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Detection of anti-helminth antibody by microenzyme-linked immunosorbent assay using recombinant antigen and anti-beta-galactosidase monoclonal antibody.

An enzyme-linked immunosorbent assay (ELISA) using antigenic beta-galactosidase-helminth derived recombinant fusion protein (FP) obtained by the recombinant DNA technique provided a useful diagnostic tool for human helminthiasis. Helminth-infected human sera reacted strongly with FP that was immobilized with anti-beta-galactosidase monoclonal antibody on microplates. However, FP did not react with sera from patients with other helminthiases. In detection of anti-helminth IgG antibody, the present ELISA system using FP and anti-beta-galactosidase monoclonal antibody was highly specific compared to that using biochemically extracted soluble antigens.

Antibodies, Helminth↗

Systemic responses to challenge infection with Haemonchus contortus in immune Merino sheep.

Some systemic responses to single-dose infection with 10,000 Haemonchus contortus infective larvae were examined in sheep already shown to have protective immunity against the parasite. The major haematological finding was a neutrophil leukocytosis that occurred after the infections became patent but not during the pre-patent period. There was no definitive eosinophilia and no discernible change in the erythrocyte parameters. Systemic hyperthermia was not conclusively evident during the pre-patent period. Enzyme-linked-immunosorbent assays (ELISA) were used to measure the secondary anti-helminth antibody response in serum during the pre-patent period when the establishment of patent infection is resisted. These ELISAs employed preparations from adult worms to represent the parasitic stages of the worm, preparations from infective larvae to represent the pre-parasitic stages of the worm, and exsheathing fluid, which is the soluble material obtained when H. contortus larvae undergo ecdysis and transform from the pre-parasitic to the parasitic phase. Antibody responses to the three preparations differed qualitatively, indicating the presence of three different but perhaps overlapping sets of antigens. The three peaks in antibody against exsheathing fluid may reflect the pulses of antigen delivered to sheep as the parasite undergoes its three moults within the host.

Animals↗

A theoretical framework for the immunoepidemiology of blocking antibodies to helminth infection.

Epidemiological evidence is widely cited in support of the hypothesis that certain antibody responses to human helminth, especially schistosome, infection exhibit blocking activity. This evidence includes positive correlations between antibody levels and the rate of re-establishment of infection following chemotherapy, antibody levels which peak in younger and more susceptible age classes, lower ratios of blocking antibodies to others in older and less susceptible age classes. In this paper simple mathematical models are used to explore expected age-specific relationships between antibody levels, parasite burdens and re-establishment rates for different combinations of protective, neutral and blocking immune responses. In general, all the above cited patterns may be generated without invoking blocking activity, especially if the abilities to produce different antibody responses have different immunological memories, i.e. persist for different lengths of time in the absence of continued exposure to antigen. None of these patterns, including a positive correlation between antibody levels and rates of re-establishment following chemotherapy, offers unambiguous evidence for blocking activity. Blocking activity is also predicted to affect the shape of the age-intensity curve and the relationship between susceptibility to infection and age in ways which are not necessarily consistent with the epidemiological evidence. The importance of the blocking activity, which has been convincingly demonstrated in vitro, to population level immunoepidemiological patterns in the field therefore remains uncertain.

Adolescent↗

Prevalence of viral antibodies and helminths in field populations of house mice (Mus domesticus) in southeastern Australia.

A 13-month study of wild mice (Mus domesticus) in wheatlands in southeastern Australia contrasted changes in the seroprevalence of antibody to 13 viruses and the occurrence of helminths with changes in their population dynamics. Mice were seropositive for mouse hepatitis virus (MHV), rotavirus, minute virus of mice (MVM), mouse adenovirus (MAdV), reovirus (reo 3), and murine cytomegalovirus (MCMV). The seroprevalences of all but rotavirus varied significantly with time and increased with host density. Near the end of the study, host density declined rapidly and the seroprevalence of MVM and reo 3 increased significantly. These two viruses had low seroprevalence when host survival was high and high seroprevalence when host survival was low, indicating they may play a role in regulating mouse populations. In the case of MVM, there was evidence of a viral epizootic during the decline in mouse abundance. The prevalence of four helminths (Taenia taeniaeformis, Syphacia obvelata, and Vampirolepis spp.) differed significantly with time but showed no apparent association with host density. These findings highlight the need for further study on the effect of viruses on the population dynamics of mice.

Animals↗

Recognition of gut-associated antigens by immunoglobulin M in the indirect fluorescent antibody test for schistosomiasis mansoni.

The specific reactivity of immunoglobulin M antibodies, as measured by a commonly used immunodiagnostic technique for schistosomiasis mansoni, the immunofluorescence assay (IFA) on Rossman-fixed sections of adult worms, was analyzed with monoclonal antibodies. Using monoclonal antibodies directed against 3 major gut-associated antigens, circulating anodic antigen (CAA), circulating cathodic antigen (CCA) and the 32 kDa antigen in blocking experiments, it was demonstrated that the measured IFA reaction was primarily due to reactivity with CCA. After blocking with anti-CCA monoclonal antibody, a mean decrease of fluorescent antibody titres of 95.8%, 92.9% and 83.8% was observed for sera from groups of patients with a recent infection (0-6 months), a static infection (6 months - 5 years), and a chronic infection (over 5 years), respectively. Titres of the group of recently infected patients were, after blocking, significantly lower (P less than 0.05) than those of the group of chronically infected patients.

Animals↗

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.

Animals↗

Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni.

This study describes the production and characterization of a monoclonal antibody (mAb) against the alkaline phosphatase of Schistosoma mansoni from splenocytes of chronically infected mice. Convenient selection of the mAb was achieved using the catalytic activity of the antigen in a developed enzyme-antigen immunoassay. The mAb was of the IgG1 subclass and it specifically recognized the alkaline phosphatase in adult worm sections by indirect immunofluorescence. Preincubation of the antibody with partially purified adult alkaline phosphatase did not result in inhibition of the enzyme activity and it did not mediate complement-dependent cytotoxicity against mechanically transformed schistosomula in vitro. The mAb was able to immunoprecipitate under reducing conditions a polypeptide of 65 kDa, similar in size to the monomeric subunit of the schistosome enzyme. The specificity of the mAb was assessed by competitive inhibition with antibodies of infected human sera in an immunoadsorption assay. Periodate treatment of the antigen resulted in altered electrophoretic mobility of alkaline phosphatase, which confirmed the presence of carbohydrate in the molecule, but this did not prevent binding by the mAb. Although the use of the mAb in capture assays for detection of circulating alkaline phosphatase in infected host sera was unsuccessful, the production of this antibody confirmed that the enzyme is exposed by adult worms to the host and that it is immunogenic; additionally, a monoclonal probe is available for further characterization of the structure and function of this important parasite surface molecule.

Alkaline Phosphatase↗

Identification of a laminated layer-associated protein in Echinococcus multilocularis metacestodes.

Echinococcus multilocularis is a cestode parasite that predominantly infects red and arctic foxes as definitive hosts. Ingestion of E. multilocularis eggs and subsequent post-oncospheral infection with the larval stage (metacestode) of the parasite results in alveolar echinococcosis (AE), a life-threatening hepatic disease concerning humans and other intermediate hosts such as small rodents. The primary fluid-filled vesicles of the asexually proliferating metacestode are comprised of an inner germinal layer, a syncytial tegument, and an outer, acellular, so-called laminated layer. This laminated layer may play an important role in protecting the developing E. multilocularis metacestode from host immune reactions, and laminated layer-associated components represent potential targets for intervention during the course of AE. We have used an in vitro cultivation technique for the long-term maintenance and proliferation of E. multilocularis metacestodes in order to generate premature (protoscolex-free) parasite vesicles. A polyclonal antiserum was raised against this host-free parasite tissue. Subsequent immunoblot analysis of parasite fractions obtained by Triton X-114 extraction lead to the identification of a 116 kDa protein (named EmP2) within the Triton-insoluble fraction. The characterization of EmP2 by SDS-PAGE, Western blotting, and by immunofluorescence revealed that EmP2 is a laminated layer-associated protein.

Animals↗

Distribution of 28.5 kDa antigen in the tegument of adult Fasciola gigantica.

Monoclonal antibody (MoAb) specific to 28.5 kDa tegumental antigen (TA) was used to localize this antigen in various tissues of adult Fasciola gigantica by means of indirect immunofluorescence, immunoperoxidase and immunogold techniques. The indirect immunofluorescence and immunoperoxidase detections revealed that this antigen was concentrated in the tegument particularly in its outer rim, tegumental cells and their processes, epithelial linings of the oral sucker and the proximal part of digestive tract. It was also detected at a moderate concentration in spermatogenic cells in the testes, cells of Mehlis' gland, oocytes within the ovary, and ovum within the egg of adult parasites. At TEM level, the immunogold detection showed deposit of gold particles specifically in G(2) tegumental granules and on the surface membrane. Thus, this antigen is expressed in the tegument and associated structures of adult parasites, and it could be a major component of the G(2) granules which are shown to fuse with the surface membrane and contribute material to replace the casted-off membrane. This process is a part of membrane turnover that prevents the parasite from being attacked by the host immune effector cells.

Animals↗

Onchocerca volvulus. Monoclonal anti-idiotype antibody as antigen signal for the microfilaricidal cytotoxicity of diethylcarbamazine-treated platelets.

Over the past 35 yr, diethylcarbamazine (DEC) has been the most widely used agent for the treatment of filarial diseases, particularly in onchocerciasis. The microfilaricidal action of DEC has been recently shown to be mediated by blood platelets with the additional triggering of a filarial excretory Ag (FEA). This FEA could be detected by using mAb in the serum of infected patients. By using one mAb (IA2(23] directed against Onchocerca volvulus and recognizing circulating Ag (Ab1), we purified by affinity chromatography the target molecule of IA2(23) (an O. volvulus glycoprotein recognized by IA2(23) mAb). This compound had a dose-dependent effect on the cytotoxic action of DEC-treated platelets. We subsequently produced an anti-idiotype mAb to Ab1 (Ab2), and considered the possibility of replacing the O. volvulus glycoprotein recognized by IA2(23) mAb by Ab2. Ab2 was selected according to its ability to inhibit the binding of radioiodinated Ab1 to the filarial target Ag. It induced the production of anti-O. volvulus antibodies (Ab3) in rats. At a constant concentration of DEC platelets, the addition of increasing amounts of Ab2 led to a dose-dependent cytotoxic effect against parasite larvae. Experiments performed with Ab2 on detergent solubilized surface proteins of platelets identified four bands of Mr 18, 26, 43.5, and 100 kDa, supporting the idea of the presence of binding sites on the platelets for a FEA required for the microfilaricidal cytotoxicity of DEC-treated platelets.

Animals↗

Secretory granule proteins from the subventral esophageal glands of the potato cyst nematode identified by monoclonal antibodies to a protein fraction from second-stage juveniles.

Sodium dodecyl sulfate-extracted proteins from second-stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.

Animals↗