• Parasites, Hosts and Diseases
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• v.41 no.1
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• pp.35-39
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• 2003

Although stool examination is the standard diagnostic method of clonorchiasis, serodiagnosis by ELISA using crude antigen is now widely used because of its convenience. However, ELISA diagnosis still suffers from cross-reactions, and therefore there is a need to improve the present conventional ELISA. The present study was undertaken to evaluate the diagnostic value of ELISA using excretory-secretory antigen (ESA) instead of crude antigen (CA) of Clonorchis sinensis. The diagnostic sensitivity of ELISA using excretory-secretory antigen was 92.5%, which was higher than that of ELISA using crude Clonorchis sinensis antigen (88.2%). In addition, the specificity of excretory-secretory antigen was found 93.1% while that of crude antigen was 87.8%. In summary, Clonorchis sinensis ESA was found to be a better serodiagnostic antigen than CA for ELISA.

• Parasites, Hosts and Diseases
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• v.47 no.1
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• pp.65-68
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• 2009

Excretory-secretory products of Toxocara canis larvae have been considered as a major functional antigen in immune responses against toxocariasis. We studied ultrastructural localization of T. canis second-stage larval antigen using a seropositive human serum under immunogold electron microscopy. High-density gold particles were observed in the secretory cells, excretory duct, intestinal epithelium, and cuticle of the larval worm sections. The distribution of the positive reactions in the larval worms suggests that the nature of the antigen is excretory-secretory antigen including waste metabolites and secretory enzymes.

• Korean Journal of Veterinary Research
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• v.39 no.1
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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.

• Applied Microscopy
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• v.27 no.4
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• pp.403-416
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• 1997

In order to observe the localization of the specific antigenic protein in the tissue of Paragonimus westermani metacercaria, immunogoldlabeling method was applied using IgG of the dog which were infected with Paragonimus westermani metacercaria and IgG of rabbits which were immunized with purified 23 kDa protein from metacercaria of the Paragenimus westermani. The metacercaria worm tissues obtained from Cambaroides similis were embedded in Lowicryl HM20 medium, treated with infected and immunized IgG and protein A gold complex (particle size; 12 nm) and observed by electron microscope. In the tissue antigen of Paragonimus westermani metacercaria, the content of excretory bladder which was highly dense electron density was constituted in the excretory bladder of the parenchymal tissue. In the metacercaria tissues antigen reacted with IgG of infected dog. Labeled gold particles distributed on the interstitial matrix of parenchymal cells, fibrous granules of parenchymal tissue and the content of excretory bladder. High antigenicity was observed on content of excretory bladder. It was found to be specifically distributed at the tissue of Paragonimus westermani metacercaria. In the tissues antigen reacted with IgG of immunized rabbit. Labeled gold particles randomly distributed on the interstitial matrix and fibrous granules of parenchymal tissue but in the content of excretory bladder of Paragonimus westermani metacercaria, gold particles were richly labeled. Therefore, the 23 kDa protein contained with Paragonimus westermani metacercaria was found protein which was specifically constituted at the content of excretory bladder of Paragonimum westermani metacercaria. The 23 kDa protein was commonly contained from of Paragonimus westermani metacercaria to adult and showed strong antigenicity against the immunized and infected IgG.

• Parasites, Hosts and Diseases
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• v.45 no.1 s.141
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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.

• Biomedical Science Letters
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• v.3 no.2
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• pp.89-94
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• 1997

The authors characterized the proteins of the crude antigen obtained from Clonorchis sinensis worm and excretory-secretory and billis from rabbits, experimentally infected for 3 months. Protein composition was observed after adding a cysteine proteinase inhibitor E-64 and a serine proteinase inhibitor PMSF, respectively. SDS-PAGE of the crude antigen from C. sinensis recovered from the infected rabbits, the crude antigen from the adult worm excretory-secretory, and the crude antigen from billis of the rabbits resolved 26, 27 and 19 profiles between 200-9 kDa, respectively. When E-64 supplemented 29, and 22 bands, respectively. More study should be carried out in the future on the immunological characteristics and the monoclonal antibody of the each antigen.

• Korean Journal of Veterinary Research
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• v.40 no.1
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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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• v.36 no.1
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• pp.37-46
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• 1998

A Czonorchis sinensis-specific antigen in excretory-secretory product of C. sinensis (CsE) was assessed in human clonorchiasis by immunoblot. Thirty and 7 kDa antigens of CsE2, one of four different batches of CsEs reacted strongly with infection sera from clonorchiasis patients; however, the antigens reacted weakly with 6-month post- treatment sera from praziquantel-cured cases, but were still highly detected by the sera from praziquantel∼failed patients, indicating that the 30 and 7 kDa antigens can detect antibodies during an active infection. The 30 kDa antigen showed some cross reactions with sera from patients with Pcragonimus westemani and Metcfonimw vokogcujci, while the 7 kDa antigen did not, suggesting that the 7 kDa antigen has high specificity. The 30 kDa antigen reacted with some past clonorchiasis sera, whereas the 7 kDa antigen did not, supporting that antibodies to the 7 kDa antigen are not present in sera from past clonorchiasis patients. In an endemic area, 92% (23/25) of active clonorchiasis patients and 91% (10/11) of mixed infection patients with C. sinensis and M. Wokosawai had IgG antibodies to the 7 kDa antigen, while 40% (6/15) of past clonorchiasis individuals and 43% (3/7) of metagonimiasis patients cross-reacted to the antigen. These data suggest that the 7 kDa antigen in an excretory-secretory antigen may serve as a marker of an active clonorchiasis with reliable specificities in past clonorchiasis, paragonimiasis and metagonimiasis.

• Applied Microscopy
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• v.37 no.1
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• pp.43-52
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• 2007

In order to observe the localization of excretory, purified and infected antigenic protein in the tissue of Trichinella spiralis larvae, immunogoldlabeling methodology using IgG and protein A-gold complex was implemented. T. spiralis larvae obtained from rat muscle were initially cultured in medium, and secreted excretory antigen was collected for 1 or 3 days. Purified antigenic protein was obtained from homogenized T. spiralis larvae. Rabbits were then immunized with 1 or 3 days secreted excretory protein and purified 45 kDa protein, and IgG was purified from collected serum. Serum, against infected antigen, collected from rat on 1 and 4 weeks after infection with T. spiralis larvae, and IgG was purified from collected serum. T. spiralis larvae were embedded in Lowicryl HM20 medium. Then they were finally treated with immunized IgG and protein A-gold complex (particle size; 15 nm) and observed under electron microscope. In T. spiralis larvae tissue, the tissue antigen reacted with rabbit IgC antigen Day 1 secreted excretory protein, infected antigenic protein and purified 45 kDa protein. But different distribution pattern of labeled gold particles were observed. When Day 1 secreted excretoy protein was used, gold particle labeling was observed specifically on the cuticle, basal layer, esophagus interstitial matrix (EIM) and ${\alpha}_0,\;{\alpha}_1$ granules of stichocyte of the worm. In a separate group of tissue, the antigen reacted with rabbit IgG against Day 3 secreted excretory protein. Labeled gold particles were specifically distributed on the surface layer of cuticle, EIM and ${\alpha}_0$ granules of stichocyte of the worm. In case of using infected antigenic protein, gold particle labeling was specifically distributed on the cuticle and EIM of the worm. When purifed 45 kDa protein was used gold particle labeling was specifically distributed on the cuticle, basal layer, EIM and ${\alpha}_0,\;{\alpha}_1$ granules of stichocyte of the worm. Therefore, excretory antigens appeared to originate from the cuticle and ${\alpha}_0,\;{\alpha}_1$ granules of stichocyte for the first day but the cuticle layer associated with globular proteins and ${\alpha}_0$ granules of stichocyte after 3 days and infected antigens appeared to originate from the cuticle for 1 and 4 weeks after infection. These results suggest that excretory and infection specific antigens are secreted into the cuticle, basal layer, EIM and ${\alpha}_0,\;{\alpha}_1$ granules of stichocyte and 45 kDa protein may be contained these specific antigens.

• Journal of agricultural medicine and community health
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• v.16 no.1
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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.