results bring to light the importance of paying close attention to tick-borne infections such as Lyme disease during clinical diagnosis. This infectious disease should be included as a differential diagnosis for patients who participate in outdoor activity from spring to fall or who have thrombocytopenia or leucopenia.

• Tuberculosis and Respiratory Diseases
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• v.41 no.5
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• pp.452-461
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• 1994

Background: Tumor necrosis factor(TNF)-$\alpha$ and Interleukin(lL)-$1{\beta}$ are thought to play a major role in the pathogenesis of the septic syndrome, which is frequently associated with adult respiratory distress syndrome(ARDS). In spite of many reports for the role of TNF-$\alpha$ in the pathogenesis of ARDS, including human studies, it has been reported that TNF-$\alpha$ is not sensitive and specific marker for impending ARDS. But there is a possibility that the results were affected by the diversity of pathogenetic mechanisms leading to the ARDS because of various underlying disorders of the study group in the previous reports. The purpose of the present study was to evaluate the roles of TNF-$\alpha$ and IL-$1{\beta}$ as a predictable marker for development of ARDS in the patients with septic syndrome, in which the pathogenesis is believed to be mainly cytokine-mediated. Methods: Thirty-six patients of the septic syndrome hospitalized in the intensive care units of the Asan Medical Center were studied. Sixteens suffered from ARDS, whereas the remaining 20 were at the risk of developing ARDS(acute hypoxemic respiratory failure, AHRF). In all patients venous blood samples were collected in heparin-coated tubes at the time of enrollment, at 24 and 72 h thereafter. TNF-$\alpha$ and IL-$1{\beta}$ was measured by an enzyme-linked immunosorbent assay (ELISA). All data are expressed as median with interquartile range. Results: 1) Plama TNF-$\alpha$ levels: Plasma TNF-$\beta$ levels were less than 10pg/mL, which is lowest detection value of the kit used in this study within the range of the $mean{\pm}2SD$, in all of the normal controls, 8 of 16 subjects of ARDS and in 8 in 20 subjects of AHRF. Plasma TNF-$\alpha$ levels from patients with ARDS were 10.26pg/mL(median; <10-16.99pg/mL, interquartile range) and not different from those of patients at AHRF(10.82, <10-20.38pg/mL). There was also no significant difference between pre-ARDS(<10, <10-15.32pg/mL) and ARDS(<10, <10-10.22pg/mL). TNF-$\alpha$ levels were significantly greater in the patients with shock than the patients without shock(12.53pg/mL vs. <10pg/mL) (p<0.01). There was no statistical significance between survivors(<10, <10-12.92pg/mL) and nonsurvivors(11.80, <10-20.8pg/mL) (P=0.28) in the plasma TNF-$\alpha$ levels. 2) Plasma IL-$1{\beta}$ levels: Plasma IL-$1{\beta}$ levels were less than 0.3ng/mL, which is the lowest detection value of the kit used in this study, in one of each patients group. There was no significant difference in IL-$1{\beta}$ levels of the ARDS(2.22, 1.37-8.01ng/mL) and of the AHRF(2.13, 0.83-5.29ng/mL). There was also no significant difference between pre-ARDS(2.53, <0.3-8.34ngfmL) and ARDS(5.35, 0.66-11.51ng/mL), and between patients with septic shock and patients without shock (2.51, 1.28-8.34 vs 1.46, 0.15-2.13ng/mL). Plasma IL-$1{\beta}$ levels were significantly different between survivors(1.37, 0.4-2.36ng/mL) and nonsurvivors(2.84, 1.46-8.34ng/mL). Conclusion: Plasma TNF-$\alpha$ and IL-$1{\beta}$ level are not a predictable marker for development of ARDS. But TNF-$\alpha$ is a marker for shock in septic syndrome. These result could not exclude a possibility of pathophysiologic roles of TNF-$\alpha$ and IL-$1{\beta}$ in acute lung injury because these cytokine could be locally produced and exert its effects within the lungs.