Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed an enzyme immunoassay (ELISA) for the quantification of the schistosome circulating cathodic antigen (CCA), a glycoprotein associated with the syncitium lining the gut of the parasite. A mouse monoclonal antibody of IgG3 isotype was used as coating (antigen-capture) antibody, while a biotinylated mouse monoclonal IgM was used as second (antigen-detecting) antibody. Streptavidin-alkaline phosphatase was used as enzyme label. The lower detection limit of the assay was 1.0 ng of the trichloroacetic acid soluble fraction of adult worm antigen (AWA-TCA) per ml, which corresponds to approximately 0.2 ng CCA per ml. The ELISA showed a linear range from 1.0 to 62.5 ng AWA-TCA per ml. Serum and urine samples of 16 individuals infected with Schistosoma mansoni (egg counts ranging from 5 to 4820 eggs per gram of faeces) were tested in the assay. Antigen titres ranged from less than 4-8192. This assay represents a considerable advantage in diagnosis of Schistosoma infections as it allows the detection and quantification of CCA in serum and urine in even lightly infected individuals.
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PMID:Detection of the schistosome circulating cathodic antigen by enzyme immunoassay using biotinylated monoclonal antibodies. 212 84

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.
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PMID:Production of a mouse monoclonal antibody against the alkaline phosphatase of adult Schistosoma mansoni. 216 96

Due to the lack of de novo purine nucleotide biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential enzyme in the human parasite Schistosoma mansoni for supplying guanine nucleotides and has been proposed as a potential target for antiparasitic chemotherapy. While the enzyme can be purified from adult schistosome worms, yields are too low to allow extensive structural and kinetic studies. We therefore cloned and sequenced the cDNA and gene encoding the schistosomal enzyme but were unable to positively identify the amino-terminal sequence of the enzyme from the DNA sequence. Knowledge of the exact amino terminus was necessary before accurate expression of active enzyme could be attempted. Therefore, we purified the HGPRTase from crude extracts of the adult worms. The purified enzyme has a subunit molecular mass of 26 kDa and an amino-terminal sequence of Met-Ser-Ser-Asn-Met. This sequence matched one of the potential initiation sites predicted from the cDNA and gene sequence. We next expressed the correct size cDNA of the S. mansoni HGPRTase in Escherichia coli using a vector that is regulated by a bacterial alkaline phosphatase promoter and uses an E. coli signal peptide for secretion of expressed product into the periplasmic space. Using this expression system, some of the recombinant enzyme is secreted and found to have a correct amino terminus. That remaining in the cytoplasm has part of the signal peptide attached to the amino terminus. The recombinant schistosomal HGPRTase isolated from the periplasm of the transformed E. coli was purified and found to have kinetic and physical properties identical to those of the native enzyme.
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PMID:The hypoxanthine-guanine phosphoribosyltransferase of Schistosoma mansoni. Further characterization and gene expression in Escherichia coli. 219 39

From a panel of mouse monoclonal antibodies reactive with a repeating epitope of the schistosome circulating anodic antigen, an IgG1 monoclonal antibody was selected. This monoclonal antibody was applied in a sandwich enzyme-linked immunosorbent assay as capture antibody and as alkaline phosphatase labeled conjugate. This assay allowed a sensitive quantitation of circulating anodic antigen in serum samples of infected individuals, detecting less than 1 ng antigen/ml serum. In Schistosoma mansoni infected individuals from Zaire, the level of antigen in serum correlated with fecal egg output. The lower detection level of the immunoassay corresponded to a level of about 10 eggs/gm feces.
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PMID:Sensitive determination of circulating anodic antigen in Schistosoma mansoni infected individuals by an enzyme-linked immunosorbent assay using monoclonal antibodies. 249 98

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.
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PMID:Role of host antibody in the chemotherapeutic action of praziquantel against Schistosoma mansoni: identification of target antigens. 249 7

The previously shown antigenicity of Schistosoma mansoni (JL venezuelan strain) alkaline phosphatase (Mg2+, pH 9.5) allowed its use in an immunodiagnosis assay, that consisted in the immunoadsorption of the enzymatic activity. Protein-A coated polyvinyl plates were used as solid phase to capture IgG from sera of infected human patients. After buffered saline washings, the plates were incubated with an enzyme-rich fraction (a n-butanol aqueous extract of adult worm obtained from pairs). Immunoadsorbed alkaline phosphatase (AP) was revealed by adding rho-nitrophenyl phosphate. Anti-AP antibodies were detected in 93% of coproparasitologically proven S. mansoni-infected venezuelan patients but not in parasite-free control sera and sera from patients infected with parasitosis other than schistosomiasis. The APIA did not correlate with cure but the anti-AP antibody response was progressively reduced after treatment. The use of an AP substrate amplifying system allowed an improvement of the assay sensitivity without loss of specificity. The data suggest that the APIA could be used as a marker of infection by S. mansoni.
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PMID:Immunodiagnosis of Schistosomiasis mansoni with APIA (alkaline phosphatase immunoassay). 251 56

We report the development of a time-resolved immunofluorometric assay (TR-IFMA) for the quantitative determination of the schistosome circulating anodic antigen (CAA). A mouse monoclonal antibody (line 120-1B10-A), recognizing a repetitive epitope on CAA, was used as both antigen-capture antibody and as Europium-labelled antigen-detecting antibody. The lower detection limit of the assay was 20 pg of trichloroacetic acid soluble fraction of adult worm antigen (AWA-TCA) per ml, with a nearly linear measuring range from 20 pg to 130 ng AWA-TCA per ml. The TR-IFMA was compared with an enzyme-linked immunosorbent assay (ELISA), in which the same monoclonal antibody was used as antigen-capture antibody and as alkaline phosphatase-conjugated antibody. The lower detection limit of the TR-IFMA was tenfold lower than that of the ELISA, while the linear range of the TR-IFMA exceeded that of the ELISA one hundred-fold. Serum samples of 80 Burundese individuals infected with Schistosoma mansoni (egg counts ranging from 4 to 2583 eggs per gram of faeces) were tested in both assays. Antigen concentrations in serum of individuals infected with S. mansoni ranged from 0-500 ng AWA-TCA per ml. The correlation between antigen levels measured by TR-IFMA and ELISA was good: Spearman's p = 0.92. Whereas in the ELISA the samples had to be titrated, the wide linear range of the TR-IFMA allowed the assay to be performed at a single serum dilution, at which an exact estimation of the antigen concentration was possible.
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PMID:Time-resolved immunofluorometric assay (TR-IFMA) for the detection of the schistosome circulating anodic antigen. 251 32

Localization of the SM alpha family of repeated DNA and the rDNA repeat on the chromosomes of Schistosoma mansoni by in situ hybridization is presented. Biotinylated DNA was hybridized to target chromosomes and hybridization was detected using either alkaline phosphatase-labeled avidin or fluorescein-labeled avidin and biotinylated anti-avidin antibody. Hybridization detection using a fluorescein conjugate was more specific and sensitive with less background noise than detection with alkaline phosphatase conjugates. SM alpha hybridizing sequences were found dispersed throughout the genome, hybridizing to the sex chromosomes and autosomes. The SM alpha probe showed specific hybridization to the euchromatic gap region within the large heterochromatic block of the short arm of the W chromosome. This specific hybridization coupled with the lack of chiasma formation in this region of the ZW bivalent (presumably due to the heterochromatinization of this region) may explain the pattern of sex-specific hybridization reported for the SM alpha family. The rDNA repeat was localized to the secondary constriction of the short arm of chromosome 3. Specifically, the rDNA probe hybridized with the stalk of the secondary constriction and with parts of both side regions, the satellite and the short arm proper.
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PMID:Schistosoma mansoni: chromosomal localization of DNA repeat elements by in situ hybridization using biotinylated DNA probes. 275 21

In fresh water snails, amebocytes are the principal cells that react to parasitic infection. Ultrastructurally, amebocytes resemble mammalian macrophages. To clarify the relationship between amebocytes and macrophages, we compared the histochemical staining for seven enzymes in Biomphalaria glabrata snail amebocytes, both in the amebocyte-producing organ (APO) and in the encapsulation reaction formed around parasite sporocysts with the staining in macrophages from the lymph nodes of patients with sarcoid or tuberculosis. Snails were infected with Echinostoma paraensei and Schistosoma mansoni miracidia. APOs and ventricular tissue with encapsulated parasites were fixed and embedded in glycol methacrylate monomer. Hardened blocks were sectioned at 2 micron and stained for alkaline phosphatase, acid phosphatase, alpha-naphthyl acetate esterase (ANAE), ATPase, peroxidase, 5'nucleotidase, and chloroacetate esterase. The amebocyte-producing organ contained cells that were positive for acid phosphatase, ANAE, and ATPase. Amebocytes in the capsules formed around echinostome sporocysts showed stronger staining for the same three enzymes. Capsules did not form around schistosome sporocysts, but the connective tissue around them contained numerous amebocytes that were also positive for these three enzymes. The amebocyte enzyme histochemistry resembled that in human granuloma macrophages, but differed from that in neutrophils. The increased expression of enzymes in amebocytes involved in the encapsulation reaction as compared to those in the APO was reminiscent of the alterations observed when human monocytes convert to tissue macrophages. These studies support the hypothesis that the amebocyte is an "invertebrate macrophage."
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PMID:Enzyme histochemical comparison of biomphalaria glabrata amebocytes with human granuloma macrophages. 298 47

Two enzymes, alkaline phosphatase and acetylcholinesterase (AChE), have been shown previously to be components of the surface of the trematode parasite Schistosoma mansoni. In this study we report that both these enzymes and other serine hydrolases are susceptible to release from the S. mansoni tegumental membrane by a phosphatidylinositol-specific phospholipase C (PIPLC) of bacterial origin. These data suggest that AChE and alkaline phosphatase of S. mansoni, as in higher organisms, are anchored to the membrane via covalently attached phosphatidylinositol. The release of AChE from the vesicular fraction of the parasite with PIPLC occurs in a concentration-dependent manner. Sucrose gradient centrifugation of the PIPLC-released AChE showed a single 8.3 S molecular form, similar to that observed for AChE solubilized by Triton X-100. PIPLC removed large amounts of AChE from the surface of intact schistosomula in culture, with no impairment of the viability of the parasite. In this case, an increase in the overall levels of AChE in the intact parasite was observed after addition of PIPLC.
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PMID:Acetylcholinesterase in Schistosoma mansoni is anchored to the membrane via covalently attached phosphatidylinositol. 313 66


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