• Parasites, Hosts and Diseases
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• v.37 no.3
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• pp.163-169
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• 1999

House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophogoides pteronyssinus, were produce. Four monoclonal antibodies produced wee species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides, A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p2 in house dust. The sensitivity limit was 4ng/ml with rDer p2 and $8{\;}\mu\textrm{g}/ml$ with the d. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p2 could be useful for the environmental studies and for the standardization of mite allergen extracts.

• Food Science of Animal Resources
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• v.20 no.4
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• pp.303-310
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• 2000

In vitro 소화시 계란 성분이 anti-Y ruckeri IgY 항세 활성을 안정화시키는데 어떡해 관여하는지를 SDS-PAGE와 ELISA로 조사한 결과는 다음과 같다. Anti-Y ruckeris IgY 항체와 난황 및 난백으로 혼합 후 펩신으로소화시킨 후 경우 1시간 후 난황 및 난백이 혼합된 시료에서 모두 anti-Y ruckeri IgY 항체의 heavy chain과 light chain이 분해는 되었지만 어느 정도 밴드를 관찰할 수있었으며, anti-Y, ruckeri IgY 항체 활성 측정한 결과 난황시료는 35%, 난백시료는 61%의 항체 활성을 유지시켰다. Anti-Y ruckeri IgY 항체와 오보알부민, 오보뮤우신, 라이소자임 및 오보뮤코이드를 혼합 한 후 펩신으로 소화시킨 경우 1시간 우에는 anti-Y, ruckeri IgY항체의 heavy chain의 밴드르 fdjsm 정도 볼 수 있는 형태로 나타났으며, anti-Y, ruckeri IgY 항체의 light chain는 오보뮤우신 및 오보뮤코이드가 홉합되어 펩신으로 소화시킨 경우 1시간 후에는 밴드를 거의 볼수 없었으나 오보알부민 오보트란스훼린 및 라이소자임에 혼합된 시료에서 밴드를 관찰할수 있었다. 특히 라이소자임과 오보트란스훼린의 경우 펩신 호화 2시간 우에도 anti-Y, ruckeri IgY 항체의 light 밴드가 관찰되었다. 펩신소화 1시간 후에 난백성분이오보알부민, 오보뮤우신, 라이소자임, 오보트란스웨린 및 오부뮤코이드 중에서 단지 오보트란스훼린만이 38%의 anti-Y, ruckeri IgY 항체 활서을 보인후 2시간 후에도 15% 정동의 활성을 나타내었다. anti-Y, ruckeri IgY항체와 난백 및 전란을 혼합한 다음 무지개송어 위 추출액으로 소화시 2시간 후에 난백은 14%, 전란은 69%로 anti-Y, ruckeri IgY 항체 활성을 유지시킨 것으로 나타났다.X> $e_{I}$ WPi_BE_QE]]]]]로 상징하며 WLWQ에 적용되는 몇 가지 제약을 관찰하고 이를 일반적인 언어원리로 설명한다. 첫째, XP는 주어로만 해석되는데 그 이유는 XP가 목적어 혹은 부가어 등 다른 기능을 할 경우 생략 부위가 생략의 복원 가능선 원리 (the deletion-up-to recoverability principle)를 위배하기 때문이다. 둘째, WLWQ가 내용 의문문으로만 해석되는데 그 이유는 양의 공리(the maxim of quantity: Grice 1975) 때문이다. 평서문으로 해석될 경우 WP에 들어갈 부분이 XP의 자질의 부분집합에 불과하므로 명제가 아무런 정보제공을 하지 못한다. 반면 의문문 자체는 정보제공을 추구하지 않으므로 앞에서 언급한 양의 공리로부터 자유롭다. 셋째, WLWQ의 XP는 주제어 표지 ‘는/-은’을 취하나 주어표지 ‘가/-이’는 취하지 못한다(XP-는/-은 vs. XP-가/-이). 이는 IP내부 에 비공범주의 존재 여부에 따라 C의 음운형태(PF)가 시성이 정해진다는 가설로 설명하고자 했다. WLWQ에 대한 우리의 논의가 옳다면, 본 논문은 다음과 같은 이론적 함의를 기닌다. 첫째, WLWQ의 존재는 생략에 대한 두 이론 즉 LF 복사 이론과 PF 삭제 이론 중 전자의 입장을 지지한다. 둘째, WP를 XP로부터 복원할 때 부분 자질만 복사된다. 이는 어휘가 통사층위로 들어온 이후에도 어휘 자질들이 완전히 동결되는 것이 아니라 계속 지시될 수 있다는 가설을 지지한다.

• IMMUNE NETWORK
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• v.6 no.1
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• pp.33-42
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• 2006

Background: Calcineurin plays a crucial role in T cell activation, cell growth, apoptosis, and angiogenesis, and its over-expression has been implicated in the pathogenesis of cardiomyopathy and stroke. However, the expression and function of calcineurin in the pathologic lesion of chronic inflammatory diseases, like rheumatoid synovium, remain to be defined. This study was aimed to determine the role of calcineurin in inflammatory arthritis and investigate the expression and function of calcineurin in the rheumatoid synovium and synoviocytes, the actual site of chronic inflammation. Methods: Immuno-histochemical staining using specific antibody to calcineurin was perfomed in the synovium of rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients were isolated from RA and OA patients, and cultured with IL-1${\beta}$ and TNF-${\alpha}$ in the presence or absence of cyclosporin A, a calcineurin inhibitor. The calcineurin expression was assessed by phosphatase assay and Western blotting analysis. IL-6, -10, -17, matrix metalloproteinase (MMP)-1, -2, -3, and -9 released into the culture supernatants were measured by ELISA. After transfection with GFP-Cabin 1 gene into synoviocytes, the levels of IL-6 and MMPs were measured by ELISA. Results: Calcineurin was highly expressed in the lining layer of synovium and cultured synoviocytes of RA patients. The elevated calcineurin activity in the rheumatoid synoviocytes was triggered by proin flammatory cytokines such as IL-1${\beta}$ and TNF-${\alpha}$. In contrast, IL-10, an anti-inflammatory cytokine, failed to increase the calcineurin activity. The targeted inhibition of calcineurin by the over-expression of Cabin 1, a natural calcineurin antagonist, inhibited the production of IL-6 and MMP-2 by rheumatoid synoviocytes in a similar manner to the calcineurin inhibitor, cyclosporin A. Conclusion: These data suggest that abnormal activation of calcineurin in the synoviocytes may contribute to the pathogenesis of chronic arthritis, and thus provide a potential target for controlling inflammatory arthritis.

• Parasites, Hosts and Diseases
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• v.50 no.3
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• pp.191-197
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• 2012

Seroepidemiological changes of Toxoplasma gondii infection among the residents of the islands of Gangwha-gun, Incheon for 2 years were surveyed and evaluated by ELISA using a crude extract antigen. In 2010, sera of 919 adult residents in Gyodong-myeon and 313 adults in Samsan-myeon were collected and checked for IgG antibody titers, which showed 14.5% (133 sera) and 19.8% (62 sera) positive rates, respectively. In 2011, sera of 955 adults in Gyodong-myeon and 341 adults in Samsan-myeon were examined, which showed an increase of positive rates to 23.8% (227 sera) and 31.7% (108 sera), respectively. Totally, the seroprevalence of the first year was 15.8% and it increased rapidly to 25.8% in the second year. The positive rates of both sexes increased simultaneously with the significant ratio of males to females by 1.7-2.2 fold (P<0.05). In both myeons, 661 sera were collected every year and showed changes in optical density (OD) in 177 sera; newly found as positives in 73 persons (11.0%), negative conversion in 10 persons (1.5%), and maintained or increased in 94 persons (14.2%). This rapid increase in the prevalence of toxoplasmosis in Gangwha islands may be due to in part peculiar changes in the toxoplasmic environment of the islands and presumably the consumption of the pork bred domestically within the islands or imported from high endemic nations. It is necessary to find out symptomatic toxoplasmic patients and confirm the risk factors for further infection in the islands of Gangwha-gun.

• Korean Journal of Veterinary Research
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• v.46 no.4
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• pp.347-353
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• 2006

Serum samples of 30 chickens per flock from 6 grand parent stock (GPS) farms and 70 parent stock (PS) farms were collected for seroprevalent study of pullorum disease-fowl typhoid (PD-FT) infection by serum plate agglutination test (SPA). The incidence of PD-FT infection in GPS flocks and PS flocks were 0% and 15.7%, respectively. Especially PS flocks infected with PD-FT showed age dependent patterns that 22.2% of flocks between 20 to 30 weeks of age and 38.9% of flocks between 30 to 40 weeks of age were positive. The incidence of GPS flocks and PS flocks using Salmonella (S.) gallinarum 9R (SG9R) live vaccine were 33.3% and 58.6%, respectively. The sero-positive rate of 11 flocks were 6.7-83.3% by SPA and 2.9-55.6% by enzyme linked immunosorbent assay (ELISA), and ELISA showed more lower antibody levels than SPA. Furthermore, specific antibodies produced by SG9R vaccination were detectable by SPA using SG9R antigen without cross-reaction with the PD-FT infection.

• Parasites, Hosts and Diseases
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• v.27 no.1
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• pp.1-8
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• 1989

Serodiagnosis of parasitic infections is widely used, since parasites or their eggs are not always detected by ordinary methods. The sensitive tests such as ELISA are highly dependent on the purity of antigens used. To solve this problem. many workers have tried to find species-specific components of antigens, The present study was performed to determine the antigenic profile of crude saline extracts of 3, 5, 8 and 12-week old p. westermani worms, which were collected from experimentally infected cats, based on SDS-PAGE and immunoblot technique. The results were as follows: 1. The SDS-PAGE showed at least 30 Protein bands ranging from 229 kDa to 10 kDa molecular weight. The protein components of p. wsstermani changed chronologically during its developmental period. The 229 kDa band was recognized only in 12-week old worms ($$SEP_{l2}$). 2. Analysis by ELISA showed a significant increase in antibody levels at 3 weeks in infected cats using crude saline extract antigens ($SEP_3,{\;}SEP_5,{\;}SEP_8,{\;}SEP_{l2}$). 3. By EITB using $SEP_3$ and $SEP_5$ infected cats recognisea major protein bands with molecular weight of 60, 35, 28, 25 or 21 kDa at 3~12 weeks of infection, and 3 additional antigens, 19, 13 and 10 kDa, were detected at 8~12 weeks of infections. 4. Using $SEP_8$ 5 antigens, 91, 85, 31, 25 and 21 kDa, were consistently detected by all infected sera tested. In addition, 3 antigens of lg. 13 and 10 kDa were detected at 8~12 weeks of infection. Using $SEP_12$, similar results were obtained with that by using $SEP_8$ and 1 additional antigen of 229 kDa, specifically reacting with the sera from 12 weeks of in(traction, was recognized.

• Journal of Food Hygiene and Safety
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• v.33 no.3
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• pp.193-199
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• 2018

In this study, PAT protein of genetically modified maize was prepared from the recombinant E. coli strain BL21 (DE3), and mice were immunized with the recombinant PAT protein. After cell fusion and cloning, two hybridoma cells (PATmAb-7 and PATmAb-12) were chosen since the monoclonal antibodies (Mabs) produced by them were confirmed to be specific to PAT protein in the indirect enzyme-linked immunsorbent assay (ELISA) and western blot tests. There were no cross-reactions of either Mabs to other GM proteins or to the extracts of non-GM maize. The ELISA based on the PATmAb-7 can sensitively detect 0.3 ng/g PAT protein in corn. These results indicate that the developed Mabs can be used as bio-receptors in the development of immunosensors and biosensors for the rapid and simple detection of GM corn adulterated in foods.

• Journal of Life Science
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• v.24 no.9
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• pp.995-1000
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• 2014

The studies of marine viruses in terms of viral isolation and detection have been limited due to the high mutation rate and genetic diversity of marine viruses. Of the modern methods currently used to detect marine viruses, serological methods based on enzyme-linked immunosorbent assay (ELISA) are the most common. They depend largely on the quality of the antibodies and on highly purified suitable antigens. Recently, a new experimental system for using viral capsid protein as an antigen has been developed using the yeast surface display (YSD) technique. In the present study, the capsid protein gene of the red-spotted grouper nervous necrosis virus (RGNNV) was expressed and purified via YSD and HA-tagging systems, respectively. Two regions of the RGNNV capsid protein gene, RGNNV1 and RGNNV2, were individually synthesized and subcloned into a yeast expression vector, pCTCON. The expressions of each RGNNV capsid protein in the Saccharomyces cerevisiae strain EBY100 were indirectly detected by flow cytometry with fluorescently labeled antibodies, while recognizing the C-terminal c-myc tags encoded by the display vector. The expressed RGNNV capsid proteins were isolated from the yeast surface through the cleavage of the disulfide bond between the Aga1 and Aga2 proteins after ${\beta}$-mercaptoethanol treatment, and they were directly detected by Western blot using anti-HA antibody. These results indicated that YSD and HA-tagging systems could be applicable to the expressions and purification of recombinant RGNNV capsid proteins.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.77-86
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• 2001

Background: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell. Methods: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. Results: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patients captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. Conclusion: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.

• KSBB Journal
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• v.21 no.5
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• pp.325-330
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• 2006

This study presents the characterization of an integrated portable microfluidic electrical detection system for fast and low volume immunoassay using polystyrene microbead, which are used as immobilization surfaces. In our chip, a filtration method using the microbead was adopted for sample immobilization and immunogold silver staining(IGSS) was used to increase the electrical signal. The chip is composed of an inexpensive and biocompatible Polydimethylsiloxane(PDMS) layer and Pyrex glass substrate. Platinum microelectrodes for electric signal detection were fabricated on the substrate and microchannel and pillar-type microfilters were formed in the PDMS layer. With a fabricated chip, we reacted antigen and antibody according to the procedures. Then, silver enhancer was injected to increase the size of nanogold particles tagged with the second antibody. As a result, microbeads were connected to each other and formed an electrical bridge between microelectrodes. Resistance measured through the electrodes showed a difference of two orders of magnitude between specific and nonspecific immuno-reactions. The detection limit was 10 ng/ml. The developed immunoassay chip reduced the total analysis time from 3 hours to 50 min. Fast and low-volume biochemical analysis has been successfully achieved with the developed microfilter and immuno-sensor chip, which is integrated to the microfluidic system.