Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were... more
Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties.
The aim of this study was to analyze the presence of 62 kDa proteinase and anti-62 kDa proteinase antibody in clinical samples of symptomatic and asymptomatic infected women. Proteinase was detected in all the swabs vaginal of infected... more
The aim of this study was to analyze the presence of 62 kDa proteinase and anti-62 kDa proteinase antibody in clinical samples of symptomatic and asymptomatic infected women. Proteinase was detected in all the swabs vaginal of infected women. Significantly, amounts of antigen (mean optical density (OD) values) were detected in swabs vaginal of symptomatic as compared to asymptomatic women. This protein was not detected in the group of patients with Trichomonas vaginalis-culture-negative results and in the groups of samples infected with other agents. Antibody to 62 kDa was detected in the swabs vaginal the only 66.6% of the symptomatic and 55.5% of the asymptomatic infected women. Antibody to 62 kDa was also detected in 7/30 of the swabs vaginal from uninfected women. No significant difference was observed in mean OD values of vaginal swabs of T. vaginalis-infected symptomatic as compared to asymptomatic women. The presence of proteinase in 100% of T. vaginalis-infected women suggested that 62 kDa proteinase could be a virulence factor.
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Fasciolosis is one of the food-borne neglected trematodioses that has reemerged as a human disease while its effects on domestic animal health remains of significant economic consideration. Being snail-borne disease, the accurate and... more
Fasciolosis is one of the food-borne neglected trematodioses that has reemerged as a human disease while its effects on domestic animal health remains of significant economic consideration. Being snail-borne disease, the accurate and time-saving epidemiological surveillance of the transmission foci where infected lymnaeid snails occur could be essential to effectively focus or redirect control strategies. For this purpose, the first monoclonal antibody-based immunoenzymatic assay to detect Fasciola hepatica-infected snails (FasciMol-ELISA) was recently developed and showed a high sensitivity and specificity when tested in an experimental F. hepatica - Galba cubensis system. Here, we surveyed populations of G. cubensis occurring in western Cuba for the assessment of the FasciMol-ELISA in determining natural F. hepatica infection in this intermediate host. A multiplex PCR, previously developed to detect F. hepatica in G. cubensis, was used for sample classification. Snail dissection m...
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In former papers we have provided evidence on the protective effect of disodium cromoglycate (DSCG) and ketotifen (Ke) against the contractions induced by various agonists (histamine, serotonin, acetylcholine, prostaglandins) in ileal and... more
In former papers we have provided evidence on the protective effect of disodium cromoglycate (DSCG) and ketotifen (Ke) against the contractions induced by various agonists (histamine, serotonin, acetylcholine, prostaglandins) in ileal and tracheal smooth muscle of guinea pigs. To offer an insight into the mode of action of these antiallergic drugs we quantified concentrations of cyclic AMP and GMP in these tissues. We observed that DSCG (10(-3)-10(-2) M) significantly diminished the concentration of cyclic AMP in ileum and trachea of guinea pigs, but it increased the level of cyclic GMP in ileum. Ketotifen (10(-6)-10(-4) M) does not modify cAMP and cGMP in ileum, however, in trachea it significantly decreases the concentrations of cGMP. We also studied the effects of oxatomide, ICI 74.917 and BRL 10.833 on cyclic nucleotides concentrations in guinea pig ileum BRL 10.833 increases cAMP at a relatively high dose (10(-4) M). ICI 74.917 at the same dose decreases cGMP. Oxatomide (10(-6)...
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The mechanism of inhibitory action of disodium cromoglycate (DSCG) and Ketotifen (Ke) on smooth muscle against various spasmogens remains uncertain. In an attempt to elucidate it, we have studied the effects of both drugs on cumulative... more
The mechanism of inhibitory action of disodium cromoglycate (DSCG) and Ketotifen (Ke) on smooth muscle against various spasmogens remains uncertain. In an attempt to elucidate it, we have studied the effects of both drugs on cumulative dose response curves (CDRC) to CaCl2 in guinea pig ileum. We found that they induce a shift to the right and a non surmountable depression of the maximal response of these CDRC which suggests non competitive antagonism. Furthermore, DSCG inhibits 45Ca++ uptake by guinea pig ileum after acetylcholine stimulation which constitutes strong evidence that inhibition of calcium ion utilization by smooth muscle cells plays a major role in the mode of action of DSCG.
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The role of lysosomal enzyme released by macrophages was examined in relation to the toxic effect caused by food yeast. Mouse peritoneal macrophages exposed to yeast in culture showed marked release of N-acetyl glucosaminidase,... more
The role of lysosomal enzyme released by macrophages was examined in relation to the toxic effect caused by food yeast. Mouse peritoneal macrophages exposed to yeast in culture showed marked release of N-acetyl glucosaminidase, beta-galactosaminidase and beta-glucuronidase below the median lethal dose (LD50). LD50 was measured from the dose response curves of the cytoplasmic lactate dehydrogenase enzyme. Saccharomyces cerevisiae showed the highest LD50 followed by Kluyveromyces fragilis and Candida utilis yeast. LD50 values obtained as well as the in vitro lysosomal release by mouse peritoneal macrophages may be relevant to assess the toxic capacity of food yeast intended for human consumption.
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... Annu Rev Physiol 2002;64:47-67. Pubmed 3. Wu H, Devi R, Malarkey WB. ... Ann NY Acad Sci 1998;839:239-43. Pubmed 9. Freeman ME, Kanycska B, Lerant B, Nagy G. Prolactin: structure, function and regulation of secretion. ... CABRERA... more
... Annu Rev Physiol 2002;64:47-67. Pubmed 3. Wu H, Devi R, Malarkey WB. ... Ann NY Acad Sci 1998;839:239-43. Pubmed 9. Freeman ME, Kanycska B, Lerant B, Nagy G. Prolactin: structure, function and regulation of secretion. ... CABRERA OLIVA, VM a ; SARRACENT PÉREZ, J b. ...
An ultramicro ELISA, which uses 10 microliter of reaction volume developed for quantitation of human alphafetoprotein (AFP), has been adapted to use in screening hybridoma products in order to take advantage of the efficiency and... more
An ultramicro ELISA, which uses 10 microliter of reaction volume developed for quantitation of human alphafetoprotein (AFP), has been adapted to use in screening hybridoma products in order to take advantage of the efficiency and sensitivity of this system. Positive samples were detected with a low background level.
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The control of fasciolosis, as that of other vector-borne diseases, must be related to the control of the lymnaeid snails, the intermediate hosts of the parasite. Thus, an accurate epidemiological surveillance of the transmission foci... more
The control of fasciolosis, as that of other vector-borne diseases, must be related to the control of the lymnaeid snails, the intermediate hosts of the parasite. Thus, an accurate epidemiological surveillance of the transmission foci where the infected mollusks occur is essential. For this purpose, immunoassays could be a useful tool. However, information regarding specific proteins of intramolluscan larvae and previous studies concerning monoclonal antibody generation against asexual stages of trematodes are scarce. Therefore, we explored the antigenic features of intramolluscan rediae of Fasciola hepatica to evaluate three antigenic preparations in order to use the most promising one for developing specific monoclonal antibodies. Mouse antiserum was generated against each antigen for assessing the polyclonal antibody response against the crude extract of rediae and the cross-reactivity against lymnaeids. The specific C-terminal of F. hepatica cytochrome c oxidase subunit I (first...
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The aim of this study was to analyze the presence of 62 kDa proteinase and anti-62 kDa proteinase antibody in clinical samples of symptomatic and asymptomatic infected women. Proteinase was detected in all the swabs vaginal of infected... more
The aim of this study was to analyze the presence of 62 kDa proteinase and anti-62 kDa proteinase antibody in clinical samples of symptomatic and asymptomatic infected women. Proteinase was detected in all the swabs vaginal of infected women. Significantly, amounts of antigen (mean optical density (OD) values) were detected in swabs vaginal of symptomatic as compared to asymptomatic women. This protein was not detected in the group of patients with Trichomonas vaginalis-culture-negative results and in the groups of samples infected with other agents. Antibody to 62 kDa was detected in the swabs vaginal the only 66.6% of the symptomatic and 55.5% of the asymptomatic infected women. Antibody to 62 kDa was also detected in 7/30 of the swabs vaginal from uninfected women. No significant difference was observed in mean OD values of vaginal swabs of T. vaginalis-infected symptomatic as compared to asymptomatic women. The presence of proteinase in 100% of T. vaginalis-infected women suggested that 62 kDa proteinase could be a virulence factor.
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This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens... more
This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens in serum and stool samples of cattle and humans with fasciolosis, using a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA). The epitope was characterized by periodate oxidation, alkaline borohydride reduction, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, and enzymatic proteolysis. These results, together with those of the 2-site ELISA, lectin immunoassays, and beta-galactosidase digestion, showed that MAb ES78 reacts with a partly protein/partly carbohydrate antigenic determinant that is found on several ES molecules of adult specimens of F. hepatica and contains at least 1 disulfide bond and beta-galactose probably as galactose-beta(1-3)-N-acetylgalactosamine disaccharide.
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... la malaria Lázara Rojas Rivero,1 Jorge Sarracent Pérez1 y Luis Fonte Galindo1 1 Labortaorio de Parasitología, Instituto de Medicina Tropical Pedro Kourí, Ciudad de La Habana, Cuba. ... 1999;77:62440. 2. Alonso PL, Sacarlal J, Aponte... more
... la malaria Lázara Rojas Rivero,1 Jorge Sarracent Pérez1 y Luis Fonte Galindo1 1 Labortaorio de Parasitología, Instituto de Medicina Tropical Pedro Kourí, Ciudad de La Habana, Cuba. ... 1999;77:62440. 2. Alonso PL, Sacarlal J, Aponte JJ, Leach A, Macete E, Milman J, et al. ...
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The effects of clofazimine on macrophages obtained from mice fed by gavage with various drug concentrations were studied. The results obtained demonstrated an increase in the activity of various lysosomal enzymes and in the amount of... more
The effects of clofazimine on macrophages obtained from mice fed by gavage with various drug concentrations were studied. The results obtained demonstrated an increase in the activity of various lysosomal enzymes and in the amount of labeled immune complexes phagocytosed at drug concentrations of 1 mg/kg and 10 mg/kg body weight. This confirms and extends the effects reported by us of clofazimine's action on the lysosomal apparatus.
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RESUMEN Introducción: la fasciolosis en Cuba es una enfermedad enzoótica en el ganado y en los últimos años existe un incremento en el número de casos en humanos. El diagnóstico coproparasitológico de la fasciolosis es poco sensible y... more
RESUMEN Introducción: la fasciolosis en Cuba es una enfermedad enzoótica en el ganado y en los últimos años existe un incremento en el número de casos en humanos. El diagnóstico coproparasitológico de la fasciolosis es poco sensible y laborioso, por lo que es importante el uso de los métodos inmuno-enzimáticos sobre todo aquellos que son capaces de detectar antígenos de este parásito en las heces. En el Instituto de Medicina Tropical "Pedro Kourí" se cuenta con un sistema de detección de antígenos denominado FasciDIG® con una sensibilidad de 10 ng/mL. Objetivo: aumentar la sensibilidad del FasciDIG® realizando modificaciones a este método diagnóstico. Métodos: en un sistema simulado se evaluaron FasciDIG® y FasciDIG modificado, utilizando diluciones seriadas dobles de antígenos a concentraciones desde 1 000 ng/mL hasta 1,95 ng/mL. El FasciDIG se modificó utilizando como segundo anticuerpo el obtenido en conejos contra antígenos de excreción-secreción de Fasciola hepatica c...
Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the... more
Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451bp), as an internal control of the reaction. The assay was able to detect up to 100pg of the parasite even at high concentr...
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frequent relapses and high lethality of criptococcosis has encouraged the search for new therapeutic strategies. to evaluate the effect of the monoclonal antibody 4B3 on the cryptococcal infection in Balb/c mice. the kinetics in serum... more
frequent relapses and high lethality of criptococcosis has encouraged the search for new therapeutic strategies. to evaluate the effect of the monoclonal antibody 4B3 on the cryptococcal infection in Balb/c mice. the kinetics in serum concentration of the studied monoclonal antibody was determined for intraperitoneal administration (500 microg) by quantitative sandwich ELISA. In order to assess its protective capability, were administered 500 microg of 4B3 and innoculated 2 x 10(22) cells/mL of Cryptococcus neoformans. The survival of mice was recorded and the yeast dissemination to the main target organs was evaluated. Macrophages P338.D1 cell lines were used to measure the effect of the monoclonal antibody 4B3 on the phagocytosis and lysis of the microorganism. the used dose helped to keep high values (38 microg/m) of 4B3 in serum for at least 46 days. It was found that the monoclonal antibody does not give protection, which makes the microorganism dissemination possible, along wi...
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The capacity of the 4B3 monoclonal antibody, previously obtained by immunization of BALB/c mice with the capsular polysaccharide of Cryptococcus neoformans was studied to recognize this structure as part of the intact cell of this yeast.... more
The capacity of the 4B3 monoclonal antibody, previously obtained by immunization of BALB/c mice with the capsular polysaccharide of Cryptococcus neoformans was studied to recognize this structure as part of the intact cell of this yeast. With this aim, 4B3 was evaluated by cellular ELISA and indirect immunofluorescence, using as an antigen a cell suspension of the 028 LMIPK C. neoformans strain. Serum from the mouse used in the fusion for obtaining 4B3 monoclonal antibody was used as a positive control, whereas an anti-dengue monoclonal antibody was used as a negative control. Both methods demonstrated that the above mentioned monoclonal antibody is capable of recognizing the native antigen, which will allow its future evaluation for diagnostic and therapeutic purposes.
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The ES-78 monoclonal antibodiy recognizes excretory-secretory antigens of Fasciola hepatica. This monoclonal antibody is used for diagnosis of humans and cattle fasciolosis. The production of 4 anti-idiotype monoclonal antibodies (3G5,... more
The ES-78 monoclonal antibodiy recognizes excretory-secretory antigens of Fasciola hepatica. This monoclonal antibody is used for diagnosis of humans and cattle fasciolosis. The production of 4 anti-idiotype monoclonal antibodies (3G5, 5G1, 3H4 and 1H6) inhibiting the antigen-monoclonal antibody ES 78 reaction was reported. This antibody recognizes excretion-secretion antigens of Fasciola hepatica and it is used for the diagnosis in faeces of fascioliasis in the bovine cattle and in humans. When the 3G5 was used in rabbits as an immunogen there was a response of antibodies against the excretion-secretion antigen of Fasciola hepatica without a previous contact wih it. The utilization of this anti-idiotype monoclonal antibody as an immunogen mimmicking a protective epitope against fascioliasis together with appropriate adjuvants may be a new way of reducing the parasitic burden in experimental models of fascioliasis.
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fasciolosis is an endemic disease in cattle in Cuba and there is an increase in the number of reported human cases in recent years. The coproparasitological diagnosis of fasciolosis has low sensitivity and is hard-working; for that... more
fasciolosis is an endemic disease in cattle in Cuba and there is an increase in the number of reported human cases in recent years. The coproparasitological diagnosis of fasciolosis has low sensitivity and is hard-working; for that reason, it is important to use immunoenzymatic methods mainly those that can detect this parasite antigens in the feces. A system for antigen detection called FasciDIG, with a reported sensitivity of 10 ng/mL has been developed in "Pedro Kouri" Institute of Tropical Medicine. to increase the sensitivity of FasciDIG through some modifications to this diagnostic method. two foul dilutions (concentrations of antigen 1 000 ng/mL- 1.95ng/mL in H20 Tween-20) were evaluated in a simulated system using FasciDIG and modified FasciDIG. The FasciDiG was modified using the secondary antibody obtained from rabbit against excretory-secretory antigens of Fasciola hepatica combined with biotin and then adding commercial conjugated extravidine peroxidase. Feces ...
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In order to determine the IgA levels in secretions, the authors engaged themselves to obtain monoclonal antibodies against this immunoglobulin and to show their possible utilization in the development of quantitation methods. 6 monoclonal... more
In order to determine the IgA levels in secretions, the authors engaged themselves to obtain monoclonal antibodies against this immunoglobulin and to show their possible utilization in the development of quantitation methods. 6 monoclonal antibodies were obtained: one against the secretory component (SC) and five against the heavy-chain (HC) of IgA. IgA purified from human colostrum was used as immunogen (HC). The immunoglobulin isotypes (IgG2a and IgG1) were determined in 2 monoclonal antibodies, one against SC and the other against HC. Polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sulphate and b-mercapto-ethanol. Immunotransference was also carried out to determine their specificity. Secretory IgA was detected in saliva specimens. The anti-SC monoclonal antibody obtained in-the laboratory of the "Pedro Kourí" Institute of Tropical Medicine (IPK) was compared with a commercial one (Sigma Chemical Co.), and a similar behaviour was obser...
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5-Azido-3-oxa-pentyl beta-D-galactopyranoside was prepared from diethylene glycol monochlorohydrin and used as a model of oligosaccharide hapten. After deprotection, a series of amides bearing thiophilic groups had been obtained through... more
5-Azido-3-oxa-pentyl beta-D-galactopyranoside was prepared from diethylene glycol monochlorohydrin and used as a model of oligosaccharide hapten. After deprotection, a series of amides bearing thiophilic groups had been obtained through the terminal amino function and essayed in coupling reactions with thiolated BSA. Also several Lewis human blood group oligosaccharides had been conjugated with thiolated BSA demonstrating the usefulness of the methodology.
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Mouse peritoneal and calf alveolar macrophage cultures were exposed to various concentrations of Clofazimine, 3 (p-chloroanilino)-10-p-Chlorophenyl 2, 10-dihydro-2-isopropylimino, for 120 hr and an increase of four lysosomal enzymes were... more
Mouse peritoneal and calf alveolar macrophage cultures were exposed to various concentrations of Clofazimine, 3 (p-chloroanilino)-10-p-Chlorophenyl 2, 10-dihydro-2-isopropylimino, for 120 hr and an increase of four lysosomal enzymes were found with 0 . 3 micrograms/ml of the drug. In mouse peritoneal macrophage cultures, higher concentrations were toxic. Cycloheximide inhibited the lysosomal enzyme activity increase found. No change in enzymatic activity was observed when a lysosomal enriched granular fraction was incubated with various drug concentrations. Our results strongly suggest that Clofazimine at concentrations close to therapeutic serum levels induces de novo synthesis of lysosomal enzymes in macrophage cultures.
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Research Interests: Macrophages, Mice, Female, Animals, Male, and 3 moreAnti-inflammatory agents, Exocytosis, and Lysosomes
Trichomonas vaginalis is a pathogen that infects the squamous epithelium of the human urogenital tract. Although the details of the process through which this microorganism parasites host tissues and ultimately causes disease are largely... more
Trichomonas vaginalis is a pathogen that infects the squamous epithelium of the human urogenital tract. Although the details of the process through which this microorganism parasites host tissues and ultimately causes disease are largely unknown, most authors agree that a necessary first step for establishing infection is the adhesion of the parasite to vaginal epithelial cells. High levels of proteolytic
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We report the generation of murine triomas by fusing splenocytes from mice previously immunized with HBsAg ay-subtype and a hybridoma, secreting anti-HBsAg ad-subtype monoclonal antibody, which was rendered HGPRT- by induced mutagenesis... more
We report the generation of murine triomas by fusing splenocytes from mice previously immunized with HBsAg ay-subtype and a hybridoma, secreting anti-HBsAg ad-subtype monoclonal antibody, which was rendered HGPRT- by induced mutagenesis with N-methyl-N'nitro-N-nitrosoguanidine. The fusion yielded a 83.8% of hybrids showing the antigen specificity of the parental hybridoma and a 16.1% of bi-specific monoclonal antibodies. One of them, coded as 1C8A5, showing a heavy chain isotype (IgG1/IgG2b) was used as capture reagent in an ultramicro-ELISA. As little as 0.78 I.U. of both HBsAg ad- and ay-subtypes could be realiably detected.
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This study describes the use of bromo-deoxiuridine, a non-radioactive analogue of thymidine, to determine the adhesion of 40 T. vaginalis isolates, as compared with the clinical manifestations found in patients. Parasite-labeling was... more
This study describes the use of bromo-deoxiuridine, a non-radioactive analogue of thymidine, to determine the adhesion of 40 T. vaginalis isolates, as compared with the clinical manifestations found in patients. Parasite-labeling was optimized and label detection by immunoassay was carried out. Bromo-deoxiuridine at 10 microM for 16 h produced the highest sensitivity. Once optimized, the assay was able to detect between 3.12 x 10(3) and 4 x 10(5) parasites, with a detection limit of 9.14 x 10(2), which is lower than with the use of [3H]-thymidine. The variation coefficients were 2.65% for repeatability and 3.8% for reproducibility. A correlation coefficient of 0.97 was found between the pattern curves obtained by both labeling procedures. The level of adhesion to HeLa cells was directly proportional to the severity of the clinical manifestations (P < 0.05).
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In this work we search for antigens of Trichinella spiralis in sera and stool of rats experimentally infected. The kinetic of antibodies to excretory and secretory (ES) antigens of muscle larvae (ML) was also determined. Wistar rats were... more
In this work we search for antigens of Trichinella spiralis in sera and stool of rats experimentally infected. The kinetic of antibodies to excretory and secretory (ES) antigens of muscle larvae (ML) was also determined. Wistar rats were infected with 15 ML per gram of body weight and blood samples were collected weekly for 10 weeks. Antibodies were studied using an indirect ELISA. For detection of circulating antigens and coproantigens, a sandwich ELISA was developed with the use of polyclonal rabbit antibodies obtained against the total extract of ML and an IgM monoclonal antibody (Mab) against ES antigens of ML. No reactivity was observed between Mab and the total worm antigens of Angiostrongylus cantonensis, Ascaris suum, Echinococcus granulosus, Fasciola hepatica, Strongyloides stercoralis, Taenia solium, Toxocara canis and Trichuris trichiura. The IgM Mab recognized antigens of 45, 49, and 55 kDa in ES antigens and was unable to bind ES antigens deglycosylated with trifluoromethanesulphonic acid (TFMS) indicating that a glycan structure is present in the epitope recognized by this Mab. The sensitivity of sandwich ELISA was 1 ng/mL. Circulating antigens were detected in all infected rats between 3 and 8 weeks post infection and coproantigens were found during the first two days post infection. Antibodies were detected since the third week post infection through the end of experiment. These results suggested that antigen detection by our sandwich ELISA could be a useful complementary laboratory test for antibody detection.