Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini(Opisthorchidae,Trematoda)

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection.     

Ultrastructural localization of 28 kDa glutathione S-transferase in adult Clonorchis sinensis

Glutathione S-transferase (28GST) with molecular mass of 28 kDa is an antioxidant enzyme abundant in Clonorchis sinensis. In adult C. sinensis, 28GST was localized in tegumental syncytium, cytons, parenchyma, and sperm tails examined by immunoelectron microscopy. C. sinensis 28GST was earlier found to neutralize bioreactive compounds and to be rich in eggs. Accordingly, it is suggested that 28GST plays important roles in phase II defense system and physiological roles in worm fecundity of C. sinensis.     

Identification of Toxoplasma gondii proteins binding human lactoferrin: a new aspect of rhoptry proteins function

In this paper, we report on the isolation, purification and identification of two Toxoplasma gondii membrane proteins binding human lactoferrin. Parasite membrane proteins were isolated using the commercial Mem-PER Eukaryotic Membrane Protein Extraction System. After purification by lactoferrin affinity chromatography, three protein bands were detected with the molecular mass of 74, 63 and 58 kDa, two of which (63 and 58 kDa) specifically bound biotin labeled human lactoferrin as examined by competitive inhibition. Further identification of latter proteins by ESI/MS/MS amino acid sequencing technique revealed those proteins as Toxoplasma ROP4 (band 63 kDa) and ROP2 (band 58 kDa) antigens known to be involved in many mechanisms essential for the parasite pathogenicity, including host lactoferrin acquisition as determined in this study.     

Purification and biochemical characterization of two novel antigens from Leishmania major promastigotes

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.     

Clonorchis sinensis: immunolocalization of 26 kDa glutathione S-transferase in adult worms

A mu-class glutathione S-transferase (Cs26GST) of molecular mass 26 kDa was characterized from Clonorchis sinensis. In adult C. sinensis, the distribution of the Cs26GST was investigated by immuno-histochemistry and electron microscopy. Cs26GST was localized to the tegument and parenchyma. Immunogold labeling was strong in the tegumental cell bodies and moderate in the tegument and ova in the oviduct. It is suggested that Cs26GST plays a role in the metabolism and fecundity of C. sinensis.     

Antigenemia and Specific IgM and IgG Antibody Responses in Rabbits Infected with Toxoplasma gondii

In this experiment, the correlation between antigenemia and specific antibody responses in Toxoplasma gondii-infected rabbits was assessed. We injected 1,000 T. gondii tachyzoites (RH) subcutaneously into 5 rabbits. Parasitemia, circulating antigens, and IgM and IgG antibody titers in blood were tested by ELISA and immunoblot. For detection of parasitemia, mice were injected with blood from rabbits infected with T. gondii and mice died between days 2 and 10 post-infection (PI). Circulating antigens were detected early on day 2 PI, and the titers increased from day 4 PI and peaked on day 12 PI. Anti-Toxoplasma IgM antibody titers increased on day 6 PI and peaked on days 14-16 PI. IgG was detected from day 10 PI, and the titers increased continuously during the experiment. The antigenic protein patterns differed during the infection period, and the number of bands increased with ongoing infection by the immunoblot analysis. These result indicated that Toxoplasma circulating antigens during acute toxoplasmosis are closely related to the presence of parasites in blood. Also, the circulating antigen levels were closely correlated with IgM titers, but not with IgG titers. Therefore, co-detection of circulating antigens with IgM antibodies may improve the reliability of the diagnosis of acute toxoplasmosis.     

IgG antibody responses in early experimental sparganosis and IgG subclass responses in human sparganosis

Antigenic components in the crude extracts of Spirometra mansoni plerocercoid were analyzed in early experimental infections and in IgG subclass observed in clinical sparganosis. By IgG immunoblot, sera obtained serially from experimental mice, fed 5 spargana each, were reacted with the crude extracts. Protein bands at 36-26 kDa and 103 kDa showed positive reactions since two weeks after infection. In a differential immunoblot, in which a monospecific antibody against sparganum chymase at 36 kDa was pre-treated, the reactions at 36-26 kDa disappeared, indicating that the sparganum chymase and its degradation products invoked IgG antibody reactions. When 69 patients sera of human sparganosis were examined for their IgG subclass responses, IgG4 levels showed the highest reaction which was followed by IgG1. The IgG4 antibody also reacted mainly with 36-31 kDa protease. These results indicate that 36 kDa chymase of S. mansoni plerocercoid is the main antigenic component inducing IgG antibody response in early stage of experimental sparganosis and for specific IgG subclass reactions in human sparganosis.     

Identification of novel Leishmania major antigens that elicit IgG2a response in resistant and susceptible mice

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.     

Characterization of partially purified 8 kDa antigenic protein of Clonorchis sinensis

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.     

Partial characterization of a 17 kDa protein of Clonorchis sinensis

A 17 kDa protein from Clonorchis sinensis adults was purified by a procedure including Sephacryl S-200 HR gel filtration and Q-Sepharose anion exchange chromatography. The protein was proved to be a cysteine protease as it showed hydrolytic activity toward Cbz-Phe-Arg-AMC in the presence of dithiothreitol and was inhibited by specific inhibitors such as iodoacetic acid or trans epoxy-succinly-L-leucyl-amido(4-guanidino) butane. The polyclonal antibody raised against the protein reacted to 17 kDa proteins of trematodes such as Paragonimus westermani, Fasciola hepatica, Opisthorchis viverrini, Gymnophalloides seoi, and Metagonimus yokogawai. The antibody recognized the 17 kDa and 16 kDa cysteine proteases purified from C. sinensis, P. westermani, and G. seoi as well. These results suggest that the 17 kDa protein may be a cysteine protease commonly present in trematodes.     

Identification of cyst and trophozoite antigens from Colombian Giardia duodenalis isolates recognized by IgA

Little is known about the role of IgA in the immune response against Giardia duodenalis infection. The current study identified the antigens of Colombian G. duodenalis isolates which stimulate the production of IgA anti-G. dudoenalis. Cyst and trophozoite stage proteins were separated by SDS-PAGE and their antigenicity was determined by Western blot. Without 2-mercapto ethanol (2-ME), the protein profile of the cyst stage showed 24 proteins within a molecular weight range of 23-270 kDa; with 2-ME, 35 polypeptides ranging from 22 to 241 kDa were distinguished. The trophozoite stage protein profile without 2-ME was formed by 16 proteins within the range of 24-270 kDa; with 2-ME, 45 proteins were present between 18 and 241 kDa. The identification of 20 and 29 antigens from the cyst and trophozoite stage, respectively, suggested that G. duodenalis stimulates a specific humoral immune response in the human host. The antigens of 31, 57, 110, 133, and 170 kDa recognized by anti-G duodenalis IgA in both cysts and trophozoites corresponded with G. duodenalis isolates from other geographic regions, whereas those of 35, 38, 43, 45, 49, 52, 60, 62, 65, 72, 82, 99, 145, 155, and 185 kDa seemed specific to Colombian isolates. This indicated that antigens of 57, 65, 145, and 170 kDa, recognized by anti-G. duodenalis IgA antibodies in cysts (with frequencies between 82% and 96%) and trophozoites (with frequencies between 86% and 97%) can be considered identification markers for G. duodenalis infections.     

Plasmodium berghei XAT: protective 155/160 kDa antigens are located in parasitophorous vacuoles of schizont-stage parasite

Effective blood-stage malaria vaccine candidates have been mainly developed from the proteins in exposed locations on the parasite such as the surface of free merozoites or infected red blood cells. In the present study, we identified and localized novel protective antigens derived from the blood-stage of Plasmodium berghei XAT after establishment of hybridomas producing protective monoclonal antibodies (mAbs) against the parasites. The protective antigens were expressed in schizonts but not in trophozoites, and located in the parasitophorous vacuoles in the infected erythrocyte cytoplasm. The antigens, with molecular weight of 155/160 kDa, were not identical to any merozoite/schizont antigens that have been reported as target molecules recognized by mAbs developed to rodent malaria parasites. The characterization of new malarial antigenic targets of potentially protective antibody responses following infection would give us new insights for the selection of candidate antigens for malaria vaccine.     

Analysis of antigenic specificities of Paragonimus westermani developmental stages using immunoblot technique

Serodiagnosis of parasitic infections is widely used, since parasites or their eggs are not always detected by ordinary methods. The sensitive tests such as ELISA are highly dependent on the purity of antigens used. To solve this problem, many workers have tried to find species-specific components of antigens. The present study was performed to determine the antigenic profile of crude saline extracts of 3, 5, 8 and 12-week old P. westermani worms, which were collected from experimentally infected cats, based on SDS-PAGE and immunoblot technique. The results were as follows: 1. The SDS-PAGE showed at least 30 protein bands ranging from 229 kDa to 10 kDa molecular weight. The protein components of P. westermani changed chronologically during its developmental period. The 229 kDa band was recognized only in 12-week old worms (SEP12). 2. Analysis by ELISA showed a significant increase in antibody levels at 3 weeks in infected cats using crude saline extract antigens (SEP3, SEP5, SEP8, SEP12). 3. By EITB using SEP3 and SEP5, infected cats recognized major protein bands with molecular weight of 60, 35, 28, 25 or 21 kDa at 3-12 weeks of infection, and 3 additional antigens, 19, 13 and 10 kDa, were detected at 8-12 weeks of infections. 4. Using SEP8, 5 antigens, 91, 85, 31, 25 and 21 kDa, were consistently detected by all infected sera tested. In addition, 3 antigens of 19, 13 and 10 kDa were detected at 8-12 weeks of infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules

Neutrophils contain several distinct classes of secretory granules that may sequentially fuse with the phagosome after the ingestion of particulates, or that may be differentially exocytosed after cellular activation with soluble stimuli. The exocytosis of neutrophil secretory granules has been shown to be GTP-dependent at a step distal to activation of the transductional G proteins. Inasmuch as ras-related low molecular mass GTP-binding proteins have been shown to play regulatory roles in vesicle sorting in the secretory pathway in yeast, the differential mobilization of neutrophil granules might be regulated by distinct GTP-binding proteins. We therefore explored the distribution and identity of low molecular mass GTP-binding proteins in neutrophil secretory granules and other subcellular fractions. After lysis by nitrogen cavitation, four highly resolved fractions were harvested from discontinuous Percoll gradients: a microsomal fraction enriched for plasma membranes, specific granules, primary granules, and cytosol. At least seven bands of distinct Mr were detected by probing protein blots with [32P]GTP. Microsomes contained a prominent GTP-binding band at 26 kDa and weaker ones at 24 and 22.5 kDa; specific granules contained bands at 26, 24, 22, and 20 kDa; primary granules showed bands at 24 and 23 kDa; cytosol showed strong bands at 23.5 and 19 kDa and a weak band at 26 kDa. Antiserum against ADP-ribosylation factor reacted strongly with the 19-kDa band in cytosol but with none of the membrane fractions. None of these proteins was recognized by antibodies against ras or against Sec4p. Botulinum exoenzyme C3 labeled bands of molecular mass 20 and 21 kDa in cytosol and microsomes that have distinct mobilities from all the blotted [32P]GTP-binding proteins. The highly compartmentalized subcellular distribution of the blotted [32P]GTP-binding proteins in neutrophils is consistent with a regulatory role in the differential mobilization of granule compartments during cellular activation.     

Clonorchis sinensis: glutathione S-transferase as a serodiagnostic antigen for detecting IgG and IgE antibodies

Human Clonorchis sinensis infection is endemic in East Asian countries. Glutathione S-transferases (GSTs) are anti-oxidant enzymes found in all living creatures as well as in trematodes. In this study, we examined the recombinant 26kDa GST protein of C. sinensis (Cs26GST) for its serodiagnostic antigenicity toward IgG and IgE antibodies by ELISA and immuno-enhanced chemiluminescence, respectively. In IgG ELISA, recombinant Cs26GST showed 33.3% sensitivity and 100% specificity for trematode-infected human sera. In the case of the IgE antibody, recombinant Cs26GST showed 50.0% sensitivity and 93.2% specificity for clonorchiasis infection. We propose that the recombinant Cs26GST is a potent serodiagnostic antigen for detecting C. sinensis-specific IgG and IgE antibodies, and that it be best used as an antigenic cocktail in combination with other antigens.     

Usefulness of 8 kDa protein of Fasciola hepatica in diagnosis of fascioliasis

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.     

Biochemical characterisation of the 56 and 82 kDa immunodominant gametocyte antigens from Eimeria maxima

Two immunodominant gametocyte antigens from Eimeria maxima with M(r) 56 kDa and M(r) 82 kDa have been identified previously as potential candidates for inclusion in a recombinant subunit vaccine against coccidiosis in poultry. Here, these proteins have been biochemically characterised, immunolocalised within the parasite, and sequences for their amino termini determined. These antigens co-purify by affinity chromatography suggesting an interaction with each other. However, separation of the proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of beta-mercaptoethanol did not reveal the presence of inter-chain disulphide bonds. The true masses of the 56 and 82 kDa antigens are 52450 and 62450 Da, respectively, as determined by mass spectrometry. TX-114 separations suggested that they exist, in part, as soluble proteins within the parasite, and immunolocalisation studies indicated that they were found in the wall forming bodies of macrogametocytes. Separation of the proteins by 2D SDS-PAGE revealed that they are acidic in nature and heterogeneous in charge. Cleavage by neuraminidase and O-glycosidase indicated that the presence of O-linked glycans contributed to some of the charge microheterogeneity of both proteins. The absence of these O-glycans however, did not abolish antibody recognition, suggesting that the development of a recombinant subunit vaccine is possible. A more extensive investigation of the carbohydrate moieties of these proteins revealed that they also possess glucose, fucose, mannose and galactose. There was no evidence for the presence of N-linked glycans. The 56 and 82 kDa antigens were separated from a mixture of proteins in a crude gametocyte lysate by 2D SDS-PAGE, the proteins isolated, and the N-terminus amino acid sequence determined. They showed no homology to each other at the N-terminus, or to any other previously characterised protein. Characterisation of these novel proteins has provided further insights into the molecular mechanisms of gametocyte differentiation in E. maxima.     

Organ-specific antigens of Clonorchis sinensis

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 organspecific 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.