ATP diphosphohydrolase from Schistosoma mansoni egg: characterization and immunocytochemical localization of a new antigen

Parasitology ◽  
2004 ◽  
Vol 129 (1) ◽  
pp. 51-57 ◽  
Author(s):  
P. FARIA-PINTO ◽  
M. N. L. MEIRELLES ◽  
H. L. LENZI ◽  
E. M. MOTA ◽  
M. L. O. PENIDO ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cross Reactivity ◽  
Outer Side ◽  
Western Blots ◽  
Atp Diphosphohydrolase ◽  
Antigenic Properties ◽  
Egg Shell ◽  
Net Charges ◽  
Egg Antigen

The fact that the Schistosoma mansoni egg has two ATP diphosphohydrolase (EC 3.6.1.5) isoforms with different net charges and an identical molecular weight of 63000, identified by non-denaturing polyacrylamide gel electrophoresis and immunological cross-reactivity with potato apyrase antibodies, is shown. In soluble egg antigen (SEA), only the isoform with the lower net negative charge was detected and seemed to be the predominant species in this preparation. By confocal fluorescence microscopy, using anti-potato apyrase antibodies, the S. mansoni egg ATP diphosphohydrolase was detected on the external surface of miracidium and in von Lichtenberg's envelope. Intense fluorescence was also seen in the outer side of the egg-shell, entrapped by the surface microspines, suggesting that a soluble isoform is secreted. ATP diphosphohydrolase antigenicity was tested using the vegetable protein as antigen. The purified potato apyrase was recognized in Western blots by antibodies present in sera from experimentally S. mansoni-infected mice. In addition, high levels of IgG anti-ATP diphosphohydrolase antibodies were detected by ELISA in the same sera. This work represents the first demonstration of antigenic properties of S. mansoni ATP diphosphohydrolase and immunological cross-reactivity between potato apyrase and sera from infected individuals.

scholarly journals Do intestinal parasites interfere with the seroepidemiologic surveillance of Schistosoma mansoni infection?

1996 ◽  
Vol 116 (3) ◽  
pp. 323-329 ◽  
Author(s):  
B. Alarcón De Noya ◽  
C. Colmenares ◽  
S. Losada ◽  
Z. Fermin ◽  
G. Masroua ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
False Positive ◽  
Total Population ◽  
Intestinal Parasites ◽  
Field Work ◽  
Cross Reactivity ◽  
Cross Reaction ◽  
Protein Fractions ◽  
Egg Antigen ◽  
Immunoenzymatic Assay

SUMMARYIn view of the known cross-reactivity of sera from patients with intestinal parasites to some Schistosoma mansoni antigens, field work was conducted in an area of Venezuela non-endemic for schistosomiasis using the routine immunoenzymatic assay (ELISA) with soluble egg antigen (SEA). False positive reactions represented 15·3% of the total population as determined by SEA–ELISA. SEA-immunoblotting of the false positive sera indicated that protein fractions of 91 and 80 kDa appear to be responsible for cross-reactivity. Sera from hookworm infected individuals produced a higher frequency and intensity of cross-reaction than other sera. SEA-fractions of 105, 54, 46, 42, 32, 25 and 15 kDa were the most specific.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

Purification, characterization, and localization of two ATP diphosphohydrolase isoforms in bovine heart

1997 ◽  
Vol 273 (2) ◽  
pp. H673-H681 ◽  
Author(s):  
A. R. Beaudoin ◽  
J. Sevigny ◽  
G. Grondin ◽  
S. Daoud ◽  
F. P. Levesque
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Coronary Vessels ◽  
Protective Role ◽  
Cross Reactivity ◽  
Heat Inactivation ◽  
Maximal Velocity ◽  
Migration Patterns ◽  
Atp Diphosphohydrolase ◽  
Bovine Heart ◽  
Triton X

Two ATP diphosphohydrolase (ATPDase) isoforms have been purified from the bovine heart ventricle. The purification procedure includes the following steps: differential centrifugation, sucrose cushion centrifugation, solubilization with Triton X-100, DEAE agarose ion exchange, and Affi-Gel blue-Sepharose and concanavalin A (con A)-Sepharose chromatographies. The purified enzyme has an optimum pH of catalysis of 7.5 and requires Ca2+ or Mg2+. The apparent Michaelis constant of the enzyme, with ADP as the substrate, is 29 microM, and the apparent maximal velocity is 1.6 mumol.min-1.mg protein-1. Substrate specificity, heat-inactivation curves, and copurification of adenosinetriphosphatase (ATPase) and adenosinediphosphatase (ADPase) activities confirmed the identity of the purified enzyme as an ATPDase. In addition, polyacrylamide gel electrophoresis, under nondenaturing conditions, showed identical migration patterns for the protein involved in ATPase and ADPase activities. Western blot analysis, with an antibody that specifically recognizes the NH2-terminal sequence of pig pancreas ATPDase and specifically reacts with bovine and human ATPDases, showed cross-reactivity with the purified ATPDase isoforms from the bovine heart. Immunocytochemical localization in the ventricle produced strong reactions with the plasma membrane of Purkinje fiber cells and the majority of myocardial cells. Immunoreactivity was variable, producing a mosaic-like aspect. As expected, smooth muscle cells and endothelial cells of coronary vessels were highly reactive. This ectoenzyme could play a protective role against the potentially deleterious effects of extracellular ATP. In tandem with 5'-nucleotidase, it produces adenosine, a powerful vasodilator, especially in hypoxic or ischemic conditions that favor the release of ATP.

Schistosoma mansoni: anomalous immunogenic properties of a 27 kDa larval serine protease associated with protective immunity

Parasitology ◽  
1997 ◽  
Vol 115 (3) ◽  
pp. 237-247 ◽  
Author(s):  
H. Y. DARANI ◽  
R. H. C. CURTIS ◽  
C. McNEICE ◽  
H. P. PRICE ◽  
J. R. SAYERS ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Serine Protease ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rabbit Antiserum ◽  
Molecular Size ◽  
Major Constituent ◽  
Partial Purification ◽  
Western Blots ◽  
Sds Page

A cationic Schistosoma mansoni cercarial antigen was shown to be a serine protease as it was capable of hydrolysing N-acetyl-dl-phenylalanine β-naphthyl ester (NAPBNE) after precipitation by immunoelectrophoresis, and this reaction was modulated by the serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP). The antigen in the immunoprecipitin arcs could also be radio-isotope labelled with tritiated DFP. The peptidolytic enzyme identified in immunoelectrophoresis with polyspecific sera and radio-isotope labelled with tritiated DFP had a relative molecular size of approximately 27 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), and evidence obtained after partial purification, SDS–PAGE and immunoblotting supported this size estimate for the enzyme. A rabbit antiserum raised against the peptidolytic antigen reacted against a doublet of antigens at 27/28 kDa in immunoelectrophoresis arcs and against an antigen of 60 kDa in Western immunoblots of crude cercarial homogenate. However, the latter serum precipitated the cationic antigen in immunoelectrophoresed cercarial homogenates only after pre-incubation of the homogenates with PMSF. Fractions containing the partially purified protease also degraded radio-isotope labelled human IgG. The reactivity of a range of polyspecific and monospecific rabbit antisera in Western blots with larval extracts indicated that antibody responses against the 27/28 kDa doublet may be modulated. When immunized with material which contained the 27 kDa enzyme as a major constituent, and which was secreted by S. mansoni cercariae during transformation, only 5 of 16 mice produced antibody to this antigen that was detectable in Western blots. The 5 antibody ‘responder’ mice were significantly (P<0·001) protected against challenge with a percutaneous infection of S. mansoni cercariae compared with a group of mice also immunized with CTF, but which had not produced antibodies against the 27/28 kDa doublet. The results indicate that the 27 kDa serine protease of S. mansoni larvae is a target that is sensitive to immunological attack.

ALeishmania(L.)amazonensisATP diphosphohydrolase isoform and potato apyrase share epitopes: antigenicity and correlation with disease progression

Parasitology ◽  
2007 ◽  
Vol 135 (3) ◽  
pp. 327-335 ◽  
Author(s):  
E. S. COIMBRA ◽  
S. C. GONÇALVES-DA-COSTA ◽  
B. L. S. COSTA ◽  
N. L. L. GIAROLA ◽  
F. A. REZENDE-SOARES ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Serum Levels ◽  
Igg Antibody ◽  
Western Blots ◽  
Atp Diphosphohydrolase ◽  
Amastigote Stage ◽  
Sds Page ◽  
Elisa Technique ◽  
Shared Epitopes

SUMMARYALeishmania(Leishmania)amazonensisATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58–63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of aL. (L.)amazonensisATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.

Schistosoma mansoni: Immunodiagnosis Is Improved by Sodium Metaperiodate Which Reduces Cross-reactivity Due to Glycosylated Epitopes of Soluble Egg Antigen

2000 ◽  
Vol 95 (2) ◽  
pp. 106-112 ◽  
Author(s):  
B. Alarcón de Noya ◽  
C. Colmenares ◽  
H. Lanz ◽  
M.A. Caracciolo ◽  
S. Losada ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Cross Reactivity ◽  
Sodium Metaperiodate ◽  
Egg Antigen

Periodate-sensitive immunological cross-reactivity between keyhole limpet haemocyanin (KLH) and serodiagnostic Schistosoma mansoni egg antigens

Parasitology ◽  
1999 ◽  
Vol 118 (1) ◽  
pp. 83-89 ◽  
Author(s):  
J. V. HAMILTON ◽  
P. L. CHIODINI ◽  
P. G. FALLON ◽  
M. J. DOENHOFF
Keyword(s):  
Schistosoma Mansoni ◽  
Sodium Periodate ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Immunoblotting ◽  
Diagnostic Efficacy ◽  
Keyhole Limpet Haemocyanin ◽  
Human Schistosomiasis ◽  
High Degree ◽  
Egg Antigen

Both CEF6, a cation-exchange fraction of soluble Schistosoma mansoni egg antigens (SEA), composed of the 2 antigens, alpha-1 and omega-1, and haemocyanin from the keyhole limpet, Megathura crenulata, have shown potential for immunodiagnosis of human schistosomiasis. Possible cross-reactivity between antigens in SEA and keyhole limpet haemocyanin (KLH) was explored by Western immunoblotting and enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with KLH, SEA, CEF6, alpha-1, omega-1, or egg antigen k5. Both immunoassays revealed a high degree of serological cross-reactivity between the schistosome egg antigens and KLH, much of it due to sodium periodate- sensitive epitopes. Cross-reactivity with schistosome antigens with proven diagnostic efficacy may thus, in part, explain the usefulness of KLH for the diagnosis of human schistosomiasis mansoni.

Mapping of segmental antigenic determinants on structurally related variant surface glycoproteins of Trypanosoma brucei

1988 ◽  
Vol 66 (11) ◽  
pp. 1231-1237 ◽  
Author(s):  
Vern B. Carruthers ◽  
Michael W. Clarke
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Trypanosoma Brucei ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cross Reactivity ◽  
Antigenic Determinants ◽  
Western Blots ◽  
Variant Surface Glycoproteins ◽  
Surface Glycoproteins

To study common and variant specific antigenic determinants on variant surface glycoproteins from Trypanosoma brucei, we have selected four serologically cross-reacting variant populations. Monoclonal antibodies were raised against the purified variant surface glycoproteins from each variant trypanosome population. Six monoclonal antibodies bind to segmental epitopes and one binds to a topographically assembled epitope. Amino acid compositions of these variant surface glycoproteins reveal striking conservation of certain residues including cysteine and charged amino acids. We also find that all seven monoclonal antibodies used in this study bind to protein determinants not exposed on the surface of the living trypanosome. Only one monoclonal antibody exhibits homologous specificity, while the remainder display cross-reactivity for three or all four variant surface glycoproteins. In addition, polyacrylamide gel electrophoresis peptide mapping and Western blots probed with each monoclonal antibody reveal significant peptide homologies. Furthermore, two pairs of monoclonal antibodies recognize two epitopes that are possibly immunodominant. The significance of these findings is discussed in terms of the structural similarities and differences among variant surface glycoproteins.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

Immunohistochemical and ultrastructural localization of Schistosoma mansoni soluble egg antigens processed by the infected host

Parasitology ◽  
1996 ◽  
Vol 112 (6) ◽  
pp. 537-543 ◽  
Author(s):  
J. J. P. M. Bogers ◽  
H. A. M. Nibbeling ◽  
A. M. Deelder ◽  
E. A. E. Van Marck
Keyword(s):  
Schistosoma Mansoni ◽  
Indirect Evidence ◽  
Ultrastructural Localization ◽  
Carbohydrate Epitopes ◽  
Eosinophilic Granulocytes ◽  
Antigenic Material ◽  
Egg Shells ◽  
Secondary Lysosomes ◽  
Antigen Antibody ◽  
Egg Antigen

SUMMARYThe detection of egg-derived antigens in the serum and urine of Schistosoma mansoni-infected individuals and experimental animals would provide an alternative method to assess the tissue egg burden. The detected levels are, however, not only a function of the amounts of antigen produced, but also of the processing or clearance by the host. In the present study the immunolocalization pattern of antigens using 2 recently described monoclonal antibodies to repetitive carbohydrate epitopes of S. mansoni soluble egg antigen (114–5B1–A and 114–4D12–A) in various organs of the host was investigated. In the liver strong immunoreactivity could be detected around the entrapped eggs and in egg-shells, as well as in Kupffer cells accumulating both antigen and schistosomal pigment. In the spleen, immunohistochemistry revealed antigen in the plasma as well as in secondary lysosomes of macrophages. Strong labelling was found in the vesicles of the eosinophilic granulocytes: indirect evidence perhaps for the presence of antigen–antibody complexes. In conclusion, the secreted egg antigens were sequestered in the reticulo-endothelial macrophages of the liver and the spleen as already partly described for worm-derived antigens. The presence of large quantities of antigenic material in the spleen could suggest an important role of this organ in the clearance of antigen and might even provide an additional explanation for the hepatosplenomegaly mainly present in S. mansoni-infected children.

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