• 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.

• 대한수의학회지
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• 제44권4호
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• pp.625-635
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• 2004

Trichnuris suis does not excrete eggs during larval stage as well as in particular adult stage, It is impossible to diagnose by use of fecal examination method in those periods. Therefore, serological diagnostic method can be very useful for those stages. In order to produce monoclonal antibody, specific somatic and secretory-excretory (SE) antigens of T. suis were identified and analyzed by SDS-PAGE and Western blot. Monoclonal antibody-producing hybridoma cells were cloned, which were made of popliteal lymph node of BALB/c mice immunized with a 45 kDa somatic antigen of T. suis. Five clones (1B9, 2C4, n2C5, 2D7 and 2D8) showing strong responses to T. suis antigens were selected and the isotype identified. All monoclonal antibodies were IgG1 isotype and the light chains were k chain. Established monoclonal antibodies reacted specifically to somatic and SE antigens of T. suis and did not cross-reacted to antigens of ascaris suum, trichuris vulpis, or Trichinella spiralis. The sensitivity of somatic and SE antigens against these monoclonal antibodies were significant (p<0.01) associated with those of positive and negative sera.

• 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.

• Parasites, Hosts and Diseases
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• 제32권4호
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• pp.249-258
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• 1994

톡소포자충(Tomplasmngondii)은 세포내에 기생하는 구포자충(Cuidia)의 일종으로 항원은 여러 가지 단백질로 구성되어 있으며 이들 항원의 분석은 면역학적 생화학적인 면에서 시도 해 볼 필요가 있다. 톡소포자충(RH주) tachyzoite의 용해물 및 분비항원을 SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)로 단백질 분획을 관찰하고 면역 전기영동이적법 (EITB, enzyme-linked immunelectrohansfer blot)을 시행하여 반응대를 관찰하였다. 토끼 (New Zealand산) 또는 BALB/c마우스를 톡소포자충으로 면역 또는 감염시키고 3주 또는 7주 후에 채혈 하였다. 면역 후 토끼나 마우스 혈청은 간접형광항체법(IFA)으로 항체가 1:1024 또는 1 : 128임을 확인하였다. 톡소포자충 용해물 및 분비항원으로 SDS-PAGE를 시행하였을 때 10 kDa에서 220 kDa까지 광범위한 단백질 분포대를 보였다. 톡소포자충 용해물 항원을 IgG 항혈청과 반응시켰을 때 주요 항원의 분자량은 23에서 35 kDa까지 5개의 반응대를 관찰하였으며 IgG와 IgM의 공동반응 대는 24. 27, 30. 35 kDa에서 관찰되었다. 분비항원을 IgG 항혈청과 반응시켰을 때 감염 마우스 의 복강액을 항원으로 한 경우 33(P30), 45 kDa의 반응대가 주요 항원임을 알 수 있었고 톡소포자 충을 CHL 세포로 in vitro에서 배양시킨 후 배양액의 상청액을 항원으로 EITB를 시행하였을 때 20 kDa. 30 kDa 이하의 분획에서 반응대가 관찰되었다. 이상으로 30 kDa 항원이 톡소포자충 tachyzoite의 용해물 뿐만 아니라 분비물에서도 관찰되는 중요한 항원임을 알 수 있었다.

• Parasites, Hosts and Diseases
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• 제39권4호
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• pp.307-312
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• 2001

In the course of Clonorchis sinensis infection, antigens presented to the hosts may be in a close relation to growth of the fluke. The antigenic proteins stimulating IgG antibody production were chronologically identified by immunoblot and localized by immunogistochemical staining. In the early stage of infection until 12 weeks post-infection (PI) , antigens were proteins with molecular mass larger than 34 kDa which were derived from the tegument, testes and intrauterine eggs. After 20 weeks PI antigens recognized were 29, 27 and 26 kDa proteins from the intestine, excretory bladder and reproductive organs. It is suggested that the tegumental proteins are the most potent antigens and the excretory-secretory proteins with middle molecular mass of 26-45 kDa contribute to the high level production of antibodies after 20 weeks of the C. sinensis infection.

• Parasites, Hosts and Diseases
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• 제36권1호
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• pp.37-46
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• 1998

인체 간흡충증의 현증감염 시기에 생산된 특이 IgG 항체와 반응하는 간촘충 특이항원을 규명하였다. 간흡충 성충의 조항원 및 서로 다른 조건에서 회수한 4가지 분비배설항원 (CsE)의 항원성을 면역이적법 소견으로 비교하였다 CsE2의 30. 7 kDa 항원이 간흡충 환자혈청과 강한 면역반응을 보였다. 이 항원들은 프라지콴텔 완치 후 6개월 혈청과는 반응하지 않았으나 투약 후 간흡충 충란이 검출된 환자의 치료 후 혈청과는 반응하여 현증감염의 면역반응과 관련된다고 판단하였다. 그러나. 30 Ka항원은 폐흡충 요코가와흉충 감염혈청과 교차반응하였고. 7 kDa 항원은 반응하지 않았다. 또. 30 kDa 항원은 일부 간흡충 과거 감염자 혈청과 반응하였으나 7 kDa 항원은 반응하지 않아 7 님)a 항원의 특이도가 높았다 간흡충 유행지역에서 7 kDa 항원의 민감도를 파악한 바 간흡충 현증감염자 25명 중 23명 (92%) 간홉충 요코가와흡충 중복감염자 11명 중 10명 (91%)과 반응하였다. 반면 간흡충 과거감염자 15명 중 6명 (40%), 요코가와흡충 감염자 7명 중 3명 (43%)과 반응하였다. 따라서. CsE2의 7 kDa 항원은 인체 간흡충 현증감염의 표식자가 되는 간흡충 특이 항원으로 판단된다.

• 농촌의학ㆍ지역보건
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• 제16권1호
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• pp.70-78
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• 1991

Antibody changes in experimental anisakiasis were observed by ELlSA and SDS-PAGE/EITB using various antigens : whole worm extract antigen(WWE), somatic antigen(SOM), excretory-secretory antign(ES), and hemoglobin antigen(HB) of Anisakis Type 1. The results obtained were as follows. l) Serum levels of IgG antibody by ELISA increased from 1st week of infection and reached their maximum titer at 5th week after infection, and decreased gradually thereafter. 2) The best result expressed as positive/negative ratio could be obtained when ES antigen was used. 3) Silver stained SDS-PAGE of each antigen showed at least 20 protein bands : In WWE, 286, 278, 262, 38, 18 Kd bands ; In SOM, 38 Kd band : In ES, 286, 65, 13 Kd bands ; In Hb, 61, 55, 38, 28, 26, 22, 20, 16, 15 Kd bands iepntibied as were major bands. 4) By EITB using WWE, Serum antibody recognized major protein with molecular weight of 86 Kd and 16 Kd. Using ES, 69, 59, 16 Kd bands were observed and using Hb, 28 Kd band was observed as specific band. In conclusion, excretory-secretory antigen(ES) of Anisakis larvae was most usable for ELISA.

• 대한수의학회지
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• 제40권1호
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• pp.130-137
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• 2000

In order to diagnose canine heartworm infection by antigen capture ELISA, the crude somatic(S), partial somatic(below 45kDa) and excretory/secretory(E/S) antigen of adult heartworm were identified and the antigenicity was examined by silver stain, immunoblot and ELISA. Then, production of monoclonal antibody to specific antigen carried out in this experiment. The bands to S antigen and E/S antigen were recognized between 10 and 200kDa and common bands were recognized strongly 14, 18, 28, 43kDa by silver stain. By western blot analysis, fractions to S antigen were recognized 14, 16, 18, 20, 24, 28, 32, 43, 50, 55kDa, etc. and only a 14kDa to E/S antigen in positive sera which were positive in modified Knott's test and necropsy. In ELISA, the positive sera reacted to antigens(SA, $SA_{45}$, E/S) were significantly different from negative sera by Student's t-test(p<0.05). Four hybridoma cell lines(14, 16, 17, 32kDa) than produce specific monoclonal antibodies for these antigens were obtained by immunizing BALB/c mice with a partially purified somatic antigen (below 45kDa) preparation, by fusing spleen cells with SP2/O cell myeloma cells, and by screening cell culture supernatants for antibody. In these results, it was confirmed that partial somatic antigen(below 45kDa) or E/S antigen can be used for serologic diagnosis of heartworm infection and monoclonal antibody reacting with specific antigen(14kDa) can be used for antigen capture ELISA in prepatent period of canine heartworm infection.

• Parasites, Hosts and Diseases
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• 제32권1호
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• pp.35-42
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• 1994

간흡충 특이 IgG 항체와 간흡충 분비배설항원간의 면역반응이 간흡충 감염과 치료에 의해 어떻게 변화되고 치료 후 소실되는 것이 있는지 관찰하였다. 간흡충 피낭유충 150, 450개씩 토끼에 감염시키고 감염대조군은 22개월. 프라지콴텔 치료군은 감염 4개월에 투약하였으나 완치되지 않아 이후 $4\frac{1}{2}$개월에 재투약하여 완치시킨 후 13개월까지 관찰하였다. 간흡충 분비배설항원은 SDS-PAGE에서 114, 102, 94, 80, 66, 63, 61, 59, 54, 52, 50, 47, 45, 42, 40, 38, 34, 33, 30, 27, 25, 23, 20, 19, 18, 17, 16, 12.5, 12, 11.5 kDa 단백질 구성이었다. 이중 항 원 단백질은 감염혈청과 치료후 혈청으로 Immunoblot할 경우 66, 63(A군) 54-38(B군) 34. 33(C군), 30-23(D군) 20-14(I군) 12, 12.5, 11.5(K군) kDa 단백질이었다. 특히 33. 27, 13. 12.5 kDa 항원은 감염기간 동안 가장 강한 면역반응을 나타낸 항원이었고 피낭유층 감염강도에 의한 차이는 없었다. 또한 투약은 했으나 완치되지 않은 4개월여 동안 면역반응의 변화는 없었다. 치료후 면역반응은 33 27 kDa 항원의 경우 환치후 13개월까지 지속되었고 치료전과 정도 차이가 없었다. 그러나 13, 12.5 kDa 항원의 면역반응은 완치후 1개월부터 약화되기 시작하여 6 개훨이면 소멸되는 양상이 뚜렷하였다. 12.5 MDa 항원은 SDS-PAGE상 단백질 함량이 많고 간흡충 감염의 강한 항원이면서 치료 후에는 특이항체와의 면역반응이 치료전에 비해 현격히 감소되므로 간흡충 현증 감염만을 혈청학적으로 진단해 줄 수 있는 항원으로 판단하고 간흡충 'K2-항원'이라 명명하였다.

• Parasites, Hosts and Diseases
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• 제50권4호
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• pp.349-352
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• 2012

A 94-year-old female with end-stage renal disease presents with fever, fatigue, and hematochezia. She had previously resided in Hunan Province, China, and Myanmar, and she immigrated to Taiwan 30 years ago. Colonoscopy revealed a colonic ulcer. Biopsy of the colonic ulcer showed ulceration of the colonic mucosa, and many Paragonimus westermani-like eggs were noted. Serum IgG antibody levels showed strong reactivity with P. westermani excretory-secretory antigens by ELISA. Intestinal paragonimiasis was thus diagnosed according to the morphology of the eggs and serologic finding. After treatment with praziquantel, hematochezia resolved. The present case illustrates the extreme manifestations encountered in severe intestinal paragonimiasis.