• Title/Summary/Keyword: enzyme-linked immunoelectrotransfer blot(EITB)

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Comparative antigen analysis of yrichomonus vaginulis by enzyme-linked immunoelectrotransfer blot technique (효소면역 전기영동 이적법을 이용한 질트리코모나스 항원의 비교 분석)

  • 민득영;임미혜
    • Parasites, Hosts and Diseases
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    • v.30 no.4
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    • pp.323-328
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    • 1992
  • Analysis of siR isolates of Trichemenes veginalis was carried out with sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunoelectrotransfer blot (EITB). Trichloroacetic acid-treated antigens of the 6 isolates revealed 25 protein profiles ranging 12~170 kDa of molecular weight in SDS-PAGE. In EITB, the specific immunogenic bands were visualized at 51 kDa and 96 kDa when HY-1 antigen was probed with difFerent mice sera immunized with 6 isolates of T. vaginalis. The banding patterns with different sera showed isolate-to-isolate variability. In EITB, homologous antigen (HY-1) did not show any enhanced response in reacting to homologous antiserum(HY-1) when 6 isolates of T. vaginalis were probed with a single serum (HY-1). It is assumed that the different banding patterns of six isolates show isolate-to-isolate variability and immunogenic common bands in 41, 47, 74 and 9:k kDa on EITB may connote the important significance on immune response in T. vaginalis infection.

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Immunoblot Patterns of Taenia asiatica Taeniasis

  • Jeon, Hyeong-Kyu;Eom, Kee-Seon S.
    • Parasites, Hosts and Diseases
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    • v.47 no.1
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    • pp.73-77
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    • 2009
  • Differential diagnosis of Taenia asiatica infection from other human taeniases by serology has been tested. An enzyme-linked immunoelectrotransfer blot (EITB) was applied to subjected human sera and tapeworm materials. Thirty-eight proteins reactive to serum IgG were observed between 121 and 10 kDa in adult worms, and more than 22 serum-reactive components between 97kDa and 21.5kDa were observed in eggs of T. asiatica. Antigens of adult T. asiatica revealed immunoblot bands between 120 and 21.5 kDa against T. asiatica infected sera. Antigens of adult Taenia saginata revealed 110-100, 66, 58-56, and 46 kDa immunoblot bands against T. asiatica infected sera. Antigens of adult Taenia solium also revealed 99-97, 68-66, and 46 kDa bands against T. asiatica infected sera. The immunoblot band of 21.5 kDa exhibited specificity to T. asiatica.

Identification of surface antigen of Trichomonas vaginalis (질편모충의 표면항원 분석)

  • 민득영;임미혜
    • Parasites, Hosts and Diseases
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    • v.32 no.4
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    • pp.243-248
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    • 1994
  • Plasma membrane proteins of a Korean isolate of Trichomonus vofinalis HY-1 were fractionated for antigen analysis. Homogenates of T. vaginalis were fractionated by the differential centrifugation using sucrose step-gradient method. The interface layer from the 25%/45% sucrose was collected as a plasma membrane fraction and its purity was examined by transmission electron microscopy. The antigenicity of plasma membrane fraction was analysed by enzyme-linked immunoelectrotransfer blot technique with immune rabbit serum and compared with surface antigen labelled with N. hydroxysuccinimide-biotin. The fluffy fraction of 25%/45% sucrose interface was homogeneous and membrane particles were present as extended sheet and concentric vesicles showing typical trilamellar appearance under transmission electron microscope. Seven fractions at 40, 50, 60, 110, 130, 140 and 150 kDa were identified as the antigenic membrane proteins in EITB with anti HY-1 rabbit serum. The common band at 60 kDa was detected both in antigenic fractions of plasma membrane and surface protein labelled with NHS-biotin. This result indicates that this protein is considered as a major surface antigen of T. vaginalis. The role of this surface antigen at 60 kDa should be studied further.

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Demonstration of species-specific and cross reactive components of Paragonimus tvestermani crude worm antigen by EITB (Immunoblot를 이용한 폐흡충 비항원의 특이 항원대의 증명)

  • Joo, Kyoung-Hwan;Ahn, Hyuck;Chung, Myung-Sook;Lim, Han-Jong
    • Parasites, Hosts and Diseases
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    • v.27 no.1
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    • pp.9-14
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    • 1989
  • Enzyme-linked immunoelectrotransfer blot (EITB) using crude worm antigen of adult Paragonimus westermani was performed for human patients sera to identify the species-specific components. Crude antigen was obtained by homogenizing and centrifuging 24-week old adult worms at 10,000 rpm for 60 minutes in phosphate buffered saline (PBS, PH 7.2) containing: Phenyl methyl sulfonyl auoride (PMSF). Gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) was performed and blotted electrophoretically onto a sheet of nitrocellulose paper. The sheet was cut into strips and exposed to sera diluted 1 : 200 with PBS. SDS-PAGE showed 26 protein bands ranging 229 to 10 kDa. Of them 229, 91, 60, 50, 35∼31, 27, 25, 21, 17, 11 and 10 kDa components showed positive reaction with serum antibody of patients with p. westermani. Sera of patients infected with Clcnorchis sinensis reacted with 35∼31, 19, and 11 kDa bands. Human sera from cysticercosis and diphyllobothriasis cases showed non-specific cross reactions with 229, 35∼31, 27, 25 and 17kDa bands. Protein bands of 91, 60, 21 and 10kDa showed strong positive reaction without cross reactions with sera from other helminthic infections.

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Analysis of antigenic specificities of Puragonimus westeymuni developmental stages using immunoblot technique (Immunoblot technique을 이용한 폐흡충의 발육단계별 항원 특이성 분석)

  • 주경환;홍성철
    • 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.

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Antigen analysis of Toxoplasma gondii Iysate and excretory-secretory materials by enzyme-linked immunoelectrotransfer blot (EITB) (효소면역 전기영동이적법에 의한 톡소포자충 용해물 및 분비 항원의 분석)

  • 안명희;손혁진
    • Parasites, Hosts and Diseases
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    • v.32 no.4
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    • pp.249-258
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    • 1994
  • Recently, the importance of toxoplasmosis is raised as a complication in immunosuppressed or AIDS patients. Our study focused on the identification of a variety of Toxoplasma antigens by immunoblotting. Rabbits and BALB/c mice were immunized with Toxoplosmo Iysate (RH strain) , frozen tachyzoites (RH strain) or cysts (Beverly and Fukaya strain) . Blood were collected from ear vein, heart or orbital plexus for detecting the serum antibody levels. For excretory-secretory (E.S) antigens, T gondii (RH) tachyzoite were cultured in CHL (Chinese hamster lung) cells with MEM containing of 5% FCS. After 72hrs, culture supernatant was collected. BALB/c mice were inoculated with RH tachyzoite intraperitoneally and peritoneal fluids were extracted three days later. E.S antigens were detected in culture supernatant and infected mouse peritoneal fluid by EITB. Serum IgG levels in rabbit were 1 :512 of 10 days after primary immunization, 1 : 2,048 of 10 days after secondary immunization, 1: 1,024 of 20 days after secondary immunization by IFAT, respectively. Serum IgG levels of immunized mice were 1:128 after 7 weeks. Tachyzoite antigens of the RH strain were detected 25 protein bands ranging 10 kDa-220 kDa of molecular weights with Coomassie blue stain. Toxoplcsma major antigens corresponding to n of 24 kDa, 27 kDa,30 kDa, 35 kDa, 38 kDa were recognized by IgG and IgM antibodies. Excretory-secretory antigens present in culture supernatant with M. W. of 20, 30 kDa and in infected mouse peritoneal fluid with M.W. of 33 (P30), 45 kDa. When RH tachyzoite antigen was probed with different mice sera immunized with 2 strains of T gondii, the IgG antibody bud of Fukaya and Beverly strain (8 week-serum) is identical to those of RH strain. It is considered that the 30 kDa polypeptide detected in excretory- secretory materials and Iysate was important major antigen of T gondii (RH).

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The production and characterization of anti-Naegleria fowleri monoclonal antibodies (Naegleyiu fowleri에 대한 단세포군 항체의 생산과 그 특성에 관한 연구)

  • 류재숙;임경일
    • Parasites, Hosts and Diseases
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    • v.30 no.1
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    • pp.33-42
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    • 1992
  • Naegleria fowleri, a free-living amoeba commonly found in moist soil and fresh water, enters the body via the nasal mucosa and migrates along the olfactory nerve to t he brain, where it causes acute amoebic meningoencephalitis. In the present study 7 clones secreting monoclonal antibodies (McAbs) against N. fowleri were produced and the effector function of them was investigated. Their isotopes were IgGl (Nf 1, Nf 154), 19G3 (Nf 137) and 19A (Nf 1, Nf 2, Nf 256, Nf 279). Five McAbs (McAb Nf 2, Nf 279, Nf 27, Nf 154, Nf 137) were specific for N. fowleri by ELISA and recognized the antigenic determinants located on the trophoBoite surface by IFAT and immunoperoxidase stain. These aye McAbs had capacity to agglutinate N. fowleri trophozoites and inhibited the growth of the amoeba in culture medium. McAb Nf 2 inhibited proliferation of trophozoites in vitro significantly. Also the cytotoxicity of JV. fowleri against CHO cell was reduced in the presence of McAb Nf 2 and McAb Nf 154. From these results McAb Nf 2 was confirmed to weaken the virulence of the amoeba among 7 screened McAbs.

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Studies on the Seroepidemiology of Helminthic Diseases in Korea (우리나라의 주요 기생충질환(寄生蟲疾患)에 대한 혈청역학적(血淸疫學的) 조사(調査))

  • Rim, Han-Jong;Lee, Joon-Sang;Joo, Kyoung-Hwan;Chung, Myung-Sook
    • Journal of agricultural medicine and community health
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    • v.16 no.1
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    • pp.48-60
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    • 1991
  • In a seroepidemiological study in several areas of Korea, the ELISA technique was performed to determine prevalence of some important helminthic diseases in our nation during March $15^{th}$ to June $30^{th}$. 1991. In this survey the serum antibody positive rates of anisakiasis, toxocariasis, clonorchiasis, paragonimiasis, cysticercosis, and sparganosis were measured. Among, 6,704 cases examined, 19.7% showed positive antibody titer at least one of the six items studied. Overall positive antibody rate was 8.1% in anisakiasis, 5.6% in toxocariasis, 3.6% in clonorchiasis, 1.7% in paragonimiasis, 4.5% in cysticercosis, and 2.6% in sparganosis respectively. In Pusan port southeastern part of Korea, antibody positive rate of anisakiasis was 2.9%, and clonorchiasis was 2.8% among 450 examine. In TaeJ$\check{o}$n city, central part of Korea. toxocariasis(6.7%) and anisakiasis(3.7%) showed high serologic positive rate. Of the 875 persons in Chunche$\check{o}$n gun(=province), northern central rural area of South Korea, anisakiasis was revealed as 3.4% seropositivity. In Tonghae port, eastern coast of South Korea. 9.9% of population examined showed positive antibody titer in anisakiasis. Of the 1,122 persons examined in Southern part of Cholla-Namdo(Southwestern coastal area of Korea), anisakiasis was 16.9%, cysticerocosis was 12.7% and the paragonimiasis was 3.3% respectively. In some localized area of Cholla-Pukdo, anisakiasis was 9.3% and cysticekosis was 4.3% among 702 cases examined. In some localized area of Kyungsang-Pukdo, anisakiasis was 10.6%. and toxocariasis was 16.1% among 900 cases examined. And finally, in Cheju-do, southern island of Korea, anisakiasis showed high positive rate(6.7%). Because cross reactions between related helminth group may disturb the analysis of these data, use of further developed techniques such as EITB(enzyme-linked immunoelectrotransfer blot) was considered as a essential tools for the study. We thought that probably most of the positive cases of cysticerosis were taeniasis cases. We can't rule out taeniasis even though EITB was employed as far as crude worm extract or cystic fluid of cysticercus was used as antigen. It was well Known that toxocariasis and anisakiasis also showed cross reactivity. However, the data presented here focus on seropositive rate of several helminthic diseases in Korea, not true prevalence rate of helminthiases, and to wait for more expensive purified antigen in sufficient amount for epidemiologic use is not necessary because increased immunologic sensitivity had little effect on epidemiologic sensitivity. We, here, suggest that ELISA should be applied as soon as possible to the evaluation of prevalence of tissue invading parasitic diseases, and a review of the antibody positive rate obtained in this study would be a basic data for controlling program of parasitic diseases in Korea.

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Analysis of antigen specificity using monoclonal and polyclonal antibodies to cysticercus cellulosae by enzyme-linked immunoelectrotransfer blot technique (효소면역전기영동이적법을 이용한 유조설고충 단세후군항체 및 환기혈청에 대한 항원특리성 분석)

  • Jo, Seung-Yeol;Gang, Sin-Yeong;Kim, Seok-Il
    • Parasites, Hosts and Diseases
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    • v.25 no.2
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    • pp.159-167
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    • 1987
  • To analyse the antigen specificity of patients sera from 24 confirmed neurocysticercosis and a monoclonal antibody, SDS-PAGE using 10~15% linear gradient gel and EITB were done. Cystic fluid, saline extracts of scolex and of whole worm of C. cellulosae, saline extracts of sparganum, hydatid cyst fluid, saline extracts of Fasciola, Clonorchis and Paragonimus were used as antigen. Of protein bands in cystic fluid of C. cellulosae, patient sera reacted frequently to bands of 152, 94, 64, 48, 24, 15, 10 and 7kDa proteins. To saline extracts of scolex and whole worm of C. cellulosae, patients sera reacted frequently to 94, 64, 52, 39, 34, 15 and 10kDa bands. Two bands in sparganum extract (130 and 64kDa) and two bands in hydatid cyst fluid (52 and 27kDa) were cross-reacting bands with sera from cysticercosis patients. Saline extracts of Fasciola, ClonorchiJ and Paragonimus did 'not exhibit cross-reacting bands. Monoclonal antibody to cystic fluid of C. cellulosae was found to react with low molecular weight proteins of 15, 10 and 7kDa.

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