A western blot characterization of Mycobacterium bovis antigens recognized by cattle sera

The immune response to Mycobacterium bovis in cattle was assessed by Western blot. The antibody recognition pattern to M. bovis whole cell extracts and culture supernatant antigens was studied by using sera from M. bovis-infected (n=62) and healthy (n=38) cattle. Although the recognition patterns were highly variable, some proteins were regularly detected, mainly those with molecular masses of 17, 23, 28, 42, 66, 71 and 80 kDa in cellular extracts, and with molecular masses of 23 and 33 kDa in supernatants. Whole cell extract antigens were more frequently recognized than culture supernatant antigens. Healthy controls produced only a week antibody response.The antibody response was variable, depending on tuberculosis stage. In early stages very few antibodies were detected. A response against the 66-kDa stress protein was mounted in intermediate tuberculosis and remained stable in more advanced disease. In late diseases, the preferentially recognized antigens were a 28-kDa cellular protein and supernatant antigens.The 28-kDa protein was studied in some detail. As determined by using monoclonal antibodies, the 28-kDa protein is different from superoxide dismutase. This protein aggregated in stored cell extracts and was not totally transferred to nitrocellulose.The principal conclusions of this work are: (i) whole cell extract proteins are more frequently recognized than the secreted proteins and (ii) a 28-kDa protein is a major antigen in late disease.     

Monoclonal antibodies against rat glutathione S-transferase isoenzymes 2-2 and 3-3

Monoclonal antibodies were prepared against rat glutathione S-transferase isoenzymes 2-2 and 3-3. Three clones were capable of specifically differentiating their respective antigens from other rat isoenzymes as well as human isoenzymes, in ELISA and on Western blot. One clone produced antibodies specific for isoenzyme 2-2, and 2 hybridomas were specific for isoenzyme 3-3. Balb/c mice did not respond to immunization with glutathione S-transferase isoenzymes 1-1 and 4-4. However, an immune response was obtained in some other strains of mice, with differential H-2 haplotypes, notably CBA/BrARij mice and CBA/CaHRij-T6 mice for isoenzyme 1-1 and CBA/BrARij mice for isoenzyme 4-4, which offers perspectives for obtaining additional specific monoclonal antibodies against these glutathione S-transferases.     

Role of host antibody in the chemotherapeutic action of praziquantel against Schistosoma mansoni: identification of target antigens

We have demonstrated previously in a mouse model that effective chemotherapy against Schistosoma mansoni with praziquantel (PZQ) is dependent upon an intact host antibody response. In the same study, it was found that worms recovered from PZQ-treated animals display surface-bound antibodies. In order to identify the target antigens of the antibodies involved in the synergy between PZQ and the immune response, monoclonal antibodies (mAbs) and polyclonal antisera recognizing different tegumental components were tested by indirect immunofluorescence (IF) assay for their ability to bind in vitro to the surface of 6-week-old schistosomes perfused from nude (athymic) mice 1 h after PZQ treatment. Nude mice were used as hosts because worms from these animals were found to lack bound anti-schistosome antibodies. Only 5 of the 21 antibodies tested reacted with drug-treated worms. This indicated that the damage caused by PZQ to the schistosome tegument is restricted to specific tegumental components. Of the positive reactions, one group of antibodies gave IF patterns different from, whereas the other group gave IF reactions similar to those seen with worms perfused from immunologically intact mice. Antibodies against a schistosome esterase and alkaline phosphatase produced reaction patterns in the former category. In contrast, two out of three monoclonal antibodies recognizing different epitopes on a 200-kDa glycoprotein abundant in worm tubercles gave IF patterns very similar to those observed on schistosomes from drug-treated, intact mice. The biological significance of these reactions was confirmed by demonstrating that transfer of one of the positive monoclonal antibodies to 6-week-infected, B cell-depleted (mu-suppressed) mice reconstitutes the efficacy of PZQ treatment to normal levels. The above results suggest that the antibodies involved in the mechanism of action of PZQ react with a limited set of antigens. Furthermore, they implicate the 200-kDa tubercle protein as a major target of this response in naturally infected hosts.     

A monoclonal antibody blocking the Schistosoma mansoni 28-kDa glutathione S-transferase activity reduces female worm fecundity and egg viability

The protective effects of two different monoclonal antibodies (mAb) raised against the Schistosoma mansoni 28-kDa glutathione S-transferase (Sm 28 GST) were investigated. Two mAb of the same isotype (IgM) have been selected according to the blocking effect on Sm 28 GST enzymatic activity (S13) or the lack of blockade (H12). When passively transferred into Fischer rats, both S13 and H12 significantly reduced the worm burden. In BALB/c mice clear effects on female worm fecundity and egg viability were observed when the S13 mAb was transferred; these effects included significantly reduced loads of intestinal eggs, reduced egg hatching rates and an increased proportion of non-living eggs. No effect on egg production and egg hatching was observed in H12-treated mice. In addition, worm pairs recovered from S13-but not H12-treated mice laid significantly fewer eggs in vitro, and normal worm pairs incubated in vitro with the S13 mAb produced significantly fewer eggs than those incubated with H12 mAb. The impairment of egg hatching ability was also reproduced in vitro by the S13 mAb. These data suggest the existence of two different effector mechanisms induced by immunization with Sm 28 GST. The effect on the schistosome worm burden appears to be independent of GST activity whereas the effect on S. mansoni female fecundity and egg viability seems to be significantly linked to the inactivation of the enzymatic site.     

Inter-species variation of schistosome 28-kDa glutathione S-transferases.

The 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) is a candidate vaccine antigen. To evaluate the antigenic and phylogenetic variations between the 28-kDa GSTs from 4 species of schistosome, we have cloned and sequenced the 28-kDa GSTs from Schistosoma haematobium (Sh28GST) and Schistosoma bovis (Sb28GST). Sb28GST and Sh28GST are more similar to each other (97%) than to Sm28GST (90%) and particularly to the 28-kDa GST from Schistosoma japonicum (Sj28GST, 77%). Antisera directed against the major Sm28GST epitopes revealed differences in the recognition of the 28-kDa GSTs from the other schistosome species suggesting that these regions have been subjected to evolutionary pressure. The consequences of such species-specific epitopes on the development of a multi-species anti-schistosome vaccine are discussed.     

Allergen profiles of dog hair and dander, body fluids and tissues as defined by immunoblotting

The sera from 25 patients with clinical type I allergy against dogs were investigated by means of immunoblotting, using extracts of dog hair/dander, skin, hair, saliva, salivary gland, serum and liver. 96% of the patients' sera showed IgE antibodies reactive with 19- and 23-kilodalton (kDa) proteins in the hair/dander extract. The 23-kDa IgE-binding protein was preferentially detected in the hair extract and saliva but not in skin, salivary gland, serum and liver extracts. The 19-kDa band was strongly expressed in skin, but not in hair, serum and liver. Inhibition experiments using the 23-kDa containing extract prepared from hair and the 19-kDa containing extract prepared from skin revealed that these two proteins are likely to be immunologically independent allergens.     

An Eikenella corrodens toxin detected by plaque toxin-neutralizing monoclonal antibodies.

Bacterial plaque from the gingival region of teeth contains cytotoxic agents which lyse undifferentiated human HL60 cells. A small panel of monoclonal antibodies (MAbs) was found to abrogate much of this activity and to detect antigens in certain strains of Streptococcus mitis and Eikenella corrodens. The aim of this study was to determine whether these bacterial antigens might be involved in HL60 cells cytolysis. Saline extracts were obtained by homogenizing washed, stationary-phase cells in 65 mM NaCl with a tight-fitting Potter-Elvehjem homogenizer. The extracts of E. corrodens were toxic to HL60 cells, whereas similar extracts of S. mitis were nontoxic. Adding plaque toxin-neutralizing MAb 3hE5 blocked the toxic effect of E. corrodens extract S. mitis extracts contained a single, strongly reactive antigen of 140 kDa (s140K antigen) detected on Western blots (immunoblots) by three MAbs from the panel. Rabbit antibodies raised to this antigen excised from the gel (anti-s140K serum) detected larger antigens in addition to s140K. E. corrodens extracts contained a number of antigens detected by the MAbs. Immunoglobulin G (IgG) was purified from anti-s140K serum by passage through DE52 cellulose. A 100-fold excess (by weight) of the purified IgG to E. corrodens protein specifically cross-precipitated an 80-kDa antigen plus a nonantigenic 16-kDa protein, presumably attached noncovalently. The remaining supernatant fraction had no toxic activity. A similar ratio of control IgG (from nonimmunized rabbits) did not precipitate these proteins, and the supernatant fraction had the same activity as the extract not treated with IgG. The proteins of 80 and 16 kDa were also detected in the anti-s140K immunoprecipitate by rabbit IgG antibodies to E. corrodens whole cells. The 80-kDa antigen, alone or complexed with the 16-kDa protein, may be involved in mediating the toxic activity in E. corrodens and plaque extracts.     

Western blot screening for monoclonal antibodies against human separase

Separase is a cysteine protease that participates in separation of sister chromatids during mitosis. Human separase is a 230-kDa enzyme that is inhibited by binding to its protein inhibitor securin, specific phosphorylation, and subcellular localization. To further characterize human separase, we raised monoclonal antibodies specific against a C-terminal fragment of the protein. A critical step in monoclonal antibody production procedure is the primary screening of hybridoma supernatants. Here we report primary screening protocol utilizing Western blot analysis. The described screening protocol is carried out using fusion of a human separase fragment with two different purification tags, maltose-binding protein (MBP) and glutathione S-transferase (GST). Immunization by MBP-fusion was followed by primary screening with both MBP- and GST-separase fusions combined in the same preparation separated in SDS-PAGE. This highly sensitive screening approach reduced the number of positive signals by eliminating antibodies specific for the purification tag used in the immunization procedure. The described separase-specific antibodies were suitable for detection of endogenous separase in crude extracts, immunoprecipitation, and immunofluorescent cell staining experiments. The presented procedure is fast, reproducible and could be adopted as a primary screening scheme for a variety of protein antigens.     

The schistosome glutathione S-transferase P28GST,a unique helminth protein,prevents intestinal inflammation in experimental colitis through a Th2-type response with mucosal eosinophils

Intestinal helminth parasites are potent inducers of T helper type 2 (Th2) response and have a regulatory role, notably on intestinal inflammation. As infection with schistosomes is unlikely to provide a reliable treatment of inflammatory bowel diseases, we have investigated the beneficial effect of a schistosome enzymatic protein, the 28-kDa glutathione S-transferase (P28GST), on the modulation of disease activity and immune responses in experimental colitis. Our results showed that immunization with recombinant P28GST is at least as efficient as established schistosome infection to reduce colitis lesions and expression of pro-inflammatory cytokines. Considering underlying mechanisms, the decrease of inflammatory parameters was associated with the polarization of the immune system toward a Th2 profile, with local and systemic increases of interleukin (IL)-13 and IL-5. Dense eosinophil infiltration was observed in the colons of P28GST-immunized rats and mice. Depletion of eosinophils by treatment with an anti-Siglec-F monoclonal antibody and use of IL-5-deficient mice led to the loss of therapeutic effect, suggesting the crucial role for eosinophils in colitis prevention by P28GST. These findings reveal that immunization with P28GST, a unique recombinant schistosome enzyme, ameliorates intestinal inflammation through eosinophil-dependent modulation of harmful type 1 responses, representing a new immuno-regulatory strategy against inflammatory bowel diseases.     

Ascaris lumbricoides: heterogeneity in ABO epithopes expression

Ascaris lumbricoides and its hosts can present the same ABO System epithopes. The aim was to study ABO epithopes expression in A. lumbricoides by Inhibition Agglutination Test using different monoclonal antibodies and policlonal sera. We worked with 27 parasite extracts (14 extracts from A Group patients, 2 AB Group 1, B Group and 10 from unknown ABO Group patients). Inhibition Agglutination Test was made facing the extracts from A, AB and unknown ABO Group patients against monoclonal antibodies and policlonal serum of A specificity and the extracts from B, AB and unknown ABO Group patients against monoclonal antibodies and policlonal serum of B specificity. The Semiquantitative Test was made with the extracts which inhibited the agglutination of any antibody. The 50% of the parasite extracts faced against A specificity antibodies and the 46% of the parasite extracts faced against B specificity antibodies significantly inhibited the agglutination in the Semiquantitative Test. No extract inhibited the totality of the antibodies. All the antibodies reacted with some extract, except one antibody of B specificity. The ABO activity in the extracts was independent of the antibody's immunoglobulin class, titre and concentration and it only was dependent on the antibody union with the epithope at which the antibody is directed. The heterogeneity in the ABO epithopes expression of A.lumbricoides might be involved in the escape of the host's immune response. The used Inhibition Tests are sensitive, simple, rapid and economic. We conclude that the use of different antibodies alows the best definition of the antigen-antibody specificity.     

Passive immunization of chickens against Eimeria maxima infection with a monoclonal antibody developed against a gametocyte antigen

Eimeria maxima gametocytes contain two major antigens with molecular masses of 56 and 82 kilodaltons (kDa) which are recognized by convalescent sera from immune chickens. Preparations enriched in these two antigens were used to immunize mice, and several monoclonal antibodies which specifically reacted with the 56-kDa antigen were produced. One of these monoclonal antibodies of the immunoglobulin M subclass, along with immune chicken sera raised against affinity-purified 56- and 82-kDa antigens, was used to passively immunize chicks. On the basis of the parameter of total oocyst output, it was found that these antibodies provided partial protection (40 to 50% inhibition) against E. maxima challenge infections.     

Immune response to Schistosoma mansoni phosphoglycerate kinase during natural and experimental infection: identification of a schistosome-specific B-cell epitope.

A cDNA clone encoding Schistosoma mansoni phosphoglycerate kinase (SmPGK) was previously identified by affinity-purified antibodies which are specific for 3-h-old schistosomula tegumental antigens. Antibodies to the recombinant SmPGK which has enzymatic activity were localized to various tissues including the tegument of the 3-h-old schistosomula and 42-day-old adult worms. In this study, we show that SmPGK is an immunogenic molecule in both natural infection in humans and experimental vaccination in animals. To understand the role that a highly conserved molecule like SmPGK played during schistosome infection, we affinity purified antibodies to SmPGK from patients with chronic schistosomiasis and demonstrated that they did not cross-react with human PGK. However, affinity-purified rabbit anti-SmPGK antibodies did show immunoreactivity to both human PGK and rabbit PGK. Thus, during natural infection antibodies that cross-react with human PGK are not produced; however, as a result of active immunization with an intact conserved molecule, such cross-reacting antibodies are produced. Immunological analysis of cyanogen bromide digests of SmPGK with monoclonal antibodies that recognize SmPGK but not human PGK identifies a B-cell epitope on a 12.2-kDa fragment represented by amino acids 61 to 174.     

Cockroach allergen detection and cockroach allergens of allergic Thai patients

Hybridomas secreting monoclonal antibodies (MAb) specific to American cockroach (Periplaneta americana) were produced through a fusion of immune splenocytes of a BALB/c mouse immunized with crude cockroach (CR) extract and mouse myeloma cells. Two hybridomas namely 38G6 and 3C2 were established. These specific hybridomas secreted IgG1 monoclonal immunoglobulins with antigenic specificities to CR protein components of over 207 to 72 kDa and 45 to 40 kDa, respectively. The monoclonal antibodies were applied to select their specific epitopes out of the crude CR extract using affinity chromatography. A Prausnitz-Kustner test revealed that these epitopes were allergens which caused wheals and flares of the skin of a guinea-pig previously sensitized with a pool of serum samples from CR allergic patients. The monoclonal antibodies were also used in a capture ELISA to detect specific IgE in serum samples of allergic Thai patients. It was found that 72% and 76% of the patients had IgE antibodies to the epitopes of MAb 38G6 and MAb 3C2, respectively, indicating that the two epitopes are major CR allergens among the CR allergic Thai patients. An antibody-sandwich ELISA was developed for quantitative detection of CR allergens using the two monoclonal antibodies as a capture reagent and rabbit polyclonal antibodies to crude CR extract as a detection reagent. The assay could detect allergenic epitopes contained in as little as 122 pg of crude cockroach extract, and has high potential for direct measurement of the marker allergens in extracts of environmental samples.     

Recognition of mycobacterial antigens by sera from patients with leprosy.

Mycobacterium leprae sonic extracts prepared from armadillo-derived bacteria were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) procedures and probed with serum or plasma samples from 20 patients with lepromatous leprosy and 14 healthy endemic controls. Five proteins of 33, 25, 18, 15, and 12 kilodaltons (kDa) were frequently recognized; the 33- and 15-kDa proteins were, respectively, recognized with high intensity by 16 and 13 of the 20 samples from patients with leprosy, whereas only one healthy donor had antibodies that recognized the 15-kDa protein. By the use of M. leprae-specific murine monoclonal antibodies it was demonstrated that the 33-, 25-, and 15-kDa antigens were different from those bound by the available murine monoclonal antibodies. The 18- and 12-kDa proteins detected had molecular masses similar to those detected by the corresponding murine monoclonal antibodies. The serum and plasma samples from patients with leprosy were also used to probe Western blots of a soluble extract of M. tuberculosis. They recognized, among others, antigens with molecular weights similar to those detected in the M. leprae antigenic preparations, although with less intensity and at a lower frequency.     

Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione S-transferase

The NH2-terminal amino acid sequence of the Mr 26 000 glutathione S-transferase (EC 2.5.1.18) of Schistosoma japonicum (Sj26) has been deduced by RNA and protein sequence analysis. Using this information, a bacterial plasmid has been constructed that directs the synthesis of the entire Sj26 molecule in Escherichia coli. Recombinant Sj26 exhibits glutathione S-transferase activity and can be readily purified from bacteria in a one-step procedure under non-denaturing conditions. The availability of recombinant Sj26 in essentially unlimited quantities will aid its assessment as a candidate vaccine molecule in schistosomiasis and could eventually lead to the rational design of a drug targetted on schistosome glutathione S-transferases.     

Characterization of the low-molecular-mass proteins of virulent Treponema pallidum.

We previously demonstrated that Treponema pallidum cells incubated in vitro in the presence of heat-inactivated normal rabbit serum (HINRS) synthesize, in very small quantities, several pathogen-specific, low-molecular-mass proteins that appear to be localized extracellularly. In this study, we have taken advantage of our ability to metabolically radiolabel T. pallidum cells to high specific activity to further characterize these antigens. We found that the low-molecular-mass proteins are not related to the 15- and 17-kDa detergent-phase proteins (J. D. Radolf, N. R. Chamberlain, A. Clausell, and M. V. Norgard, Infect. Immun. 56:490-498, 1988). The low-molecular-mass proteins did not incorporate 3H-labeled fatty acids and were not precipitated by rabbit immunoglobulin G (IgG) antibodies directed against glutathione S-transferase fusions to the nonlipidated 15- and 17-kDa proteins. We prepared polyclonal antisera to the low-molecular-mass proteins by immunizing two rabbits with the concentrated supernatant of T. pallidum cells. IgG antibodies present in the sera of both rabbits precipitated a 21.5-kDa protein from solubilized extracts of T. pallidum supernatant and cells. IgG antibodies in the serum of the second rabbit precipitated an additional 15.5-kDa low-molecular-mass protein only from solubilized extracts of supernatant. While investigating the effect of eliminating HINRS from the extraction medium, we observed that the low-molecular-mass proteins remained associated with treponemal cells that were incubated in the absence of HINRS. These proteins could be eluted from the cells by the addition of HINRS or rabbit serum albumin, suggesting that they are located on or near the treponemal cell surface. The 15.5- and 21.5-kDa low-molecular-mass proteins were not washed off treponemal cells with buffer containing 1 M KCl. Experiments employing selective solubilization of the T. pallidum outer membrane with 0.1% Triton X-114 and proteinase K accessibility indicated that the 15.5-kDa protein, but not the 21.5-kDa protein, is cell surface exposed.     

Identification of a Plasmodium chabaudi antigen present in the membrane of ring stage infected erythrocytes

A novel antigen of asexual blood stages of the rodent malaria parasite Plasmodium chabaudi, was detected by means of a modified indirect immunofluorescence assay (IFA), using glutaraldehyde fixed and air dried monolayers of P. chabaudi infected erythrocytes. P. chabaudi hyperimmune sera gave a distinct surface immunofluorescence of erythrocytes infected with early stages of the parasite. Fixation and drying of the erythrocytes was necessary for the antigenic activity to be exposed. The antigens were species specific as P. chabaudi hyperimmune serum only stained P. chabaudi but not P. yoelii or P. falciparum infected erythrocytes. The antigenic activity involved in the IFA was resistant to trypsin, phospholipases and neuraminidase but not to pronase, suggesting that the antigens were polypeptides. The surface immunofluorescence was inhibited by an extract of parasitized erythrocytes, but not by similar extracts of normal erythrocytes. The inhibitory antigens were soluble and heat stable (100 degrees C, 5 min). For identification and characterization of the antigens, antibodies were isolated by acid elution from monolayers of infected erythrocytes and monoclonal antibodies were produced. Probing in immunoblotting of extracts of parasitized erythrocytes separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the eluted antibodies, showed that they reacted consistently with a polypeptide of Mr 105 000 (Pch105). The Pch105 antigen shares many characteristics with Pf155, a P. falciparum antigen considered as a candidate for a vaccine against that parasite.     

Production and characterization of monoclonal antibodies to a 58-kilodalton antigen of Aspergillus fumigatus

Eight monoclonal antibodies that recognize a serodiagnostically important 58-kDa antigen of Aspergillus fumigatus were produced and partially characterized. 2-7, 2-12, and 2-14 are of the immunoglobulin M class, and 2-2-1, 2-2-4, 2-2-6, 2-2-9, and 2-2-13 are all immunoglobulin G1(kappa) antibodies. Immunoblot analysis with A. fumigatus mycelial extract demonstrated that all of the monoclonal antibodies recognize a major 58-kDa antigen. The antigen was also detected by immunoblot analysis of 4- and 7-day culture filtrate preparations. 2-2-1, 2-2-4, and 2-2-6 cross-reacted with an antigen of approximately 55 kDa from an extract of Candida albicans. 2-7, 2-12, 2-14, and 2-2-4 formed a precipitin band with immunoaffinity-purified 58-kDa antigen by immunodiffusion. Results from indirect immunofluorescence assays with 2-7 and 2-2-9 showed fluorescent staining mainly on the surfaces of conidia and hyphae, indicating that the 58-kDa antigen may be cell wall associated. 2-2-9 and 2-2-13 and antibodies in patient and immune rabbit sera precipitated the [35S]methionine-labeled 58-kDa antigen. The 58-kDa antigen immunoprecipitated by each of the antibodies was enzymatically cleaved by Staphylococcus aureus V8 protease; one cleavage product, a 35-kDa fragment, was generated, indicating that the precipitated antigens share primary structure. Immunoblot analysis with an immunoaffinity-purified 58-kDa antigen showed that sera from patients with invasive aspergillosis reacted with the same antigen as that recognized by the monoclonal antibodies.     

Schistosoma mansoni: Detection and characterization of schistosome derived antigens by inhibition enzyme-linked immunosorbent assay (IELISA) utilizing monoclonal antibodies

Monoclonal antibodies were used in an inhibition enzymelinked immunosorbent assay (IELISA) to detect a variety of schistosome derived antigens. Preparations were obtained from various stages ofSchistosoma mansoni and from the eggs ofS. japonicum. Using appropriate titers of monoclonal antibodies it was possible to detect less than 0.01 g/ml of schistosome antigens. The sensitivity of IELISA was dependent upon the type and concentration of the monoclonal antibody used, as well as upon the source of the antigens. Specificity studies showed that some of the monoclonal antibodies recognized species specific antigenic determinants, while others reacted against genus specific antigens. Furthermore, certain antibodies interacted with antigens which were neither genus or species specific. Fractions ofS. mansoni andS. japonicum egg antigen extracts, which have been previously considered to be relatively pure, were compared using certain monoclonal antibodies. The results indicate that these fractions are very heterogenous with regard to the unique antigenic specificities. For example, most antigenic determinants in crude soluble egg antigen are not retained on concanavalin-A lectin affinity columns and major serologic antigens have more than one determinant with different distributions. The expression of these antigenic determinants appears to be a function of both the concentration of the specific antigen and the mode of expression of the moiety within the antigenic matrix.     

Comparative antigen analysis of different life stages of Schistosoma mansoni by crossed immunoelectrophoresis

Two-dimensional (crossed) immunoelectrophoresis was used for analysis of soluble antigen extracts obtained from the three developmental stages, cercariae, adult worms and eggs, of Schistosoma mansoni by using homologous hyperimmune sera produced in sheep. The antigenic relationships between the three stages as well as the possible relationship to the intermediate snail host were studied. Seven antigen components were shown to be shared between all three life stages of S. mansoni. Furthermore, one antigen was common to adult worm and snail, and one other antigen was shared between cercaria and snail. By using an intermediate gel containing lectin in the antigen-antibody system or by enzyme staining of the immune precipitates it was possible to identify schistosome antigens possessing lectin reactivity or enzyme activity. Characterization of enzyme activities revealed three individual precipitating antigens in adult worm of S. mansoni possessing esterase, leucyl-glycyl-glycine peptidase and phenylalanyl-leucine peptidase activities, respectively. One further precipitinogen with malate dehydrogenase activity was identified for all three life stages.