• 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.

• Applied Microscopy
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• 제39권2호
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• pp.117-124
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• 2009

간흡충(Clonorchis sinensis)은 우리나라에서 높은 감염률(2.9%)을 나타내는 기생충으로(KAHP, 2004), 간흡충에 감염된 담관은 간흡충의 흡반에 의한 물리적 자극과 대사산물 및 분비물 등의 화학적 자극에 의해 담관염이 일어나고, 간흡충이 성장할 때 충체 주위의 담관 상피세포의 증식, 탈락, 담관 주위의 염증 및 섬유화가 일어난다. 담관 점막에 분포하는 섬유모세포는 결합조직을 구성하는 세포의 한 종류로서 세포질돌기들이 잘 발달된 형태적 특징이 있으며, 세포질 내에 세포의 형태 유지, 신호전달, 인접세포와의 연접 등에 관여한다. 또한 조직을 발달시키고, 조직이 손상된 부위에서 콜라겐 층을 형성하여 손상된 조직이 복구되도록 하기도 한다. 상처의 반흔(scar) 형성과 지방축적, 염증(inflammation) 발생 과정에서 섬유모세포의 작용이 제대로 조절되지 못하면 섬유증(fibrosis)이 발달하게 된다는 연구 보고도 있다. 본 연구에서는 정상 흰쥐에서 분리된 담관 섬유모세포와 간흡충 감염 흰쥐에서 분리된 담관 섬유모세포를 배양하고, 각각의 실험군에 간흡충 분비배설 물질(Cs excretory-secretory product, ESP)을 첨가하여 배양하였다. 배양된 섬유모세포의 미세구조 변화와 세포 표면에 존재하는 sialic acid 및 actin의 분포를 전자현미경으로 관찰하여, 간흡충 감염에 따른 섬유모세포의 변화 및 간흡충 분비배설 물질의 자극에 따른 섬유모세포의 변화를 관찰하여 흰쥐의 담관 섬유모세포와 간흡충 감염과의 연관성을 알아보고자 하였다. 정상 흰쥐 담관에서 분리한 섬유모세포(G1)에 비하여 간흡충에 감염된 흰쥐 담관에서 분리한 섬유모세포(G2)와 간흡충 분비배설 물질을 첨가하여 배양한 섬유모세포(G1-1, G2-1)의 증식속도가 느린 것이 확인되었다. 세포질돌기의 수는 간흡충에 감염된 흰쥐의 담관으로부터 분리된 섬유모세포(G2)에서 가장 많은 수가 관찰되었고, 배양배지에 분비배설 물질을 첨가하면 정상 담관의 섬유모세포에서도 세포질돌기가 증가하였다. 따라서 간흡충 대사물질은 섬유모세포의 세포질돌기형성을 촉진시키는 것으로 생각된다. 간흡충에 감염된 흰쥐 담관에서 분리한 섬유모세포(G2)의 소포체는 정상 담관에서 분리된 섬유모세포(G1)의 것에 비하여 감소하는 양상을 나타내었고, 여기에 분비배설 물질을 첨가하면 섬유모세포의 소포체가 증가하는 것이 관찰되었다. 그리고 세포표면에 분비되는 sialic acid는 주로 세포질의 소포낭 주변에서 관찰되었으며, 정상 섬유모세포(G1, G1-1)보다 감염된 섬유모세포(G2, G2-1)에서 증가하였다. Actin은 세포표면과 세포질돌기에서 주로 관찰되었으며, 정상 섬유모세포(G1, G1-1)보다 감염된 섬유모세포(G2, G2-1)에서 반응이 증가하였고, 간흡충 분비배설 물질을 첨가하면 G1-1은 반응이 증가하고, G2-1은 반응이 감소하는 것이 관찰되었다. 이상의 결과로 간흡충에 감염된 흰쥐 담관의 섬유모세포는 세포질돌기들이 매우 발달하며, actin단백과 sialic acid가 증가하여 세포변형을 초래하게 된다. 또한 간흡충 감염으로 손상된 담관의 섬유모세포로 구성된 결합조직은 정상으로 회복되지 않으며, 세포질 부피 및 세포질돌기의 증가는 이루어지지만 간흡충 대사물질의 영향으로 섬유모세포의 분열 및 성장 속도가 억제되는 것으로 확인되었다. 이 결과 간흡충 감염으로 손상된 숙주의 담관 결합조직과 섬유모세포들은 간흡충 대사물질에 의하여 변형을 일으키고, 세포 활성 및 증식이 저하되므로 팽대된 담관은 간흡충이 사멸된다 하더라도 원상회복이 불가능할 것으로 생각된다.

• 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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• 제49권2호
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• pp.139-144
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• 2011

The present study was performed to estimate the seroprevalence of larval Anisakis simplex infection among the residents health-examined in 3 hospitals in southern parts of Korea. A total of 498 serum samples (1 serum per person) were collected in 3 hospitals in Susan Metropolitan city, Masan city, and Geoje city in Gyeongsangnam-do (Province) and were examined by IgE-ELISA and IgE-western blotting with larval A. simplex crude extract and excretory-secretory products (ESP). The prevalence of antibody positivity was 5.0% and 6.6% with ELISA against crude extracts and ESP, respectively. It was also revealed that infection occurred throughout all age groups and higher in females than in males. A specific protein band of 130 kDa was detected from 10 patients with western blot analysis against crude extract and ESP among those who showed positive results by ELISA. Our study showed for the first time the seroprevalence of anisakiasis in Korea. The allergen of 130 kDa can be a candidate for serologic diagnosis of anisakiasis.

• Parasites, Hosts and Diseases
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• 제49권4호
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• pp.373-380
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• 2011

We have reported that a 24 kDa protein (22U homologous; As22U) of Anisakis simplex larvae could elicit several Th2-related chemokine gene expressions in the intestinal epithelial cell line which means that As22U may play a role as an allergen. In order to determine the contribution of As22U to allergic reactions, we treated mice with 6 times intra-nasal application of recombinant As22U (rAs22U). In the group challenged with rAs22U and ovalbumin (OVA), the number of eosinophils in the bronchial alveolar lavage fluid (BALF) was significantly increased, as compared to the group receiving only OVA. In addition, mice treated with rAs22U and OVA showed significantly increased airway hyperresponsiveness. Thus, severe inflammation around the airway and immune cell recruitment was observed in mice treated with rAs22U plus OVA. The levels of IL-4, IL-5, and IL-13 cytokines in the BALF increased significantly after treatment with rAs22U and OVA. Similarly, the levels of anti-OVA specific lgE and lgG1 increased in mice treated with rAs22U and OVA, compared to those treated only with OVA. The Gro-${\alpha}$ (CXCL1) gene expression in mouse lung epithelial cells increased instantly after treatment with rAs22U, and allergy-specific chemokines eotaxin (CCL11) and thymus-and-activation-regulated-chemokine (CCL17) gene expressions significantly increased at 6 hr after treatment. In conclusion, rAs22U may induce airway allergic inflammation, as the result of enhanced Th2 and Th17 responses.

• Parasites, Hosts and Diseases
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• 제44권3호
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• pp.251-254
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• 2006

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3'-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5'-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.

• Parasites, Hosts and Diseases
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• 제46권1호
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• pp.29-32
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• 2008

The aim of this study is to determine the Toxocara seropositive rate among healthy people with eosinophilia. A total of 97 people residing in Seoul who were healthy and whose blood eosinophilia was over 10%, as shown by regular health check-ups in 2004, were subjected to this study. Their sera were tested by immunoblotting and ELISA with the antigen of larval Toxocara canis excretory-secretory (ES) protein. Sixty-five sera were band-positive (67.0%). The seropositve control sera were positive to band sizes of 66 kDa, 56 kDa, 32 kDa, and 13 kDa. In ELISA, 63 sera (65.0%) were positive to T. canis ES protein. There was no significant correlation between the IgG ELISA titer and the level of eosinophilia (r = 0.156, P = 0.156). As there were insufficient data to determine whether there were cross-reactions with other helminthic infections, or whether atopy occurred, further studies are required to verify the cause of the seropositive reactions against T. canis ES antigen. Toxocariasis seropositivity is suggested to be the major cause of eosinophilia, since the Toxocara seroprevalence among Korean rural adults was shown to be approximately 5%.

• Parasites, Hosts and Diseases
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• 제40권2호
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• pp.83-88
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• 2002

The 8 kDa antigenic protein of Clonorchis sinensis was partially purified by ammonium sulfate precipitation and subsequently by a column chromatographic steps. The purified protein was separated into 7 and 8 kDa protein bands through SDS-tricine gel electrophoresis, while the protein was fecund to migrate to a 8 kDa band in 7.5-15% SDS-PAGE. The molecular weight of the antigen was estimated to be 110 kDa by Superose 6 HR 10/30 gel filtration. The purified antigen strongly reacted with the human sera of clonorchiasis. The hyperimmune sera of BALB/c mice immunized against the 8 kDa protein were reacted with both the crude extract and the excretory-secretory product of adult worms, but not with the metacercarial extract. Immunohistochemical staining demonstrated that the protein was distributed to the tegument and subtegumental cells and also to the seminal receptacle. The present findings suggest that the 8 kDa protein is a partition of the multicomplek protein originating from various organs of adult C. sinenis, and that it is composed of several 7 and 8 kDa proteins.

• Parasites, Hosts and Diseases
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• 제42권4호
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• pp.169-174
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• 2004

This study was carried out to find out specific proteins from different organs of Clonorchis sinensis. Crude extract, organ-specific and excretory-secretory (ES) proteins were analyzed by immunoblot with infected human sera. The bands of 7- and 17 -kDa were main component of intestinal fluid and ES protein and commonly found in all organ-specific proteins. The 17-kDa protein was observed from ES antigen, intestinal fluid, eggs and sperms, 26- and 28-kDa proteins were from the uterus, vitellaria, and ovary, and 34-, 37-, 43- and 50-kDa proteins were mainly from the testis and sperms. Serum of mice immunized with sperms reacted to the 50-kDa protein by immunoblotting and immunohistochemical staining showed a positive reaction at the seminal receptacle and seminiferous tubule. The present results show that the 7-kDa protein is a common antigen of every part or organ of C. sinensis, but different organs express their specific antigenic protein bands.

• Parasites, Hosts and Diseases
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• 제58권4호
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• pp.475-479
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• 2020

Tegumental and excretory-secretory proteins are reported as diagnostic antigens for human opisthorchiasis. Rhophilin associated tail protein1-like (OvROPN1L) protein of Opisthorchis viverrini sperm tail showed potential as a diagnostic antigen. The OvROPN1L recombinant fragments were assayed for diagnostic antigenicity for human opisthorchiasis using indirect ELISA. The strongest antigenic region was a N-terminus peptide of M1 - P56. One synthetic peptide (P1, L3-Q13) of this region showed the highest antigenicity to opisthorchiasis. Sera from other parasitic infections including Strongyloides stercoralis, hookworm, Taenia spp, minute intestinal flukes, Paragonimus spp showed lower reactivity to P1. Peptide P1 is located in the disordered N-terminus of ROPN1L supporting its suitability as linear epitope. In the Platyhelminthes the N-terminal sequence of ROPN1L is diverging with taxonomic distance further suggesting that peptide P1 has potential as diagnostic tool in the genus Opisthorchis/Clonorchis. It should be further evaluated in combination with peptides derived from other O. viverrini antigens to increase its diagnostic power.