• The Journal of Korean Society of Virology
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• v.28 no.1
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• pp.31-38
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• 1998

In Korea, all domestic made test systems for detecting antibodies in HIV-1 contain the antigens from human immunodeficiency type 1 (HIV-1) subtype B. However, because HIV-1 subtype O is significantly different in amino acid sequences from all other subtypes of HIV-1, there has been a need for developing a test for detecting antibodies in subtype O. For this purpose, the entire nucleotide sequence corresponding to the extracellular domain of the transmembrane glycoprotein of HIV-1 subtype O was synthesized with consideration of Escherichia coli condon usage. Various regions of the extracellular domain were cloned into E. coli expression vectors and tested for levels of protein production. The nucleotide sequence, named ECTM, that can encode a 129 amino acid-long peptide, was found to be expressed at a high level in E. coli. The protein of approximately 17 kDa specifically reacted with sera from individuals infected with HIV-1 subtype O. The ECTM protein was purified to near homogeneity by the CM-T gel chromatography, using concentrated, denatured inclusion bodies. In Western blot analysis, the purified viral antigen reacted with sera from individuals infected with subtype O more efficiently than subtype B. The enzyme linked immunoabsorbent assay (ELISA) system was developed using the subtype O viral protein and compared with the commercially available kit lacking the antigens from subtype O. The ELISA kit containing the subtype O antigen ECTM alone efficiently reacted with sera from individuals infected with subtype O. The subtype O antigen-containing kit produced a positive absorbence even when sera were diluted 512-fold, suggesting a high sensitivity. The commercially available kit also reacted with subtype O sera, but produced a negative result at a dilution of 8-fold. Our results suggest that the currently available kit may not be able to efficiently detect subtype O sera and that the viral protein developed in this study may be added to the current system to maximize the detection of sera from individuals infected with subtype O.

• Parasites, Hosts and Diseases
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• v.24 no.2
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• pp.171-176
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• 1986

In order to obtain the most specific and sensitive antigen from crude antigens of Fasciola hepatica for the immunodiagnosis of bovine fascioliasis by the enzyme linked immunosorbent assay (ELISA), phosphate buffered saline extract of F. hepatica was prepared. The crude extract was fractionated into 7 antigens using sephadex G-100 column chromatography. Seven fractionated antigens were applied to ELISA, precipitation test and intradermal test, respectively. Results obtained are as follows: 1. The specificity (95% confidence interval in negative sera of bovine fascioliasis; Mean+$2{\times}SD$ of absorbence) of the first (MW>150,000) and the second antigens(MW 120,000) were 93.7%, but those of others including crude antigen showed 100%. 2. The sensitivity(positive sera of bovine fascioliasis having higher values with compared to the criterion) of the first, the sixth (MW 16,000) and the seventh antigen (MW<5,000) were 91.6%, 87.5% and 0%, respectively, but those of others showed all 100%. 3. The absorbance by ELISA using the fifth antigen (MW 26,000) was 8. 43-folds higher in the positive sera than that in the negative sera. This could be used as one of the most specific antigens for the immunodiagnosis of bovine fascioliasis. 4. In Ouchterlony test, precipitin lines were not found in the sera naturally infected with F. hepatica, but some were found in the sera of rabbits immunized with the crude antigens. The numbers of precipitin lines in the sera of rabbits were different in the different fractionated antigens. They were 6 in the crude, 2 in the second and the third antigens, 1 in the fourth, the fifth and the sixth antigens and absent in the seventh antigen, respectively. 5. The wheal size for bovine infected with F. hepatica was $2.46{\pm}0.15cm$ in the intradermal test antigen(saline extract of F. hepatica) supplied by the Veterinary Research Institute, Rural Development Administration, Korea. The wheal size of the first, the second and the third antigens were larger than that. of intradermal test antigen, whereas those of the fouth, the fifth, the sixth and the seventh antigens showed smaller than that of the intradermal test antigen. The results suggest that the fifth antigen may be specific antigen for the immunodiagnosis of bovine fascioliasis.

• Development and Reproduction
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• v.13 no.4
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• pp.217-226
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• 2009

Effects of sera from several fish species on monolayer formation, viability and functions of catfish hepatocytes were investigated to establish a primary hepatocyte culture system for screening endocrine disruptors. Hepatocytes of Korean catfish (Silurus asotus) were attached and formed monolayer using the media supplemented with their own serum or sera from eel and tilapia, but not with fetal bovine serum (FBS). The amount of fish sera (0.5~3%) for monolayer culture of the catfish hepatocytes was less than 1/10 of FBS (5~20%) that is commonly used for primary culture of hepatocytes of other species. The results indicate that FBS can be replaced with sera from some fish species and the fish sera are more effective than FBS in maintaining the shape and functions of the hepatocytes. The primary culture of catfish hepatocytes was maintained monolayer with fish sera for at least 10 days, which makes possible to be used for screening the activities of endocrine disruptors. In conclusion, the primary culture system of hepatocytes with fish sera in the present study could be a useful tool for screening and studying endocrine disruptors.

• Parasites, Hosts and Diseases
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• v.31 no.1
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• pp.43-48
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• 1993

Antibody test is sometimes necessary for the diagnosis of acute human metagonimiasis because eggs may not be detected in stool. The antibody test (ELISA) was evaluated for its significance by reacting human sera from clinically diagnosed metagonimiasis, fascioliasis, clonorchiasis and paragonimlasis with 4 crude extracts of Metosonimn vokognwai (metacercariael , adults of Fosciola hepatica Cronorchis sinenis and Paragonimus westermoni. By ELISA, 10 of 11 metagonimlasis sera showed the highest absorbance (abs.) to the homologous antigen. Cross reactions to M. yokogawai antigen occurred most frequently in clonorchiasis sera. The antigenic protein fractions in M. vokogawai metacercarial extract were observed by SDS-PAGElimmunoblot using patients and control sera together with experimental cat sera. Out of 14 protein bands In the extract, 11 bands were reacting. Cross reacting bands to other trematodiasis sera were frequently observed. Of the reacting bands, 66 and 22 kDa proteuls were recognized as specific for metagonimiasis.

• Biotechnology and Bioprocess Engineering:BBE
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• v.4 no.1
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• pp.63-65
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• 1999

Although many pharmaceutically useful proteins are produced in E. coli expression system, it is very are rare for the system to be used in the production of diagnostic antigen due to a major problem, i.e., false-positive reaction of e. coli host-derived proteins contaminating purified diagnostic antigen with human sera. The N (nucleocapsid) protein of Seoul virus causing haemorrhagic fever with renal syndrome (HFRS) was produced in E. coli BL21 (DE3), and used for the detection of N protein-specific antibodies in human sera. Using the N protein as a diagnostic antigen of HFRS, the false-positive reaction was cleared by merely mixing the test sera with the extract of E. coli host strain not harboring expression plasmid.

• Parasites, Hosts and Diseases
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• v.54 no.2
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• pp.239-241
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• 2016

Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.16
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• pp.7149-7153
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• 2015

Background: Inflammation plays a major role in the development and progression of gastric and other gastrointestinal tumors. The IL-17 family of cytokines has been under investigation as targets of immunotherapy. Materials and Methods: We investigated the levels of IL-17A inflammatory cytokine in the sera of 57 patients with gastric cancer (GC) and 90 healthy age/sex matched controls using ELISA methods. Results: In only 5 (8.8%) of the patients' sera was IL-17A detectable. No IL-17A was apparent in the sera of healthy controls. The maximum concentration of IL-17A in patients was 7.004 pg/ml. Vascular and lymphatic invasions were only seen in one of the 5 positive cases. Although all of them were in the age group >60 years, no correlation was seen between age and IL-17A level. These results are somewhat different from our findings for colorectal cancer (CRC) in the same population. Conclusions: It is possible that the inflammopathology of CRC and GC are rather different, at least in Fars, a southern province of Iran.

• Parasites, Hosts and Diseases
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• v.41 no.3
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• pp.175-177
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• 2003

An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the C-terminal of the molecule was expressed as a $6{\;}{\times}{\;}His$ tagged protein (Ncp43p) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1％) were found to react with Ncp43p positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43p could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T.gondii infections in other mammals.

• Parasites, Hosts and Diseases
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• v.46 no.1
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• pp.29-32
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• 2008

The aim of this study is to determine the Toxocara seropositive rate among healthy people with eosinophilia. A total of 97 people residing in Seoul who were healthy and whose blood eosinophilia was over 10%, as shown by regular health check-ups in 2004, were subjected to this study. Their sera were tested by immunoblotting and ELISA with the antigen of larval Toxocara canis excretory-secretory (ES) protein. Sixty-five sera were band-positive (67.0%). The seropositve control sera were positive to band sizes of 66 kDa, 56 kDa, 32 kDa, and 13 kDa. In ELISA, 63 sera (65.0%) were positive to T. canis ES protein. There was no significant correlation between the IgG ELISA titer and the level of eosinophilia (r = 0.156, P = 0.156). As there were insufficient data to determine whether there were cross-reactions with other helminthic infections, or whether atopy occurred, further studies are required to verify the cause of the seropositive reactions against T. canis ES antigen. Toxocariasis seropositivity is suggested to be the major cause of eosinophilia, since the Toxocara seroprevalence among Korean rural adults was shown to be approximately 5%.

• Parasites, Hosts and Diseases
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• v.37 no.3
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• pp.157-162
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• 1999

Serum from mouse orally ingested with tissue cyst forming stain (Me49) of Toxoplasma gondii was assayed by Western blot and immunofluorescene assay (IFA) to establish early responses in antigenicity of the parasite in mouse model of foodborne toxoplasmosis. Sera were collected weekly to blot the RH antigen transferred onto nitrocellulose paper after being separated by 12% SDS-PAGE. With the second week serum, 34 kDa protein (p34) was detected uniquely, and all antigens of T.gondii were detected with the sera from 3 or 4 weeks. p34 was not a member of the major surface membrane proteins and confirmed to be localized in the rhoptry by IFA. It was secreted into parasitophorous vacuolar membrane (PVM) during the entry into host cells. 10.3% of sera detected p34, while all the ELISA positive sera detected the band. It has diagnostic usefulness of presumed T.gondii infection. We suggest the name of the p34 protein as ROP9.