• 한국임상수의학회지
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• 제27권6호
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• pp.704-712
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• 2010

Porcine circovirus-2 (PCV2) 는 돼지에서 여러 형태의 질병과 증후군의 발생과 관련이 되어있어 현재는 PCV-associated diseases (PCVAD)로 총괄적으로 분류된다. PCVAD에 의한 높은 경제적 손실 때문에 많은 양돈장들이 PCV2의 감염을 확인하기 위하여 혈청을 검사하고 있다. 하지만, 기존의 혈액채취법은 비용이 높고 많은 인력이 소요되므로 큰 규모의 병원체 검사에는 어려움이 있었다. 이에 본 연구에서는 혈액채취법을 이용한 돈군의 PCV2 검사법에 대한 대체방법으로 돈방 단위의 구강액채취법의 유용성을 실제 농장에서 평가하였다. 세 곳의 다른 양돈 농장들에서 각각 6개의 25두 규모의 돈방들을 선정하여 생후 3, 5, 8, 12, 16주에 돈방 마다 하나의 구강액과 5개의 혈청을 채취하였다. 모든 시료들은 real-time PCR을 이용하여 PCV2 DNA를 검사하였고 IgG 또는 IgA 간접형광항체 검사법및 세 가지의 ELISA 검사법 (blocking ELISA, indirect ELISA, and IgG/IgM sandwich ELISA)을 이용하여 PCV2에 대한 항체를 검사하였다. 구강액에서 PCV2 DNA는 8주까지는 간헐적으로 검출이 되다가 16주에는 모든 돈방에서 검출이 되었으며, 모체유래 PCV2 특이 IgG는 3주부터 검출이 되었고 모든 농장에서 5-8주까지 지속이 되었다. 16주에는 한 농장 (Site 1)의 모든 돈방에서 감염에 의한 IgG와 IgA가 검출되었다. 혈청에서의 PCV2 DNA와 PCV2 항체의 검출은 구강액에서의 검출과 높은 상관관계를 보였다. 따라서 구강액은 돈군의 PCV2 감염을 모니터링 하기 위해 혈청대신 사용할 수 있는 저비용, 고효율의 시료가 될 수 있을 것으로 사료된다.

• The Plant Pathology Journal
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• 제36권5호
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• pp.509-514
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• 2020

Satsuma dwarf virus (SDV) seriously damages citrus production by reducing the quality and yield of fruit. To avoid contamination with SDV, mother trees are checked to be SDV-free in advance of nursery tree distribution. In this study, we compared an immunochromatographic assay (ICA) kit with double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for large-scale diagnosis of SDV in orchardgrown trees in Shizuoka Prefecture, Japan. The two methods gave conflicting results for 11 of 1,705 samples, all of which were negative by DAS-ELISA but positive by ICA. The samples scored as positive by either DAS-ELISA or ICA were analyzed by reverse transcription polymerase chain reaction and all were confirmed to be positive. These results validate the use of ICA as a screening method for large-scale diagnosis. Strain discrimination revealed that 16 of 22 isolates belonged to SDV, while citrus mosaic virus (CiMV) infection only and co-infection (SDV and CiMV) were in a minority.

• 환경생물
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• 제19권4호
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• pp.302-312
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• 2001

Immunoglobulin G was purified by 40% $(NH_4)_2SO_4$ precipitation, DEAE-Sephadex, Sephadex G-150 column chromatographies from rabbit antiserum against V. vulnificus ATCC 27562 O antigen and used for immunological test for V. vulnificus isolates. The profiles of cell lysate total protein and outer membrane protein from the isolates were analyzed by SDS-PAGE and densitometry. The overall profiles in all isolates were similar. Distict protein band was observed in comparison with V. parahaemolyticus. Western Blotting with rabbit Immunoglobulin G against cell lysates and OMP of V. vulnificus isolates showed a strong antigenic response to antigen 66, 60, 54, 48, 33 and 26 kDa which were common to all strains examined. The 26 kDa antigen showed V. vulnificus specific antigen in comparison with Vibrio parahaemolyticus. A sandwich enzyme-linked immunosorbent assay was developed by using rat anti-V. vulnificus ATCC 27562 polyclonal antibodies as capture antibody, a purified rabbit IgG antibody as detector antibody, and goat anti-rabbit IgG-alkaline phosphatase conjugate as developer antibody. When four V. vulnificus isolates were tested, the reactivity showed from 50 to 70% by sandwich ELISA.

• International Journal of Industrial Entomology and Biomaterials
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• 제29권2호
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• pp.214-219
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• 2014

We prepared antibodies from insect glycosaminoglycans (GAGs) and assayed the titer. Nine polyclonal antibodies against insect GAGs were raised for development of an ELISA in biological fluids (mice serum). The 3th booster collection of antiserum of BALB/c mice as a primarily antibody was assayed for titer determination by ELISA method. In sandwich ELISA of GAGs derived from Isaria sinclairii or other insects, antiserum from insect GAGs gave satisfactory results for so potent antibody(100: 1~1000:1) raising (manufacturing) agent in range of 10 ng/ml.

• BMB Reports
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• 제30권6호
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• pp.390-396
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• 1997

A sandwich-type enzyme-linked immunosorbent assay (ELISA) for the quantification of human apolipoprotein A-I (apoA-I) was developed using monoclonal antibodies. For this assay, we used three monoclonal antibodies to trap and detect apo A-I. HDAI16 and HDA15 monoclonal antibodies were used for trapping apoA-I and HDAI8 monoclonal antibody was for detecting apoA-I. These three monoclonal antibodies were produced by immunizing mice with high density lipoprotein (HDL) isolated from human plasma. By immunoblot analysis, these three monoclonal antibodies were specific to apoA-I and showed no cross-reactivities with other plasma proteins. The results of competition assays for epitope cross-reactivity test also verified that these monoclonal antibodies identified separate and distinct epitopes on HDL and apoA-I. Affinity constants of monoclonal antibodies were measured by ELISA. Their association constants ranged from $10^7$ to $10^8$ $M^{-1}$. For this assay, pure apoA-I was isolated by affinity chromatography using monoclonal antibodies. In this sandwich assay, the amount of HRP-labeled HDAI8 bound to apoA-I trapped by HDAI16 and HDAI5 was proportional to apoA-I concentration in the range of 0 to 500ng/ml. ApoA-I concentration in plasma was calculated from the linear regression equation of standard curve. The precision and reliability of the assays are reflected in the low intra-and interassay coefficients of variation that averaged 3.25% and 4.30%, respectively. This assay is sensitive, simple, reproducible, convenient in incubation interval, and does not use radioisotope: thus it can be widely applied in clinical laboratories.

• 대한한방내과학회지
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• 제30권3호
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• pp.582-593
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• 2009

Background and Objective : Allergy is defined as an altered reactivity to an antigen, which can result in pathologic reactions upon subsequent exposure to that particular antigen. This study was to evaluate the effect of oriental materia medica on IL-5 secretion in the mouse spleen cell. Methods : We used the splenocytes of mouse 8 weeks after its birth, and then cultivated those into the 2 experimental groups and a control group for 48 hours. The culture media of the experimental groups were made of $1{\mu}g/ml$, $10{\mu}g/ml$ oriental materia medica, representative. And the culture media of control group was given no oriental materia medica. Then, we assayed the quantity of cytokine-expression by the sandwich ELISA. The quantities of cytokine-expression of the experimental groups were compared with that of the control group, which was standardized. These methods were used for all of the oriental materia medica treated. Results : Some oriental materia medica inhibit the secretion of IL-5 in both $1{\mu}g/ml$ and $10{\mu}g/ml$ culture media. These were Acori Rhizoma, Luffae Fasciculus Vascularis, Amomi Rotundi Fructus, Schisandrae Fructus, Biotae Semen, Clematis armandii, Dioscoreae Sativa Rhizoma, Coicis Semen, Sophorae Flos, Oroxyli Semen, Aurantii Semen, Pini Nodi Lignum. Conclusion : This study indicates that some oriental materia medica inhibit the secretion of IL-5 and are beneficial for allergic disease.

• Parasites, Hosts and Diseases
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• 제25권2호
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• pp.141-148
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• 1987

실험적으로 폐홉춘을 감염시킨 백서의 혈청내 항원을 ELISA 법으로 측정하였다. 시험용 혈청은 폐흡충의 피낭유충을 마리당 25개씩 먹인 총 22마리의 백서에서 감염후 12주가 될 때까지 채혈하여 얻었다. 폐흡충에 대한 특이항체는 폐흡충 감염 고양이의 혈청으로부터 ammonium sulfate 침전과 anionezchange chromatography를 이용, IgG를 분리한 후 affinity chromatography를 이용하여 순수분리하였다. 항원을 찾기 위한 ELISA는 소위 "double antibody sandwich method"를 사용하였다. 대조로서 사용한 감염전 혈청의 O.D.치 평균값은 0.04(표준편차 0.04)이었으며 감염후 O.D.치 (표준편차)의 변화를 보면 0.5주(3일)는 0.03(0.01), 1주는 0.55(0.50), 1.5주는 0.69(0.45), 2주는 0.20(0.19), 2.5주는 0.13(0.10)이었고 감염 3주 이후는 대조 혈청의 값으로 떨어졌다. 대조혈청의 $평균간+3{\times}$ 표준편차, 즉 0.16이하를 이 system에서의 비특이적 수치로 잡았을 때 이 이상의 수치를 나타낸 경우는 감염 후 1주부터 2.5주까지의 혈청에서만 관찰되었다. 한편 감염 12주 후에 총 10마리의 백서를 희생시켜 유충을 회수하여 보았다. 폐와 흉강에서는 평균 2.2마리, 근육에서는 평균 6.2마리의 유충을 회수할 수 있었다. 폐흡충 감염 백서에 있어 충체가 생산하는 항원은 감염 1주 후부터 혈청 내에서 검출되었다. 그러나 감염 3주후부터는 검출되지 않았는데 이것은 항원-항체 복합체 형성에 의한 반응 저해 현상에 의한 것으로 생각되었다.

• The Plant Pathology Journal
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• 제32권5호
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• pp.452-459
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• 2016

The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-${\beta}$-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

• Journal of Microbiology
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• 제35권2호
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• pp.87-91
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• 1997

Monoclonal antibodies were prepared in order to an assay method for Vibrio vulnificus. Sixteen mouse ybridoma cell lines were established by immunization of whole cell antigen to BALB/c mice, fusion with SP2/O myeloma cells, and cloning. Most of them secreted IgM.lambda. antibodies. A sandwich enzyme-linked immunosorbent assay was developed with rabbit anti-V. vulnificus polyclonal antibodies as capture antibody, an IgM monoclonal antibody as detector antibody, and goat anti-mouse IgM-alkaline phosphatase conjugate as developer antibody. The range of detection was 10$\^$4/ to 10 V. vulnificus cells per microplate well. When four related Vibrio species were tested for cross-reactions, V. parahaemolyticus showed 3.5% reactively and V. carchariae, V. fluvialis, and V. furnisii showed negligibal (<1%) cross-reactivity.

• 대한미생물학회지
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• 제22권4호
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• pp.377-392
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• 1987

Monoclonal antibody to the Escherichia coli heat-stable enterotoxin(ST) was produced to develop a rapid and convenient diagnostic method to the ST. The toxin was purified from culture supernatant of enterotoxigenic E. coli O148H28($ST^+/LT^+$) and conjugated to bovine serum albumin(BSA). The ST-BSA conjugate was used to immunize BALB/c mice and the immune spleen cells from these mice were fused with $P3{\times}63$ Ag8.V653 plasmacytoma cells. Hybridomas were screened by ELISA and positive hybridomas were cloned by limiting dilution. Finally, one stable clone (AS36) specific to ST was selected for further growth and characterization. Antibody titers of culture supernatant and ascitic fluid from BALB/c mice were 1:1,024 and 1:20,480 respectively in ELISA. The isotype and subclass of monoclonal antibody was IgG1 in sandwich ELISA. To test the neutralizing effect of monoclonal antibody on toxin activity of ST, mixture of ascitic fluid and ST was assayed by infant mouse assay and this monoclonel antibody was proved to be a neutralizing antibody. The titer of ascitic fluid which completely neutralized biological activity of 4 units of ST was 1:4. Purified ST was quantitatively measured by competitive ELISA and minimum amount of ST detectable by this assay was 250pg, which was an amount six-fold smaller than that detectable by infant mouse assay. Four reference strains of enterotoxigenic E. coli from WHO were detected by competitive ELISA and highly specific, sensitive and reproducible result was obtained.