• Journal of Veterinary Science
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• 제23권4호
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• pp.55.1-55.12
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• 2022

Background: ASF was first reported in Kenya in 1910 in 1921. In China, ASF spread to 31 provinces including Henan and Jiangsu within six months after it was first reported on August 3, 2018. The epidemic almost affected the whole China, causing direct economic losses of tens of billions of yuan. Cause great loss to our pig industry. As ELISA is cheap and easy to operate, OIE regards it as the preferred serological method for ASF detection. P54 protein has good antigenicity and is an ideal antigen for detection. Objective: To identify a conservative site in the African swine fever virus (ASFV) p54 protein and perform a Cloth-enzyme-linked immunosorbent assay (ELISA) for detecting the ASFV antibody in order to reduce risks posed by using the live virus in diagnostic assays. Method: We used bioinformatics methods to predict the antigen epitope of the ASFV p54 protein in combination with the antigenic index and artificially synthesized the predicted antigen epitope peptides. Using ASFV-positive serum and specific monoclonal antibodies (mAbs), we performed indirect ELISA and blocking ELISA to verify the immunological properties of the predicted epitope polypeptide. Results: The results of our prediction revealed that the possible antigen epitope regions were A23-29, A36-45, A72-94, A114-120, A124-130, and A137-150. The indirect ELISA showed that the peptides A23-29, A36-45, A72-94, A114-120, and A137-150 have good antigenicity. Moreover, the A36-45 polypeptide can react specifically with the mAb secreted by hybridoma cells, and its binding site contains a minimum number of essential amino acids in the sequence 37DIQFINPY44. Conclusions: Our study confirmed a conservative antigenic site in the ASFV p54 protein and its amino acid sequence. A competitive ELISA method for detecting ASFV antibodies was established based on recombinant p54 and matching mAb. Moreover, testing the protein sequence alignment verified that the method can theoretically detect antibodies produced by pigs affected by nearly all ASFVs worldwide.

• 한국임상수의학회지
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• 제27권4호
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• pp.402-406
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• 2010

Bovine leukemia virus (BLV) is a delta-retrovirus which causes chronic lymphocytosis in cattle. BLV infections have been divided into two groups such as enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL) according to the clinical symptoms in infected cattle. The conventional detection method of BLV was hematological procedure which is determining lymphocytosis in the suspected animals. Recently several sensitive methods were developed to detect antibody to BLV and nucleic acid of the BLV from infected cattle. In this study we have compared the difference of positive rates between agar gel immunodiffusion (AGID) and enzyme linked immunosorbent assay (ELISA) which are using for BLV antibody detection methods. The positive detection rate of ELISA test was 7.4% greater than the positive rate of AGID. The discrepancy of the positive rate between ELISA and AGID were showed in the group of age over one year old to under three year old group. The result from each test agreed very well in the group of over 5 year old cattles. The serological test is very useful method to select the infected cattle for the eradication or control of the disease in the infected herd. But it has a limit by interference of the maternal antibody from the cow of under 6 month old. This study shows that 16.2% of these ages group showed BLV gene positive by polymerase chain reaction (PCR) method. The result suggests that ELISA test need to be used with PCR to clarify misinterpretation of positive animals by antibody response due to the natural infection from maternally derived antibody in calves of under 6 months old.

• Journal of Veterinary Science
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• 제21권2호
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• pp.14.1-14.10
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• 2020

African swine fever (ASF) is a highly contagious disease of domestic pigs and wild boars (WBs). Without a vaccine, early antibody and antigen detection and rapid diagnosis are crucial for the effective prevention of the disease and the employment of control measures. In Sardinia, where 3 different suid populations coexisted closely for a long time, the disease persists since 1978. The recent ASF eradication plan involves more stringent measures to combat free-ranging pigs and any kind of illegality in the pig industry. However, critical issues such as the low level of hunter cooperation with veterinary services and the time required for ASF detection in the WBs killed during the hunting season still remain. Considering the need to deliver true ASF negative carcasses as early as possible, this study focuses on the evaluation and validation of a duplex pen-side test that simultaneously detects antibodies and antigens specific to ASF virus, to improve molecular diagnosis under field conditions. The main goal was to establish the specificity of the two pen-side tests performed simultaneously and to determine their ability to detect the true ASF negative carcasses among the hunted WBs. Blood and organ samples of the WBs hunted during the 2018/2019 hunting seasons were obtained. A total of 160 animals were tested using the pen-side kit test; samples were collected for virological and serological analyses. A specificity of 98% was observed considering the official laboratory tests as gold standards. The new diagnostic techniques could facilitate faster and cost-effective control of the disease.

• Journal of Veterinary Science
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• 제23권3호
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• pp.50.1-50.12
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• 2022

Background: There is an urgent need to find reliable and rapid bovine tuberculosis (bTB) diagnostics in response to the rising prevalence of bTB worldwide. Toll-like receptor 2 (TLR2) recognizes components of bTB and initiates antigen-presenting cells to mediate humoral immunity. Evaluating the affinity of antigens with TLR2 can form the basis of a new method for the diagnosis of bTB based on humoral immunity. Objectives: To develop a reliable and rapid strategy to improve diagnostic tools for bTB. Methods: In this study, we expressed and purified the sixteen bTB-specific recombinant proteins in Escherichia coli. The two antigenic proteins, MPT70 and MPT83, which were most valuable for serological diagnosis of bTB were screened. Molecular docking technology was used to analyze the affinity of MPT70, MPT83, dominant epitope peptide of MPT70 (M1), and dominant epitope peptide MPT83 (M2) with TLR2, combined with the detection results of enzyme-linked immunosorbent assay to evaluate the molecular docking effect. Results: The results showed that interaction surface Cα-atom root mean square deviation of proteins (M1, M2, MPT70, MPT83)-TLR2 protein are less than 2.5 A, showing a high affinity. It is verified by clinical serum samples that MPT70, MPT83, MPT70-MPT83 showed good diagnostic potential for the detection of anti-bTB IgG and M1, M2 can replace the whole protein as the detection antigen. Conclusions: Molecular docking to evaluate the affinity of bTB protein and TLR2 combined with ELISA provides new insights for the diagnosis of bTB.

• Asian Pacific Journal of Cancer Prevention
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• 제16권3호
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• pp.1281-1287
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• 2015

Background: Early detection of hepatocellular carcinoma using serological markers with better sensitivity and specificity than alpha fetoprotein (AFP) is needed. Aims: The aim of this study was to evaluate the diagnostic value of serum sICAM-1, ${\beta}$-catenin, IL-8, proteasome and sTNFR-II in early detection of HCC. Materials and Methods: Serum levels of IL-8, sICAM-1, sTNFR-II, proteasome and ${\beta}$-catenin were measured by ELISA assay in 479 serum samples from 192 patients with HCC, 96 patients with liver cirrhosis (LC), 96 patients with chronic hepatitis C (CHC) and 95 healthy controls. Results: Serum levels of proteasome, sICAM-1, ${\beta}$-catenin and ${\alpha}FP$ were significantly elevated in HCC group compared to other groups (P-value<0.001), where serum level of IL-8 was significantly elevated in the LC and HCC groups compared to CHC and control groups (P-value <0.001), while no significant difference was noticed in patients with HCC and LC (P-value=0.09). Serum level of sTNFR-II was significantly elevated in patients with LC compared to HCC, CHC and control groups (P-value <0.001); also it was significantly higher in HCC compared to CHC and control groups (P-value <0.001). ROC curve analysis of the studied markers between HCC and other groups revealed that the serum level of proteasome had sensitivity of 75.9% and specificity of 73.4% at a cut-off value of $0.32{\mu}g/ml$ with AUC 0.803 sICAM-1 at cut off value of 778ng/ml, the sensitivity was 75.8% and the specificity was 71.8% with AUC 0.776. ${\beta}$-catenin had sensitivity and specificity of 70% and 68.6% respectively at a cut off value of 8.75ng/ml with an AUC of 0.729. sTNFR-II showed sensitivity of 86.3% and specificity of 51.8% at a cut off value of 6239.5pg/ml with an AUC of 0.722. IL-8 had sensitivity of 70.4% and specificity of 52.3% at a cut off value of 51.5pg/ml with AUC 0.631. Conclusions: Our data supported the role of proteasome, sICAM-1, sTNFR-II and ${\beta}$-catenin in early detection of HCC. Also, using this panel of serological markers in combination with ${\alpha}FP$ may offer improved diagnostic performance over ${\alpha}FP$ alone in the early detection of HCC.

• 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.

• 한국동물위생학회지
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• 제31권1호
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• pp.43-55
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• 2008

Recently, the major viral diseases, Newcastle disease (ND), infectious bronchitis (IB), low pathogenic avian influenza (LPAI), avian pneumovirus infection (APV), Marek's disease (MD) and infectious bursal disease (IBD), have led to huge economic losses in chicken industry of Korea. To evaluate prevalence of the major viral disease infections in broiler breeder and broiler farms, epidemiological survey has been conducted in Jeonbuk province from 2005 to 2007 by serological ELISA test for APV, PCR for MD, and RT-PCR for ND, IB, LPAI and IBD, respectively. A total of 424 cases was submitted to our laboratory for diagnosis of the major viral disease from broiler breeder and broiler farms in the above period. The diagnosed results were analysed for the detection rate of infections on basis of years, seasons and ages, respectively. This study was showed that the detection rates of ND and APV were considerably high for every years regardless of seasons and ages in both broiler breeder and commercial broiler. In comparison with detection rates of ND and APV, IB and LPAI were lower but detected around 10% for every years. Especially, detection rate of IB was significantly high in commercial broiler than in broiler breeder. Therefore, to minimize economic losses for broiler breeder and broiler farms, it will need for effective countermeasures to decrease detection rate of the viral respiratory diseases. Although the detection rates of MD and IBD were gradually decreased from 2005 to 2007 in both broiler breeder and commercial broiler, it will continually make an effort about disease control for increasing productivity in chicken industry.

• 한국건강관리협회지
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• 제2권3호
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• pp.73-96
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• 2004

Along with a development of medical technology, a variety of tests, such as laboratory tests, x-ray and endoscopies are being used in health screening tests. As the tests determine the quality of health screening, test items and methods should be carefully selected. This study was to get hold of the test items of major health screening programs in Korea Most of the health screening programmes focused upon detection of risk factors and diagnosis of life - style related diseases (diabetes, hypertension, cardiovascular diseases, hypercholesterolemia, overweight, drinking, smoking, cerebrovascular diseases, osteoporosis) ,cancers (stomach, cervix, lung, breast, liver, colon, prostate, ovary, pancreas, thyroid, esophagus) , infectious diseases (hepatitis, tuberculosis, sexually-transmitted diseases, parasites) , chronic obstructive respiratory diseases, chronic renal diseases (bacteriuria hematuria, proteinuria) , anemia, glaucoma, hearing loss, Alzheimer disease, stress, early psychiatric diseases. The health screening tests were basic physical examination, basic laboratory tests (CBC, urinalysis, liver function tests, lipid tests, glucose, HbAlc, uric acid, electrolytes, serological tests (HBsAg, HBs-Ab, HCV-Ab, HIV-Ab, VDRL) EKG, x-ray (chest PA, CT) , endoscopy (gastroscopy, colonoscopy) , sonography(abdomen, thyroid, pelvis, breast) ,cytology (cervix) , bone density, tumor markets (NMP22, alpha-FP, CEA, CA-19-9, CA12S, PSA) and eye tests. Advanced technologies, like CT, PET, MRI, MRT/Angio, molecular testings) were widely used in hospital health screening programmes .In summary, a variety of tests were utilized in health screening in Korea. Those tests were utilized by stages or according to sex and age in most of health screening programmes, however a few program used tests to excess disregarding health screening subjects.

• 한국건강관리협회지
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• 제2권1호
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• pp.9-25
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• 2004

Along with a development of medical technology, a variety of tests, such as laboratory tests, x-ray and endoscopies are being used in health screening tests. As the tests determine the quality of health screening, test items of major health screening program in Korea. Most, of the health screening programmes focused upon detection of risk factors and diagnosis of life-style related diseases(diabetes, hypertension, cardiovascular diseases, hypercholesterolemia, overweight, drinking, smoking, cerebrovascular diseases, osteoporosis), cancers(stomach, cervix, lung, breast, liver, colon, prostate, ovary, pancreas, thyroid, esophagus), infections diseases(hepatitis, tuberculosis, sexually-transmitted diseases, parasites), chronic obstructive respiratory diseases, chronic renal diseases(bacteriuria, hematuria, proteinuria), anemia, glaucoma, hearing loss, Alzheimer disease, stress and earlypsychiatric diseases. The health screening tests were basic physical examination, basic laboratory tests( CBC, urinalysis, liver function tests, lipid tests, glucose, HbA1c, uric acid, electrolytes, serological tests(HBsAg, HBs-Ab, HCV-Ab, HIV-Ab, VDRL) EKG, x-ray(chest PA, CT) endoscopy(gastroscopy, colonoscopy), sonography (abdomen, thyroid, pelvis, breast), cytology(cervix), bone density, tumor markers(NMP22, alpha-FP, CEA, CA-19-9, CA125, PSA and eye tests. Advanced technologies, like CT, PET, MRI, MRI/Angio, molecular testing were widly used in hospital based health screening programmes. In summary, a variety of tests were untilized in health screening in Korea. Those tests were utilized by stages or according to sex and age in most of health screening programmes, however a few programs used tests excessvely disregarding health screening subjects.

• 한국환경보건학회지
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• 제20권3호
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• pp.19-30
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• 1994

Regarding to Q fever which is one of the most important zoonoses in Food (Milk) Hygiene and in Environmental Public Health, a sero-epidemiological study was carried out to detect the complement fixation antibodies to Coxiella burnetii Nine Mile strain phase II antigen among the milking cows in Kyunggi Province. The results obtained were summarized as follows: 1. The overall prevalence of the CF antibodies to Q fever among 989 milking cows of 75 dairy farms in Kyunggi Province was revealed as high as 58.7% by the farms and 27.8% by the individual cows with higher prevalence in Kyunggi Central and Kyunggi East regions than any other regions in the Province. 2. Anticomplementary reactions were appeared as 7.5% (74/989) and it ranged from 1.0% to 16.0% according to the regions investigated. 3. In the titration of the positively reacted sera, the figures of 16.7%, 37.5%, 29.8%, 9.5%, 2.9% and 3.6% at the serum dilutions of 1: 10, 1: 20, 1: 40, 1: 80, 1: 160 and higher than 1: 160, respectively. 4. It was recognized that the relatively higher cumulated frequency distribution of the CF antibodies was shown in the sera collected from the regions with higher prevalence of Q fever. 5. There was a high correlation between the prevalence of Q fever CF antibodies and the age which is usually equivalent to one year older than the calving history of the milking cows.