• 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.

• Korean Journal of Veterinary Research
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• v.44 no.4
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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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• 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.

• 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).

• Parasites, Hosts and Diseases
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• v.39 no.4
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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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• 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.

• 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.

• 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.32 no.1
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• pp.35-42
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• 1994

This study was designed to evaluate the humoral immune reactions in clonorchiasis before and after praziquantel treatment. Rabbits were infected with 150 or 450 metacercariae, treated on 4 and 8111 months after infection, and observed for 13 months of posttreatment. Infection controls were maintained for 22 months. Antigen was the metabolic product of worms incubated in physiologic saline. The immune reactions of anti-clonorchis IgG were observed using SDS-PAGE/immunoblot. During the Infection and Posttreatment, the antigenic Proteins of 66, 63, 54, 52, 50,47,42, 40, 38, 34,33,30, 27, 25, 23, 20, 19, 18, 17, 16, 15, 14, 13, 12.5 and 11.5 kDa were detected. Of them, 33,27, 13, and 12.5-kDa antigens were highly antigenic and observed predominently in infection controls. After the treatment, 13 and 12.5-kDa antigens faded in 6 months after the second treatment, but 33 and 27-kDa antigens were detected until 13 months of posttreatment. The results clearly demonstrate that 13 and 12.5-kDa antigens represent attenuated host immune reactions by praziquantel treatment. As the 12.5-kDa antigen had a large amount of protein in SDS-PAGE, it was designated as'K2-Ag'of C. sinenis.

• Parasites, Hosts and Diseases
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• v.50 no.4
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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.