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Jurgen Markl - One of the best experts on this subject based on the ideXlab platform.
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haliotis tuberculata hemocyanin hth analysis of oligomeric stability of hth1 and hth2 and comparison with Keyhole Limpet hemocyanin klh1 and klh2
Micron, 2000Co-Authors: J. R. Harris, Wolfgang Gebauer, Dirk Scheffler, R Lehnert, Jurgen MarklAbstract:Abstract The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from Keyhole Limpet hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole Limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31–41; Harris, J.R., Gebauer, W., Sohngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole Limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43–56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole Limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329–339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8 MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100 mM concentrations of calcium and magnesium ions in Tris–HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100 mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100 mM calcium and magnesium ions yielded an almost 100% conversion into didecamers, whereas the HtH2 subunit produced a mixture containing large numbers of short multidecamers and relatively few didecamers, together with a considerable number of smaller diameter helical/tubular polymers. The association properties of the HtH1 and HtH2 decamers, and the subunit reassociation, firmly indicate the integrity and structural competency of the protein under the experimental conditions used. Data on the association of KLH2 decamers is also presented, which together with previously published data on the association KLH1 decamers and the reassociation of KLH1 and KLH2 subunits, enables a detailed comparison of the two hemocyanin isoforms from both molluscan species to be made. Biochemical manipulation of the oligomer states and the subunit reassociation of molluscan hemocyanins can usefully be assessed by the study of negatively stained TEM specimens.
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Keyhole Limpet hemocyanin molecular structure of a potent marine immunoactivator a review
European Urology, 2000Co-Authors: Robin J Harris, Jurgen MarklAbstract:Objectives: In this short review we present a survey of the available biochemical and electron microscopic data on Keyhole Limpet hemocyanin (KLH). Results: The b
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Keyhole Limpet hemocyanin type 2 (KLH2): detection and immunolocalization of a labile functional unit h.
Journal of structural biology, 1999Co-Authors: Wolfgang Gebauer, J. Robin Harris, Gaby Geisthardt, Jurgen MarklAbstract:Keyhole Limpet hemocyanin (KLH) is a mixture of two hemocyanin isoforms, termed KLH1 and KLH2. Within KLH1 eight oxygen-binding functional units (FUs), 1-a to 1-h, have been identified, in contrast to KLH2, which was previously thought to be organized in seven FUs (2-a to 2-g). By limited proteolysis of KLH2 subunits, isolation of the polypeptide fragments, and N-terminal sequencing, we have now identified an eighth FU of type h, with a molecular mass of 43 kDa. This is unusually small for a FU h from a gastropodan hemocyanin. It is also shown that KLH2 didecamers can be split into a stable and homogeneous population of decamers by dialysis against 50 mM Tris/HCl, pH 7.5, in the absence of divalent cations. Electron microscopic immunolocalization using a specific monoclonal antibody reveals that FU KLH2-h is located at the collar of the decamer.
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Keyhole Limpet hemocyanin (KLH): a biomedical review.
Micron (Oxford England : 1993), 1999Co-Authors: J. R. Harris, Jurgen MarklAbstract:In this review we present a broad survey of fundamental scientific and medically applied studies on Keyhole Limpet hemocyanin (KLH). Commencing with the biochemistry of KLH, information on the biosynthesis and biological role of this copper-containing respiratory protein in the marine gastropod Megathura crenulata is provided. The established methods for the purification of the two isoforms of KLH (KLH1 and KLH2) are then covered, followed by detailed accounts of the molecular mass determination, functional unit (FU) structure, carbohydrate content, immunological analysis and recent aspects of the molecular genetics of KLH. The transmission electron microscope (TEM) has contributed significantly to the understanding of KLH structure, primarily from negatively stained images. We give a brief account of TEM studies on the native KLH oligomers, the experimental manipulation of the oligomeric states, together with immunolabelling data and studies on subunit reassociation. The field of cellular immunology has provided much relevant biomedical information on KLH and has led to the expansion of use of KLH in experimental immunology and clinically as an immunotherapeutic agent; this area is presented in some detail. The major clinical use of KLH is specifically for the treatment of bladder carcinoma, with efficacy probably due to a cross-reacting carbohydrate epitope. KLH also has considerable possibilities for the treatment of other carcinomas, in particular the epithelially derived adenocarciomas, when used as a carrier for carcinoma ganglioside and mucin-like epitopes. The widespread use of KLH as a hapten carrier and generalised vaccine component represent other major on-going aspects of KLH research, together with its use for the diagnosis of Schistosomiasis, drug assay and the treatment of drug addiction. Immune competence testing, assessment of stress and the understanding of inflammatory conditions are other areas where KLH is also making a useful contribution to medical research.
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structure of Keyhole Limpet hemocyanin type 1 klh1 at 15 a resolution by electron cryomicroscopy and angular reconstitution
Journal of Molecular Biology, 1997Co-Authors: Jurgen Markl, Robin J Harris, Elena V Orlova, Prakash Dube, Erich Beckman, F Zemlin, Marin Van HeelAbstract:Abstract A three-dimensional reconstruction of Keyhole Limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to ∼45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the “functional units” within the dimeric “morphological units” of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole Limpet hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components.
Rudolf Geyer - One of the best experts on this subject based on the ideXlab platform.
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Identification and characterization of Keyhole Limpet hemocyanin N-glycans mediating cross-reactivity with Schistosoma mansoni.
The Journal of biological chemistry, 2005Co-Authors: Hildegard Geyer, Manfred Wuhrer, Anja Resemann, Rudolf GeyerAbstract:Abstract Keyhole Limpet hemocyanin (KLH) of the mollusc Megathura crenulata is known to serologically cross-react with Schistosoma mansoni glycoconjugates in a carbohydrate-dependent manner. To elucidate the structural basis for this cross-reactivity, KLH glycans were released from tryptic glycopeptides and fluorescently labeled. Cross-reacting glycans were identified using a polyclonal antiserum reacting with soluble S. mansoni egg antigens, isolated by a three-dimensional fractionation scheme and analyzed by different mass spectrometric techniques as well as linkage analysis and exoglycosidase treatment. The results revealed that cross-reacting species comprise ∼4.5% of released glycans. They all represent novel types of N-glycans with a Fuc(α1-3)GalNAc(β1-4)[Fuc(α1-3)]GlcNAc motif, which is known to occur also in schistosomal glycoconjugates. The tetrasaccharide unit is attached to the 3-linked antenna of a trimannosyl core, which can be further decorated by galactosyl residues, a xylose residue in 2-position of the central mannose and/or a fucose at the innermost N-acetylglucosamine. This study provides for the first time detailed structural data on the KLH carbohydrate entities responsible for cross-reactivity with glycoconjugates from S. mansoni.
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Characterization of Keyhole Limpet hemocyanin (KLH) glycans sharing a carbohydrate epitope with Schistosoma mansoni glycoconjugates.
Micron, 2003Co-Authors: Hildegard Geyer, Manfred Wuhrer, Tomofumi Kurokawa, Rudolf GeyerAbstract:Keyhole Limpet hemocyanin (KLH) is known to share carbohydrate epitopes with Schistosoma mansoni. In order to define the structural basis for the observed serological cross-reactivity, KLH glycans were released either by enzyme treatment or by hydrazinolysis and probed with a rabbit hyperimmune serum directed against S. mansoni egg antigen. Both major, non-reacting oligosaccharide species as well as the minor compounds recognized were isolated by two-dimensional high performance liquid chromatography and in part by lectin affinity chromatography, and characterized by mass spectrometry. The results revealed that KLH carries predominantly high mannose-type glycans as well as short sugar chains. As a characteristic feature, a number of the latter glycans contained a Gal(beta1-6)Man-unit, which has not yet been found in glycoprotein-N-glycans. Oligosaccharides cross-reacting with schistosomal glycans comprised a terminal Fuc(alpha1-3)GalNAc-motif, which appears to represent the main carbohydrate epitope mediating cross-reactivity of KLH with glycoconjugates from S. mansoni.
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A fucose-containing epitope is shared by Keyhole Limpet haemocyanin and Schistosoma mansoni glycosphingolipids.
Molecular and biochemical parasitology, 2000Co-Authors: Manfred Wuhrer, Michael J. Doenhoff, Roger D. Dennis, Rudolf GeyerAbstract:The glycolipids of Schistosoma mansoni adult worms, cercariae and eggs are recognised by schistosome infection serum and the monoclonal antibody M2D3H. The haemocyanin of the Keyhole Limpet, Megathura crenulata, is known to be immunoreactive to schistosomal infection sera and is, therefore, under investigation for the diagnosis of and vaccination against schistosomiasis. By dot-blot, inhibition-ELISA and inhibition-HPTLC immunostaining we have demonstrated that the M2D3H epitope is shared by both S. mansoni glycolipids and Keyhole Limpet haemocyanin (KLH). Analogously to the established epitopic importance of fucose to the immunorecognition of S. mansoni glycolipids, we have similarly defined the significance of the fucose residue(s) for the immunoreactivity between KLH and schistosomal infection serum and the monoclonal antibody M2D3H. Fucose was specifically removed from KLH by partial hydrolysis, monitored by ultrafiltration and carbohydrate component analysis. On removal of the fucose residue(s) the serological and immunological reactivity of KLH was greatly diminished, which implied that the fucose-containing M2D3H antigenic determinant was common to both S. mansoni glycolipids and KLH.
Elena V Orlova - One of the best experts on this subject based on the ideXlab platform.
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structure of Keyhole Limpet hemocyanin type 1 klh1 at 15 a resolution by electron cryomicroscopy and angular reconstitution
Journal of Molecular Biology, 1997Co-Authors: Jurgen Markl, Robin J Harris, Elena V Orlova, Prakash Dube, Erich Beckman, F Zemlin, Marin Van HeelAbstract:Abstract A three-dimensional reconstruction of Keyhole Limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to ∼45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the “functional units” within the dimeric “morphological units” of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole Limpet hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components.
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Three-Dimensional Structure of Keyhole Limpet Hemocyanin by Cryoelectron Microscopy and Angular Reconstitution
Journal of Structural Biology, 1995Co-Authors: Prakash Dube, J. Robin Harris, Elena V Orlova, F Zemlin, Marin Van Heel, Jurgen MarklAbstract:Abstract A three-dimensional (3-D) reconstruction of the Keyhole Limpet hemocyanin (KLH) didecamer has been obtained by single particle image analysis of cryoelectron micrographs. We exploit the random orientations of the individual particles within the vitreous ice matrix to reconstruct this oligomer in three dimensions without tilting the specimen holder and using only a single low-dose exposure of each specimen area. The symmetrical didecamer appears as a largely hollow cylinder of 370 ± 5 A × 400 ± 5 A and is apparently created by the face-to-face dimerization of two asymmetrical decamers. The molecule was reconstructed assuming D5 symmetry, which implies that a fivefold axis coincides with the cylinder axis and that five dyad axes lie within the central plane between the two decamers. The terminal domains of the subunit pair are thought to be folded into the central cavity at the "collar" region of the molecule, thus generating pronounced globular masses at each end of the cylinder. At the current level of resolution of about 45 A it is not yet possible to distinguish structurally between the two types of KLH didecamer, recently termed KLH1 and KLH2.
Robin J Harris - One of the best experts on this subject based on the ideXlab platform.
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Keyhole Limpet hemocyanin molecular structure of a potent marine immunoactivator a review
European Urology, 2000Co-Authors: Robin J Harris, Jurgen MarklAbstract:Objectives: In this short review we present a survey of the available biochemical and electron microscopic data on Keyhole Limpet hemocyanin (KLH). Results: The b
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structure of Keyhole Limpet hemocyanin type 1 klh1 at 15 a resolution by electron cryomicroscopy and angular reconstitution
Journal of Molecular Biology, 1997Co-Authors: Jurgen Markl, Robin J Harris, Elena V Orlova, Prakash Dube, Erich Beckman, F Zemlin, Marin Van HeelAbstract:Abstract A three-dimensional reconstruction of Keyhole Limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to ∼45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the “functional units” within the dimeric “morphological units” of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole Limpet hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components.
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Keyhole Limpet haemocyanin klh purification of intact klh1 through selective dissociation of klh2
Micron, 1995Co-Authors: Robin J Harris, Wolfgang Gebauer, Sabine M Sohngen, Jurgen MarklAbstract:Abstract Keyhole Limpet haemocyanin (KLH) from almost all newly captive animals contains a mixture of KLH1 and KLH2. We show that the dissociation of KLH2 can be produced during EM specimen preparation by the negative staining-carbon film (NS-CF) procedure and in solution by ammonium molybdate-PEG solutions at slightly acidic pHs. The KLH2 multidecamers split apart in the pH range 7.5-6.5 and in the pH range 6.5-6.0 the individual decamers break open and start to dissociate. At pH 5.9 the dissociation of KLH2 yields predominantly a mixture of single subunits and what appear to be subunit dimers. Over the pH range 7.0-5.7 the KLH1 didecamer remains stable. Separation of intact KLH1, in the form of didecamers and small clusters of PEG at pH 5.9 (pH 6.2 at 4°C). Assessment of the achieved separation of KLH1 and KLH2 was monitored by gel electrophoresis under native conditions and by electron microscopy of negatively stained specimens. Integrity of the multi-domain KLH1 and KLH2 subunits was assessed by SDS-PAGE. The significance of this separation for the overall understanding of the two types of KLH is that it provides for the first time purified KLH1 didecamers. Reassociation of the pH 5.9 dissociated KLH2 was achieved by prolonged dialysis against calcium-supplemented stabilizing buffer at pH 7.2 and resulted in multidecamers and slightly smaller diameter helical tubes.
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electron microscopy and biochemical characterization of a 350 kda annular hemolymph protein from the Keyhole Limpet megathura crenulata
FEBS Journal, 1994Co-Authors: Robin J Harris, Jurgen MarklAbstract:The isolation and biochemical characterization of an annular non-hemocyanin hemolymph protein from a marine gastropod, the Californian giant Keyhole Limpet (Megathura crenulata) is presented. By analytical ultracentrifugation, the protein has a sedimentation coefficient of 12S and molecular mass of approximately 350 kDa. The subunit mass, obtained by SDS/PAGE in the presence of -SH reagent and 8 M urea, is approximately 35 kDa, thereby indicating the presence of 10 subunits in the native molecule. By negative staining, the protein is revealed in one predominant image projection as a pentagonal approximately 8 nm ring-like structure with an approximately 2-nm stain-filled centre and, in another image projection, as a dimeric rectangular structure, approximately 8 nm × 14 nm. In deep stain, each of the components of the side-on dimer also shows an indication of stain penetration within a central channel. Unlike the hemolymph protein limulin/C-reactive protein from the horseshoe crab Limulus polyphemus, this Megathura protein does not possess any potential for the aggregation of phosphatidylcholine liposomes although it has an affinity for this phospholipid and causes bilayer destruction. It now appears likely that the previously reported lipid-bilayer ionic-conductance species in Keyhole Limpet hemolymph, attributed to hemocyanin, could indeed by due to the presence of this annular protein.
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immunoelectron microscopy of hemocyanin from the Keyhole Limpet megathura crenulata a parallel subunit model
Journal of Structural Biology, 1993Co-Authors: Robin J Harris, Wolfgang Gebauer, Jrgen MarklAbstract:Abstract Immunoelectron microscopy has been performed using negatively stained immune complexes of Keyhole Limpet hemocyanin (KLH) subunit 2 di- and multidecamers with domain-specific monoclonal antibodies. One antibody (KLH2 a macr 1) links the hemocyanin molecules in a side-to-side pattern, whereas the other antibody (KLH2 fg macr 1) links the molecules end-to-end. From existing knowledge of the domain sequence of KLH subunit 2, these data provide support for a parallel arrangement of subunits within each decamer. Ten N-terminal a macr: domains are then present at the noncollar region of each decamer with 10 C-terminal g macr domains at the collar region. The immunonegative staining data and the parallel KLH subunit model are discussed in relation to other models of molluscan hemocyanins from different species.
Michael J. Doenhoff - One of the best experts on this subject based on the ideXlab platform.
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A fucose-containing epitope is shared by Keyhole Limpet haemocyanin and Schistosoma mansoni glycosphingolipids.
Molecular and biochemical parasitology, 2000Co-Authors: Manfred Wuhrer, Michael J. Doenhoff, Roger D. Dennis, Rudolf GeyerAbstract:The glycolipids of Schistosoma mansoni adult worms, cercariae and eggs are recognised by schistosome infection serum and the monoclonal antibody M2D3H. The haemocyanin of the Keyhole Limpet, Megathura crenulata, is known to be immunoreactive to schistosomal infection sera and is, therefore, under investigation for the diagnosis of and vaccination against schistosomiasis. By dot-blot, inhibition-ELISA and inhibition-HPTLC immunostaining we have demonstrated that the M2D3H epitope is shared by both S. mansoni glycolipids and Keyhole Limpet haemocyanin (KLH). Analogously to the established epitopic importance of fucose to the immunorecognition of S. mansoni glycolipids, we have similarly defined the significance of the fucose residue(s) for the immunoreactivity between KLH and schistosomal infection serum and the monoclonal antibody M2D3H. Fucose was specifically removed from KLH by partial hydrolysis, monitored by ultrafiltration and carbohydrate component analysis. On removal of the fucose residue(s) the serological and immunological reactivity of KLH was greatly diminished, which implied that the fucose-containing M2D3H antigenic determinant was common to both S. mansoni glycolipids and KLH.
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Periodate-sensitive immunological cross-reactivity between Keyhole Limpet haemocyanin (KLH) and serodiagnostic Schistosoma mansoni egg antigens.
Parasitology, 1999Co-Authors: Joanne V. Hamilton, Peter L. Chiodini, Padraic G. Fallon, Michael J. DoenhoffAbstract: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.
