Single step purification of potent antigenic protein from sparganum by gelatin-affinity chromatography

Out of many component proteins in crude saline extract of Spirometra mansoni plerocercoid (sparganum), 36 kDa and 29 kDa proteins were found to be the most antigenic and were already purified by immunoaffinity chromatography using monoclonal antibody as a ligand. In this study, a single step purification of these potent antigenic proteins of sparganum extract was investigated. When the crude saline extract was charged to gelatin-Sepharose 4B affinity column, 36 kDa and 29 kDa protein fractions were bound. SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE/immunoblot confirmed that the bound protein to gelatin was serologically pure. When evaluated by ELISA with patients sera, the purified protein of 36 and 29 kDa also showed improved antigenicity.     

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.     

Protective immunity against Brugia malayi infective larvae in mice. II. Induction by a T cell-dependent antigen isolated by monoclonal antibody affinity chromatography and SDS-PAGE

A mAb directed against filarial worm secretory/excretory product and reactive with Brugia malayi larval worm surface was used in conjunction with preparative SDS-PAGE to isolate protective Ag from extracts of adult B. malayi. The IgM mAb OVH bound to a repeating carbohydrate epitope present in adult, infective, and fourth stage larvae and microfilariae of B. malayi, and on the surface of fourth stage larvae. Ag bearing this epitope were also present in the sera of hosts infected with a variety of helminths, including Brugia, Onchocerca, Dirofilaria, and Paragonimus. Affinity chromatography of SDS extract of adult Brugia, using mAb OVH immobilized on agarose beads, isolated several Ag that separated into multiple protein staining bands on SDS-PAGE. In comparing SDS-PAGE-fractionated Ag from the crude SDS extract with fractionated mAb OVH-isolated Ag for the ability to protect BALB/c mice from challenge with B. malayi-infective larvae, it was found that of the mAb OVH-isolated Ag only those at a molecular mass of 26 to 32 kDa were protective while the original SDS extract yielded protective Ag at the following molecular mass: greater than 200, 170 to 200, 40 to 44, 33 to 36, 23 to 28, 20 to 22, and 17 to 19 kDa. Although Ag isolated by mAb OVH were highly protective, they failed to induce high antibody levels against the immunogen or SDS extracts compared to crude SDS extract immunized mouse sera, as determined by immunoblot and ELISA. Transfer of nylon wool non-adherent T cells from BALB/c mice immunized with the 26- to 28-kDa fraction of mAb OVH-isolated Ag to naive mice just before challenge with infective larvae of B. malayi resulted in a 70% reduction in larvae recovered 14 days after challenge.     

Characterization of body louse midgut proteins recognized by resistant hosts

Abstract. The human body louse, Pediculus humanus , showed eighteen midgut proteins ranging between 12 and 117 kDa, when analysed by SDS-PAGE electrophoresis. Seven of them (12 kDa, 17 kDa, 29 kDa, 35 kDa, 40 kDa, 55 kDa and 97 kDa) were major bands based on their intensity of staining. The immunization of rabbits with a midgut extract elicited the production of protective polyclonal antibodies. These antibodies reacted strongly with all major midgut proteins as well as with 63 kDa and 117 kDa proteins when tested by the Western blot technique. The analysis of the proteins revealed that the 12 kDa, 25 kDa, 29 kDa, 35 kDa, 45 kDa, 87 kDa and 97 kDa proteins are glycosylated and none of them contained a lipid moiety. By electroelution, the proteins of 35 kDa and 63 kDa were purified. On trypsinization, the proteins of 35 kDa and 63 kDa produced four major fragments (F, F₂, F₃, and F₄) when resolved on a 18% SDS-PAGE. The Fj fragment of the 35 kDa protein reacted with me polyclonal antibodies by the immunoblot technique.     

Immunohistochemical localization of 36 and 29 kDa proteins in sparganum.

Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.     

Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody

The quality improvement of antigen (crude saline extract) of Spirometra mansoni pleroceroid (sparganum) was investigated by protein purification. The crude extract was fractionated by gel filtration through Sephacryl S-300 Superfine. Its third fraction was purified by affinity chromatography using a monoclonal antibody as ligand. When observed by SDS-PAGE, the purified protein was composed of 2 bands of 36 kDa and 29 kDa which were found already as the most sensitive components in the crude extract by immunoblots with patients sera. The quality of the purified antigen was evaluated in comparison with the crude extract by enzyme-linked immunosorbent assay (ELISA) for the specific (IgG) antibody in sera of human sparganosis, other parasitic and neurologic diseases, and normal control. When the purified antigen was used, the sensitivity was not altered but remained high (96.4%) while the specificity was increased from 86.8% to 96.9%.     

Purification, characterization, and localization of a major membrane protein antigen from Porphyromonas (bacteroides) gingivalis.

Humans and rats infected with P. gingivalis develop a strong immune response to a 75 kDa major membrane component of P. gingivalis and hence knowledge of the nature of this molecule may aid in understanding the host response to P. gingivalis during infection. Purification of the 75 kDa protein was achieved by repeated precipitation from a crude sonicate of P. gingivalis 2561 at pH 5.0. Homogeneity of the purified 75 kDa protein was confirmed by SDS-PAGE and Western immunoblot analysis using monoclonal and polyclonal antibodies. The purified protein revealed an apparent molecular mass of 300 kDa in native form. Although most of the strains of P. gingivalis tested showed a major membrane protein band in the range of 61 to 78 kDa, on Western immunoblot analysis only the strains which have proteins in the range of 75 to 78 kDa were reactive with anti-75 kDa protein polyclonal antibodies. Affinity purified polyclonal antibodies were used to localized 75 kDa protein on the cell surface of P. gingivalis 2561 by immunogold electron microscopy. Immunolabeling of the 75 kDa protein demonstrated specific localization of the protein along the outer cell membrane, but not on the fimbriae. Furthermore, immunogold labeling of the 75 kDa protein on the thin sections showed that the 75 kDa component was present on not only the outer membrane, but also on the cell membrane, and on membrane bound organelles. Localization of this protein suggests that the 75 kDa component is a membrane-associated protein.     

Purification and characterization of phosphoglucomutase from peas

Phosphoglucomutase (EC 2.7.5.1) was isolated from pea seeds ( Pisum sativum L. cv. Grenadier) and purified to homogeneity as determined by sodium dodecylsulfate - polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The enzyme was purified by utilizing 25% polyethylene glycol 4000 precipitation, followed by Fractogel-diethyla-minoethyl (DEAE) 650. Fractogel-TSK HW-55(s). and high pressure liquid chroma-tography (HPLC)-(PEI) column chrornatography. The resulting enzyme had a specific activity of 157 units (mg protein)-1. a 152-fold increase over that of the crude plant extract. The molecular weight of the enzyme was 128 to 136 kDa. as determined by native-PAGE and column chromatography, and when it was subjected to SDS-PAGE analysis, it was found to be composed of two subunits having molecular weights ranging from 59 to 64 kDa. Upon SDS-PAGE analysis of a sample purified through HPLC-PEI chromatography. two bands of protein were found: one having a molecular weight of 64 kDa and the other 68 kDa. A pH optimum of 8.6 was found for the enzyme while it was also found that cysleine. Mg²⁺ and glucose 1.6-bisphosphate were necessary for optimal activity Histidine and imidazole only partially fulfilled the cysteine requirement. A 20-min preincubation period in the absence of glucose 1-phosphate was necessary for optimal activity of the enzyme. Without a preincubation period, there was a pronounced lag preceding the linear portion of the reaction as well as a reduction in the V_max. An analysis of the kinetics of the reaction showed K_m values ot 3.6 × 10⁻⁵ and 1.45 × 10⁻⁵ M for glucose 1-phosphate and glucose 1.6-bisphosphate. respectively. A K., of 7.3 × 10⁻⁵ M was obtained for MgCl₂.     

Purification and characterization of a 36 kDa antigen of Mycobacterium leprae

A 36 kDa antigen of Mycobacterium leprae was purified by phenol biphasic partition followed by preparative SDS-PAGE. The purified antigen appeared as a single band in SDS-PAGE and eluted as a single peak in ion-exchange chromatography. The antigen comprised epitopes which were cross-reactive with M. tuberculosis, as well as a species-specific epitope (recognized by MAb F47-9). Different treatments of the 36 kDa antigen suggested it to be largely protein in nature; the amino acid composition of 81% of the antigen was determined. A majority of sera from leprosy patients contained antibodies recognizing the 36 kDa antigen.     

Purification of Bacillus subtilis Spore Coat Protein by Electrophoretic Elution Procedure and Determination of NH2-Terminal Amino Acid Sequences

Spore coat protein of Bacillus subtilis was purified by electrophoretic elution procedure. Solubilized coat protein components were separated on SDS-PAGE and the desired protein was recovered from the gel pieces under the optimal condition examined. Two purified polypeptides with molecular weights of about 40 kDa were obtained; each of them was in very closed size on SDS-PAGE, both retaining antigenic activity against anti-spore coat protein serum on immunoblot analysis. The N-terminal 23 and 30 amino acid sequences of them were determined, and they were not identical to each other and also not homologous in the sequences of coat proteins previously reported.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Efficacy of protein A-HRP in an immunological study of black rockfish (Sebastes schlegeli Higendorf) humoral immune responses

The efficacy of protein A-horse radish peroxidase (HRP), as compared to that of mouse polyclonal antibody raised against purified Ig, in detection of black rockfish (Sebastes schlegeli Higendorf) immunoglobulin (Ig) was examined. Protein A affinity chromatography successfully purified Ig from black rockfish serum; the purified-Ig could be visualised as two protein bands (MW 70 and 25kDa) following resolution with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. In SDS-PAGE immunoblot profiles of the purified-Ig, the mouse polyclonal antibody recognised both the light chain and heavy chains of rockfish Ig, whereas protein A-HRP immunostained only the heavy chain of rockfish Ig. These results suggest that protein A-HRP may be used to detect rockfish antibody-antigen complexes in immunoassays. In a 2-DE immunoblot assay for exploring antigenic profiles of Lactococcus garvieae KG9408, protein A-HRP successfully detected specific antibodies to antigenic proteins of L. garvieae in the rockfish Ig. In addition, enzyme linked immunosorbent assay (ELISA) showed a high correlation between the results obtained for positivity of L. garvieae when protein A-HRP and the mouse polyclonal antibody-was used to analyse samples from 25 diseased rockfish. These results collectively indicate that protein A-HRP has a high affinity for Ig, and may be useful for new investigations into the humoral immune responses of rockfish.     

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

Partial purification of the maturation-promoting factor MPF from unfertilized eggs of Xenopus laevis

A 200-fold purification of the maturation-promoting factor or MPF from unfertilized eggs of Xenopus laevis is reported for the first time. Purification was achieved by three successive column chromatographies on hydroxyapatite, trisacryl blue and L-arginine-agarose. The presence of MPF was assessed by the usual maturation criteria after injections of test material into immature stage VI unstimulated X. laevis oocytes: the precocious appearance of the maturation spot (within 45-120 min), the germinal vesicle breakdown, the presence of the first polar body and the second metaphase spindle. Purification was monitored by the decrease of the minimal amount of protein injected in a constant volume (50 nl) required to induce 50% frequency of germinal vesicle breakdown. This amount decreased from 500 ng in the crude extract to 2.5 ng in the 200-fold purified material. Analysis by SDS-PAGE of the crude extract showed about 40 Coomassie-blue-stained polypeptides with molecular masses ranging from 300 kDa to 20 kDa, whereas in the 200-fold purified MPF only 5 stained polypeptides were revealed, with molecular masses of 62, 53, 49, 39 and 37 kDa. In vitro phosphorylations for the detection of kinase activities for endogenous and exogenous substrates were monitored by analysis of autoradiograms of SDS-PAGE, after treatment of fractions with [gamma-32P]ATP. Only inactive fractions eluted from columns ahead of MPF, and fractions containing MPF activity were tested. Phosphorylation of numerous stained polypeptides was demonstrated in the crude MPF extract and exogenous substrates such as phosvitin, casein and histone type II-AS were also strongly phosphorylated. In the MPF fraction, purified on hydroxyapatite, a polypeptide of 53 kDa was more highly and specifically phosphorylated and the presence of kinase activities was observed for the above three exogenous substrates. In the 100-fold and 200-fold purified MPF, phosphorylation of endogenous substrates could not be shown and kinase activities for the above three substrates were drastically decreased as compared with the crude and purified MPF obtained after hydroxyapatite column chromatography. However, neither endogenous phosphorylations nor kinase activities with the above exogenous substrates could be shown in inactive fractions eluted ahead of MPF at the different purification steps. Some characteristics of the purified material are also described in this paper.     

Purification and characterization of S layer proteins from Clostridium difficile GAI 0714

The S layer of Clostridium difficile GAI0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8.3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0.1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses of about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine. The 32 kDa protein exhibited multiple isoelectric forms (pI 3.7-3.9), whereas the 45 kDa protein had a single form (pI 3.3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.     

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)     

聚球藻PCC 7942氢酶的分离纯化及特性研究

报道了室温、空气环境下聚球藻Synechococcus sp.PCC7942氢酶的分离纯化.经过超声破碎、超速离心、离子交换层析、疏水层析及凝胶层析等步骤,氢酶被纯化了218倍,得率为6.5%,比活为1.46U·mg^-1蛋白.纯化氢酶的SDS-PAGE图显示五条蛋白带,分子量约为83kDa,60kDa,47kDa,30kDa和27kDa.该氢酶为可溶性的双向氢酶,其催化放氢的最佳电子供体为还原态的甲基紫精,最适温度50℃,最适pH8.0.     

Isolation of 110K actin binding protein from mammalian brain and its immunocytochemical localization within cultured cells

Crude extract of young rat brain forms actin-based gels upon incubation at 25 degrees C. After boiling the gelled material, a protein fraction composed mostly of a major band of 110 kDa and a minor band of 120 kDa in SDS-PAGE was obtained by hydroxyapatite column chromatography. When the same protein fraction was prepared from bovine brains using the same procedure with two additional column chromatographies, the amounts of both proteins were nearly the same. Both proteins cosedimented with actin filaments upon centrifugation. Antibody was produced in a rabbit against the bovine fraction and affinity purified using a nitrocellulose paper onto which these proteins were transferred electrophoretically. Immunoblot analysis showed that both proteins are immunologically similar, and we refer to both proteins as 110K protein, collectively. The immunoblot analysis also revealed that the 110K protein is contained in cultured cells such as BHK, 3Y1, NRK, and MDBK. Analysis of various tissue extracts showed that brain is rich in this protein but liver, kidney, and lung contain negligible amounts. Indirect immunofluorescent analysis using cells during spreading showed preferential localization in the leading edge region and no fluorescence was detected in the stress fiber. Double immunostaining using monoclonal anti-vinculin and anti-110K protein antibodies revealed that the distribution patterns of both proteins are different from each other.     

Preparation and Characterization of Monoclonal Antibodies to Serotonin Binding Protein

Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross-reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 X 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45-kDa or 56-kDa SBP. Culture supernatants were screened for the presence of anti-SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type-specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme-linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44-10) was obtained and affinity-purified. In the presence of goat anti-mouse IgM, the partially purified 44-10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44-10 blocked the binding of [3H]serotonin ([3H]5-HT) to 45-kDa and 56-kDa SBP in a concentration-dependent manner. The 68-kDa protein was found to bind [3H]5-HT. Sites reacting with MAB 44-10 were located immunocytochemically in sections of rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fasciola hepatica: host responders and nonresponders to parasite glutathione S-transferase.

Fasciola hepatica glutathione S-transferase (FhGST) was isolated from adult worms by glutathione agarose affinity chromatography. SDS-PAGE shows three proteins of M(r) ranging from 29-27.8 kDa. Western immunoblot analyses using SDS-PAGE separated adult worm extracts and probed with a rabbit anti-FhGST antiserum reveal two bands in the same M(r) range. Mice and rabbits immunized with purified FhGST develop copious amounts of anti-FhGST antibodies. Moreover, antisera to F. hepatica adult worms and excretion-secretion products also react with FhGST. Cross-reactivity with schistosomes is evidenced in the reactivity with FhGST of anti-Schistosoma mansoni adult worm antisera and, to a lesser extent, antisera to S. mansoni-soluble egg antigens. The time of appearance of anti-FhGST antibodies in different species of animals infected with F. hepatica was determined. Sheep and a New Zealand white rabbit developed anti-FhGST antibodies detectable by ELISA as early as 2 weeks postexposure with F. hepatica. However, neither mice nor calves infected with F. hepatica developed antibodies to FhGST through the 5-10 weeks of infection tested. But mice infected with S. mansoni developed anti-FhGST cross-reacting antibodies by 6 weeks of infection. Calves immunized with a Fasciola/Schistosoma cross-reactive, cross-protective antigen complex in which a 12,000-kDa protein (Fh12) has been shown to contain immunoprophylactic activity, also developed antibodies to FhGST. Since FhGST is a novel potential vaccine, its protection-inducing capability in a multivalent vaccine combined with Fh12 clearly warrants study. In summary, it appears that hosts with fascioliasis are either responders to FhGST (rabbits, sheep) or nonresponders (mice, cattle), offering interesting models for studying the immune response.