This study was designed to detect and evaluate an antigenicity of low molecular weight proteins of Fasciola hepatica in fascioliasis. Low molecular weight protein of F. hepatica was purified by ammonium sulfate precipitation and Sephacryl S-100 HR gel filtration. The protein obtained was estimated to be 8 kDa on 7.5-15% gradient sodium dodecyl sulfate gel electrophoresis. Immunoblotting studies showed that the 8 kDa protein reacted with human fascioliasis sera, but not other trematodiasis sera. This result suggests that the 8 kDa protein of F. hepatica is one of diagnostic antigens in human fascioliasis without cross-reaction with other human trematodiasis.
Animals ; Antigens, Helminth/*isolation & purification ; Electrophoresis, Polyacrylamide Gel ; Fasciola hepatica/immunology/*isolation & purification ; Fascioliasis/blood/*parasitology ; Helminth Proteins/*isolation & purification ; Human ; Immunoblotting

OBJECTIVETo establish and optimize the proteomic analysis of protoscoleces-specific antigens from Echinococcus granulosus. To provide a foundation for identifying specific antigens in the soluble proteins of E. granulosus protoscoleces for further research.

METHODSBrood capsules were collected aseptically from fertile E. granulosus cysts from the livers of an infected patient. The fertile E. granulosus cysts were fractured, and protoscoleces were collected by centrifugation. The soluble proteins of protoscoleces were acquired using the 2D Quant kit according to the manufacturer's instructions. We employed two-dimensional electrophoresis (2-DE) combined with immunoblot assay (Western blot) to analyze the soluble components of E. granulosus protoscoleces antigens. The 2-DE and immunoblot maps obtained were analyzed with PDQuest 8.0 image analysis software.

RESULTSAbout 233 soluble protein spots were identified with Coomassie-stained gels. Most of the proteins had a molecular weight of 16,000 Da to 117,000 Da, and an isoelectric point value of 3.0 to 10.0. 2-DE immunoblot was conducted and 57 specific antigen spots were observed, among which 23 spots were identified.

CONCLUSION2-DE combined with Western blot is the key to successful proteomic analysis and presents a new possibility for searching the specific E. granulosus protoscoleces antigens.

Animals ; Antigens, Helminth ; chemistry ; metabolism ; Blotting, Western ; methods ; Echinococcus granulosus ; classification ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Regulation ; Helminth Proteins ; isolation & purification ; Proteomics ; methods

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of D1 fraction (D1A) among 5 antigenic protein fractions partially purified by DEAE-anion exchange chromatography from water-soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. D1A showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that D1A was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immuno-reactivity of two immunoglobulins (PI-Ig, D1-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with D1-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in D1 antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue.
Animals ; Antigens, Helminth/*analysis/isolation & purification ; Chromatography, Ion Exchange ; Immunohistochemistry/methods ; Microscopy, Immunoelectron ; Paragonimus/*immunology ; Support, Non-U.S. Gov't

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.
Animals ; Antibodies, Helminth/diagnostic use/isolation & purification ; Antibodies, Monoclonal/diagnostic use/isolation & purification ; Antigens, Helminth/*blood/*urine ; Diagnostic Tests, Routine/*methods ; Humans ; Immunoassay/methods ; Parasitology/*methods ; *Point-of-Care Systems ; Schistosoma mansoni/immunology/*isolation & purification ; Schistosomiasis mansoni/*diagnosis ; Sensitivity and Specificity

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 found 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 multicomplex protein originating from various organs of adult C. sinensis, and that it is composed of several 7 and 8 kDa proteins.
Animals ; Antigens, Helminth/immunology/*isolation & purification/metabolism ; Clonorchiasis/immunology ; Clonorchis sinensis/anatomy & histology/*immunology/metabolism ; Helminth Proteins/immunology/*isolation & purification/metabolism ; Human ; Mice ; Mice, Inbred BALB C ; Molecular Weight ; Support, Non-U.S. Gov't

Roundworms of Toxocara canis and Toxascaris leonina are common gastrointestinal helminths of canids over the world. Humans are infected with T. canis larvae through ingestion of infective eggs in contaminated environments or larvae by consumption of raw or uncooked meat or livers. Recently, patients of clinically diagnosed toxocariasis are increasing and require correct diagnosis in Korea. The present study investigated serological cross-reactivity between crude antigens of T. canis (TCLA) and T. leonina (TLLA) larvae. We collected serum specimens from 177 toxocariasis patients who were clinically suspected in the Seoul National University Hospital and 115 healthy controls. An ELISA method for toxocariasis was used to evaluate diagnostic efficacy of TLLA for serodiagnosis of human toxocariasis. The IgG ELISA using TLLA gave 14 (14.3%) positives of 98 TCLA positive specimens among 177 suspected toxocariasis patients. Most of them showed high absorbances with TCLA. In conclusion, there is a partial cross reaction between serum specimens of toxocariasis and TLLA.
Animals ; Antibodies, Helminth/blood ; Antigens, Helminth/*immunology ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G/blood ; Larva/immunology/metabolism ; Toxascaris/growth & development/*immunology/isolation & purification ; Toxocara canis/growth & development/*immunology/isolation & purification ; Toxocariasis/*diagnosis/parasitology

The author reported previously on separation of the outer tegument of the spargana (plerocercoids of Spirometra mansoni) using high concentration of urea solution. To determine which layer of the tegument is separated by this method, an electron microscopic analysis has been processed in this study. It was confirmed that the basement layer of the tegument is separated from the parenchyme of the sparganum. In addition, the antigenicity of the separated outer tegument against the human sparganosis patient sera was evaluated. Numerous antigenic proteins, including 16 and 55 kDa proteins, were noticed in the separated tegument; however, there were no diagnostic 31/36 kDa molecules in this tegument. The molecules reactive with the patient sera in the tegument are to be characterized in future studies.
Animal Structures/immunology/ultrastructure ; Animals ; Antigens, Helminth/chemistry/*immunology/isolation & purification ; Helminth Proteins/chemistry/*immunology/isolation & purification ; Humans ; Immunoblotting ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron ; Molecular Weight ; Sparganum/*immunology/*ultrastructure

OBJECTIVETo identify antigens which may help evaluate the therapeutic effect of angiostrongyliasis from adult worm antigen of Angiostrongylus cantonensis.

METHODSThe adult worm antigens of A. cantonensis were analyzed by Western blotting with the sera of rats infected with A. cantonensis before and after treatment. The sera of rats were tested by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe antigens with relative molecular mass between 38,000 and 78,000 reacted not only with the sera of rats before treatment, but also with that after treatment. The antigens with M(r) between 190,000 and 17,000 reacted with the sera of rats before treatment but not with that after treatment; those with M(r) between 32,000 and 24,000 antigens strongly reacted with the former, but the reaction became much weakened with the latter. The AC32-IgG antibody appeared earlier than the AC-IgG, and disappeared rapidly after treatment. Six of the 10 treated rats became negative for AC-IgG as found by ELISA.

CONCLUSIONThe antigens of adult worm antigen of A. cantonensis with M(r) of 190,000, 32,000, 24,000, 17,000 and 16,000 may serve as candidate antigens for therapeutic effect evaluation of angiostrongyliasis.

Angiostrongylus cantonensis ; immunology ; isolation & purification ; Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; blood ; immunology ; isolation & purification ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Rats ; Rats, Sprague-Dawley ; Strongylida Infections ; diagnosis ; immunology ; parasitology

The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.
Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/*diagnostic use ; Diagnostic Tests, Routine/*methods ; Egypt ; Fasciola/immunology/*isolation & purification ; Fascioliasis/*diagnosis/parasitology ; Humans ; Immunoblotting/*methods ; Parasitology/*methods ; Predictive Value of Tests ; Sensitivity and Specificity

Gnathostoma spinigerum can cause subarachnoid hemorrhage (SAH). The detection of specific antibodies in serum against G. spinigerum antigen is helpful for diagnosis of neurognathostomiasis. There is limited data on the frequency of G. spinigerum infection in non-traumatic SAH. A series of patients diagnosed as non-traumatic SAH at the Srinagarind Hospital, Khon Kaen University, Thailand between January 2011 and January 2013 were studied. CT or MR imaging of the brain was used for diagnosis of SAH. Patients were categorized as aneurysmal subarachnoid hemorrhage (A-SAH) or non-aneurysmal subarachnoid hemorrhage (NA-SAH) according to the results of cerebral angiograms. The presence of specific antibodies in serum against 21- or 24-kDa G. spinigerum antigen was determined using the immunoblot technique. The detection rate of antibodies was compared between the 2 groups. Of the 118 non-traumatic SAH patients for whom cerebral angiogram and immunoblot data were available, 80 (67.8%) patients had A-SAH, whereas 38 (32.2%) had NA-SAH. Overall, 23.7% were positive for specific antibodies against 21- and/or 24-kDa G. spinigerum antigen. No significant differences were found in the positive rate of specific antibodies against G. spinigerum in both groups (P-value=0.350).
Adult ; Aged ; Animals ; Antibodies, Helminth/*blood ; Antigens, Helminth/diagnostic use ; Brain/radiography ; Female ; Gnathostoma/immunology/*isolation & purification ; Gnathostomiasis/*diagnosis/*parasitology ; Humans ; Immunoblotting ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Serum/immunology ; Subarachnoid Hemorrhage/*diagnosis/*etiology ; Thailand ; Tomography, X-Ray Computed