• Parasites, Hosts and Diseases
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• 제21권2호
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• pp.270-280
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• 1983

임상적으로 폐흡충증이 의심되나 충란검출이 안되었던 155예(3예는 충란발견)의 피내반응 양성례에 대하여 Agar-gel Diffusion (AGD)반응, Counterimmunoelectro-phoresis(CIEP)반응 및 Enzyme-Linked Immunosorbent Assay(ELISA)를 실시하여 이들사이의 상관관계를 검토하였다. AGD 반응과 CIEP 반응은 폐흡충친항원원액(단백농도 7.56mg1m1)과 혈청원연을 사용하였고 ELISA반응에서는 상기 조항원의 40,000배 희석항원을, 그리고 혈청은 100배 및 200배 희석혈청을 사용하였다. 1. AGD 반응과 CIEP 사이의 양성반응의 일치률은 98%이었고 융성반응의 일치률은 100%이었다. 침강대는 AGD 반응에서 1∼3개가 흔히 관찰되었고 CIEP 반응에서는 1∼7개가 관찰되었으며 염색강도도 후자에서 일부의 피검혈청에서 강하게 나타났다. 2. AGD 반응과 ELISA 반응은 사성반응에서는 100배 희석혈청에서 96%, 그리고 200배 희석혈청에서 94%의 일치률을 나타내었고, 음성반응에서는 100석 희석혈청 및 200배 회석혈청에서 각각 97%와 99%의 일치율을 나타내었다. 3. CIEP와 ELISA 반응은 양성반응에서 100배 희석혈청에서는 98%, 200배 희석혈청에서는 96%의 일치률을 나타내었고 융성반응에서는 100배 희석혈청에서 97%, 200배 희석혈청에서 99%의 일치률을 나타내었다. 4. 대조혈청인 간흡충감염 혈청, 아메바감염 혈청 및 Toxoplaima형광항체반응 양성혈청에 대한 AGD 반응, CIEP 및 ELISA 반응은 전례에서 음성이었다. 이상의 결과로 볼 때 반응에 사용된 항원-항혈청의 농도로 보아서는 ELISA 반응의 감수성이 월등히 높았다. 같은 농도의 항원과 혈청을 사용한 AGD와 CIEP 반응에서는 CIEP에서 침강반응의 염색강도가 보다 강하게 나타난 것으로 보아 세가지 반응의 감수성의 높은 순위는 ELISA, CIEP, AGD라고 보여진다. 그러나 본 실험에서 AGD와 CIEP반응에서 ELISA보다 높은 농도의 항원과 항혈청을 사용하였을 때 감수성의 차이는 거의 볼 수 없는 유사한 상관관계의 반응을 나타내었다. 즉 반응방법에 따라 적절한 농도의 항원과 항혈청을 사용한다면 감수성의 차이는 없다고 본다. 세가지 반응의 유효성 으로 볼 때 가장 수기가 간편한 AGD 반응은 조항원을 사용할 때 용이하게 사용할 수 있는 방법이라 생각되며 CIEP는 AGD보다 전기영동장치가 필요한 번거로움이 있으나 보다 조기에 반응 결과를 알 수 있다. 가장 감수성이 예민한 ELISA반응은 항원을 정제하여 소동의 항원으로 다수의 집권집사를 할 수 있는 가장 바람직한 방법이라 생각된다. (본 실험을 수행함에 있어 ELISA 반응을 검사해 주신 연세의대 기생충학교실의 장재경 선생님께 감사드립니다)

• 대한수의학회지
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• 제39권1호
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• pp.159-168
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• 1999

Swine whipworm(Trichuris suis) is cosmopolitan nematode which can cause serious pathology in immature stage(larva2~larva5) of infected pigs, such as anorexia, diarrhea, anemia, and death in heavy infections. In this larval stages, it is very difficult to diagnose the infection of whipworm and to differentiate from other common swine gastrointestinal disorders such as 21 day scours which are associated with TGE virus, rota virus, coccidium, and the stress of weaning. In this experiment, the isolated antigens of Trichuris spp. were carried out to examine the structure and specificity of antigens and to select the reasonable antigens which would be used in serological diagnosis by electrophoresis, Western blotting, ELISA. The results of this experiment were as follows; 1. The common fractions of each Trichuris suis antigen were identified 28,32,45, 80kDa by SDS-PAGE with silver stain and four major fractions could be detected in positive swine sera by Western blot analysis. 2. The OD(optical density) values of somatic and excretory-secretory antigens which were reacted against positive(negative) sera from pigs infected with Trichuris suis by ELISA reader were; 1) OD values($mean{\pm}SD$) of adult somatic antigen against positive(negative) sera were $0.30{\pm}0.12(0.09{\pm}0.006)$ and third-stage larva of somatic antigen were $0.28{\pm}0.038(0.10{\pm}0.005)$. And OD values of excretory-secretory antigens of adult and third-stage larva were $0.24{\pm}0.031(0.11{\pm}0.005)$ and $0.08{\pm}0.013(0.10{\pm}0.003)$, respectively. 2) OD values of adult somatic, larval somatic antigen and adult excretory-secretory antigen response to positive sera were significantly (p<0.01) associated with negative swine sera. And the Cut-off OD values(minimum positive value) were determined to be mean negative value plus 3 SD that would minimized the risk of false positives. 3. The OD values of somatic antigens of T suis and T vulpis against swine positive(negative) sera were $0.30{\pm}0.120(0.09{\pm}0.006)$ and $0.25{\pm}0.141(0.09{\pm}0.003)$. These data mean that the somatic antigens of T suis and T vulpis were able to diagnose T vulpis infection in dogs as well as T suis infection in pigs. These results suggest that somatic antigen of third-stage larva and excretory-secretory antigen of adult T suis could be used the diagnostic antigen by serological test(ELISA) in immature Trichuris spp. infection.

• IMMUNE NETWORK
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• 제13권4호
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• pp.157-162
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• 2013

Application of vaccine materials through oral mucosal route confers great economical advantage in animal farming industry due to much less vaccination cost compared with that of injection-based vaccination. In particular, oral administration of recombinant protein antigen against foot-and- mouth disease virus (FMDV) is an ideal strategy because it is safe from FMDV transmission during vaccine production and can induce antigen-specific immune response in mucosal compartments, where FMDV infection has been initiated, which is hardly achievable through parenteral immunization. Given that effective delivery of vaccine materials into immune inductive sites is prerequisite for effective oral mucosal vaccination, M cell-targeting strategy is crucial in successful vaccination since M cells are main gateway for luminal antigen influx into mucosal lymphoid tissue. Here, we applied previously identified M cell-targeting ligand Co1 to VP1 of FMDV in order to test the possible oral mucosal vaccination against FMDV infection. M cell-targeting ligand Co1-conjugated VP1 interacted efficiently with M cells of Peyer's patch. In addition, oral administration of ligand-conjugated VP1 enhanced the induction of VP1-specific IgG and IgA responses in systemic and mucosal compartments, respectively, in comparison with those from oral administration of VP1 alone. In addition, the enhanced VP1-specific immune response was found to be due to antigen-specific Th2-type cytokine production. Collectively, it is suggested that the M cell-targeting strategy could be applied to develop efficient oral mucosal vaccine against FMDV infection.

• Parasites, Hosts and Diseases
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• 제39권2호
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• pp.171-176
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• 2001

Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), meroxoite surface protein (MSP-1), apical merozoite antigen (AMA- 1), serine repeat antigen (SERA), and exported antigen (EXP- 1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG. The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination. When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera. In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively. We obtained higher detection rates when using S antigens (91.9%) rather than using each antigen solely (55.6 - 80%), a combination of 2 (76.3 - 87.5%), 3 (85.6 - 90.6%), or 4 antigens (89.4 - 91.3%). This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria.

• Parasites, Hosts and Diseases
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• 제45권1호
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• pp.19-26
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• 2007

This study describes the isolation of a Toxocara canis species-specific excretory-secretory(ES) antigen and the development of an enzyme-linked immunosorbent assay(ELISA) based on this antigen. Analysis of the ES antigens of T. canis, Toxocara vitulorum, Ascaris lumbricoides and Necator americanus larval antigen was performed by SDS-PAGE followed by western blotting. A 57 kDa T. canis-specific antibody fraction(TcES-57) was identified by western blotting and labelling with anti-Toxocara antibodies(from experimental rabbits and human patients) and tracing with anti-human or anti-rabbit peroxidase conjugate. No protein fraction of 57 kDa was detected in ES or larval antigens collected from T. canis, T. vitulorum, A. lumbricoides and N. americanus. Using TcES-57, a specific anti-serum was produced in rabbits and a double sandwich ELISA was developed. This test was validated using known seropositive sera from toxocariasis patients, sera from A. lumbricoides or N. americanus patients, and 50 serum samples from cats. These tests revealed that TcES-57 antigen is specific to T. canis infection and does not cross react with sera of other related infections. Thus, ELISA based on TcES-57 antigen was proven to be an effective tool in the diagnosis of toxocariasis and studies on the role of T. canis in the epidemiology of human toxocariasis.

• 대한수의학회지
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• 제41권1호
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• pp.43-49
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• 2001

As compared with reaction of antibody for sonicated antigen of Brucella abortus strain RB51 and 1119-3 by Western blot analysis, Brucella field positive sera was detected strong reaction at 40~80 kDa LPS of strain 1119-3, but detected very weak reaction at strain RB51 partly. Otherwise, as we analyzed major immunogen of RB51 by antisera bled periodically during 6 months after RB51 vaccination. we detected strong immunological reaction at 17, 18 and 8 kDa antigen of RB51. Especially, reaction of 8 kDa antigen by Western blot coincided with reaction of dot-blot assay in RB51-antibody detection method. We also compared with reaction of field sera by STAT(standard tube agglutination test), dot-blot assay and Western blot (reaction of 8 kDa antigen of strain RB51). 16 sera of 4~5 months after RB51 vaccination are all negative by STAT, and 12 field brucellosis positive serum are all positive, and also 12 of 16 sera vaccinated RB51 are positive by dot-blot assay and reaction of 8kDa antigen by Western blot. but 1 of 15 Brucellosis negative sera reacted nonspecifically dot-blot assay.

• 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.

• 한국동물위생학회지
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• 제26권1호
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• pp.19-26
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• 2003

This study was attempted to survey on the prevalence of canine heartworm(Dirofilaria immitis) infection among 100 dogs(male 39, female 61) on the nine breeding farms in eastern Chungnam province in December 2002. Blood samples taken from dogs were examined for the presence of D immitis microfilaria by the modified Knott's test and an antigen test was using FASTest$\^$/ HW Antigne kit (Mega Cor A-6912 Horbranz-Austraia). 1. Eleven(11.0%) of the 100 examined dogs were microfilaria positive, while nineteen dogs(19.0%) were antigen positive, which suggested that the antigen test was more sensitive than the microfilarial test in detecting heartworm infection. 2. Infected dogs were observed higher more at 2 years older ages(4/48, 8.3%) and male(9/39, 23.1%) than young ages(4/48, 8.3%) and female(10/61, 16.4%). 3. The regional infection rates were of Gongju(15/43, 34.9%), Geumsan(4.27, 14.8%), while none of infection dogs in Yeongi(0/30, 0%). 4. Survey for hematological values of D immitis infected dogs : WBC and eosinophils were 21.4${\pm}$7.2 k/${\mu}\ell$, 3.5${\pm}$0.4 k/${\mu}\ell$, respectively. In conculsion, this study could be overemphasized the importance of control program the heartworm in eastern Chungnam province

• 대한바이러스학회지
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• 제30권2호
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• pp.151-159
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• 2000

Hepatitis C virus (HCV) is one of the important human pathogen that can cause acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Recently, the third generation radiation immuno assay (RIA) method has been developed as a very sensitive test to detect anti-HCV antibody. However, false positive is the problem with RIA test. To solve this the RIA results were compared to those of 5-antigen recombinant immunoblot assay (5-RIBA) and reverse transcription-polymerase chain reaction (RT-PCR). Among 12,767 serum samples tested from clinic visitors, total 275 (2.2%) samples were antibody positive by RIA. RIBA was performed with 148 RIA positives cases but among them was shown eighty five was antibody positive and sixty three (42.6%) was negative result. However, nested RT-PCR test was shown also carried out with 43 positive, 6 intermediates and 25 negatives of RIBA. As a result of the nested RT-PCR results, HCV antigen were detected in RIBA positive, 33.3% (2/6) RIBA intermediate and 12% (3/25). Clinical syndrome of all 148 patients as a with chronic active hepatitis (46.0%), cirrhosis (18.9%), hepatocellular carcinoma (8.1%) and others (27.0%) and they were positive in reaction by RIA test. But RIBA positive patients with 34.9% of chronic active hepatitis, 18.6% of cirrhosis, 4.6% of hepatocellular carcinoma and 41.9% of others were detected to be positive case by nested RT-PCR.

• 한국동물위생학회지
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• 제34권2호
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• pp.159-166
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• 2011

This study was carried out to develope enzyme-linked immunosorbent assay (ELISA) for detection of canine brucellosis in dogs experimentally inoculated with Brucella abortus 1119-3 and B. canis RM666. Groups A, B and C of dogs (each group consisting of three dogs) were orally inoculated with approximately $5{\times}10^9$ colony-forming units of B. abortus and B. canis, and with sterile pyrogen-free PBS, respectively. The animals were monitored at regular intervals upto the 12th week post inoculation (PI) by standard tube agglutination test (STAT), plate agglutination test (PAT), Rose Bengal test (RBT), 2-mercaptoethanol rapid slide agglutination test (2ME-RSAT) and ELISA. The induced antibody titers in group A dogs were detected from the first week PI to the eighth week PI in STAT, PAT and RBT using the inactivated whole cells of B. abortus 1119-3 as antigens, while no sera in groups B and C dogs reacted with the antigens. In 2ME-RSAT using whole cells of B. canis M-strain as antigens, the induced antibody titers in group B dogs were observed at the second week PI and persisted for the 12th week PI, while sera of groups A and C dogs did not react with the whole cells. In ELISA using cytoplasmic fractions antigen of B. abortus 1119-3, the mean optical density of antibodies in groups A and B was detected from the first and second weeks PI, respectively, and persisted for 12th week PI, while sera of group C did not cross-react with the fractions antigen. However, in ELISA using the hot saline extracts of B. canis M- as an antigen, the induced antibody titers in only group B dogs were detected from second week PI and persisted for until the end of this study. These results indicate that the ELISA using B. abortus 1119-3 cytoplasmic fractions as antigens can be a good candidate for detection of brucellosis by B. abortus as well as B. canis in dogs.