The preparation and partial characterization of antigenic fractions obtained from the mycelial walls of several Aspergillus species

Extracts with immunological activity were prepared from Aspergillus fumigatus, A. flavus, A. terreus, A. niger and A. nidulans. In each case crude mycelial wall was extracted with an aqueous solution of Triton X-100 giving detergent-soluble material. Further fractionation was achieved by removing the detergent from this solution; the resultant precipitate was removed by centrifugation, and the aqueous supernatant was used as a source of soluble antigens. The sensitivity of these preparations was compared with that of water-soluble antigenic material, prepared from whole macerated mycelium, by double diffusion and counterimmunoelectrophoresis using homologous antisera and sera from patients suffering from aspergilloma and allergic bronchopulmonary aspergillosis. The selectivity of these antigenic preparations was monitored with heterologous antisera raised in rabbits. Batch variability was analysed for one strain of A. fumigatus using chemical and immunological methods. The nature of the antigenic sites involved in these reactions was investigated by studying the susceptibility of the preparations to proteolytic hydrolysis, periodate oxidation and concanavalin A treatment. The total protein and carbohydrate content of each fraction was determined and the constituent sugars analysed in an attempt to correlate chemical composition with antigenic activity.     

Immunochemical studies of the surface and cytoplasmic membranes of Entamoeba invadens (Rodhain 1934).

The antigenic properties of cell fractions prepared from axenically grown Entamoeba invadens were investigated using various immunoelectrophoretic methods. All membrane fractions displayed varying degrees of antigenicity, the most predominant being a cytoplasmic light-vesicle fraction. The surface membrane showed the least diversified range of antigenic components and was also the least immunogenic fraction as judged by the reactivity of the antisera produced. Using tandem-crossed immunoelectrophoresis the antigenic profiles of the membrane fractions were compared. No evidence was obtained for the presence of either lipid or carbohydrate in the antigenic moieties of any of the membrane fractions. Using a series of sequential solvent extractions it was concluded that both divalent metallochelate linkages and interiorly located hydrophobic associations were principally involved in binding the major antigenic components within the light-vesicle membrane. An enzymic function was assigned to certain of the membrane antigens, the immunoprecipitates obtained showing both acid phosphohydrolase and non-specific esterase acitivity toward a variety of substrates.     

The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum.

Antiserum raised against horseradish peroxidase (HRP) recognizes a neural specific carbohydrate antigen in Drosophila and other insects. The epitopic activity of the carbohydrate moiety of HRP recognized by anti-HRP antiserum was measured by a newly developed enzyme-linked immunosorbent assay, in which HRP glycopeptides conjugated with bovine serum albumin were coated onto the wells and then reacted with goat anti-HRP antiserum. HRP sugar moieties released by almond glycopeptidase A digestion of HRP pepsin digests were subjected to pyridylamination. Pyridylamino oligosaccharides were separated into seven fractions by reverse-phase high performance liquid chromatography. The major fraction, which comprised about 80% of the total sugars, reacted strongly with anti-HRP antiserum. The carbohydrate structure of this fraction was determined by sugar composition analysis and 600-MHz 1H NMR spectroscopy as follows: Man alpha 1----6(Man alpha 1----3)(Xyl beta 1----2)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc. Analyses of reactivity with anti-HRP antiserum of various oligosaccharide derivatives obtained from the major fraction by exoglycosidase digestion and partial acid hydrolysis indicated that alpha 1----6-linked mannose and alpha 1----3-linked fucose are predominantly involved in the epitopic structure.     

Isolation and preliminary characterization of a purified pig zona antigen (PPZA) from porcine oocytes

A protocol was developed for isolation of a purified pig zona antigen (PPZA) under nondissociating conditions. Heating of isolated zona-encased porcine oocytes at 73 degrees C for 20 min resulted in optimal solubilization of zona antigen activity (ZAA) as assessed by radioimmunoassay. Subsequent fractionation of solubilized proteins by ammonium sulfate precipitation, ultrafiltration, gel filtration, and ion exchange chromatography yielded PPZA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of PPZA yielded a major diffuse band at 58,000 Mr which stained for protein and carbohydrate and which possessed ZAA. Two-dimensional gel electrophoresis confirmed the identity of the 58,000 Mr glycoprotein and one of the three major charge heterogeneous glycoprotein families of the porcine zona pellucida. These experiments established that the principal macromolecular and antigenic component of PPZA is a 58,000 Mr glycoprotein originating from the zona pellucida. The nature of PPZA antigenic determinants recognized by a rabbit antiserum to PPZA was studied by radioimmunoassay. ZAA of PPZA was sensitive to the action of mercaptans and proteases, indicating a contribution of polypeptide chain to PPZA antigenicity. Loss of ZAA upon periodate oxidation of PPZA also implicated carbohydrate residues. PPZA retains antigenic determinants of the intact zona pellucida as assessed by reactivity with antiserum to intact porcine zonae. Likewise, rabbit antiserum to PPZA reacts avidly with intact porcine zonae. These results demonstrate that PPZA is a suitable target antigen for further studies on immunocontraception.     

Vasculitis and anaphylactoid shock in mice induced by the polysaccharide fraction secreted into culture supernatants by the fungus Candida metapsilosis

The biological effects of Candida metapsilosis water-soluble fraction (CMWS), prepared using a completely synthesized medium, were examined to determine whether CMWS induces vasculitis similar to that seen in Kawasaki disease, and anaphylactoid shock, in mice. It was found that intraperitoneal injection of CMWS induces coronary arteritis and i.v. injection induces acute anaphylactoid shock in mice, similar to Candida albicans water-soluble fraction (CAWS)-induced arteritis and anaphylactoid shock. The mannan structure of the polysaccharide fraction was then analyzed by performing antiserum reactivity tests and nuclear magnetic resonance spectroscopy. The mannan structure was investigated because the present authors have recently found that the mannan moiety within the polysaccharide fraction might be responsible for these pathogenic activities. The structural analysis showed that the mannan structure within CMWS expresses α-mannan residues, but not β-mannan. In addition, the mannan structure of CMWS is quite similar to that of CAWS. The present findings indicate that the polysaccharide fraction from C. metapsilosis, which is mainly composed of mannan, contributes to coronary arteritis and acute shock, and that the mannan structure could be responsible for this pathogenicity.     

Antibodies specific for mitotic human chromosomes

The induction of premature chromosome condensation in an interphase cell immediately following fusion with a mitotic cell suggests the presence of factors in the mitotic cell that are responsible for the transformation of an interphase nucleus into prematurely condensed chromosomes (PCC). Several lines of evidence suggest that these factors are proteins present in the cytoplasm of mitotic cells. The objective of this study was to raise antibodies to the factors responsible for PCC. Cytosol from synchronized mitotic HeLa cells was injected into rabbits in order to obtain antiserum. The IgG fraction from this antiserum reacted with 98% of mitotic HeLa cells when tested by indirect immunofluorescence. Most of the fluorescence was localized on the chromosomes. About 5% of the interphase nuclei also reacted with the antiserum, but 50% of these cells were in early G1. Antigenic reactivity was induced in the condensing interphase chromatin in 31% of the interphase nuclei found in mitotic-interphase fused cells. Rodent cells did not react with the antibody by indirect immunofluorescence. Mitotic HeLa cells were able to induce antigenic reactivity in 23 % of interphase Chinese hamster ovary (CHO) cell nuclei in fused binucleate cells, whereas the converse was not true of mitotic CHO cells. Enzyme digestion and incubation with denaturing agents suggested that antigenic reactivity depended on a DNA-non-histone protein complex.     

Molecular weight analysis of soluble antigens from Toxoplasma gondii

Ultrasonicated Toxoplasma gondii (RH strain) tachyzoites were fractionated into a water-soluble and a deoxycholate-soluble fraction. Polyclonal immune mouse serum was prepared by challenging chronically-infected mice with viable RH strain tachyzoites. The parasite fractions were labelled with 125I, and the radio-labelled antigens were precipitated by the immune mouse serum or a monoclonal anti-Toxoplasma antibody (FMC 20), that reacts only in the indirect hemagglutination antibody test. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the immunoprecipitates showed that the water-soluble fraction contained 10 antigenic polypeptides, and the deoxycholate-soluble fraction contained seven antigenic peptides. The FMC 20 reacted against a 98,000-dalton antigen that was present in the water-soluble fraction only.     

Characterization of rat alpha 1 microglobulin

A molecule heterogeneous in charge with an apparent MW of 30 000 d and associated chromophore and carbohydrate was isolated from rat urine. When compared to human alpha 1-m its fluorescence spectra as well as staining pattern on agarose gel electrophoresis or electrofocussing were very similar. Furthermore, the specific antiserum prepared against the molecule detects an antigenic determinant also detected by monoclonal antibodies against human alpha 1-m. For these reasons this protein can be considered as the rat equivalent of human alpha 1-m.     

Immunological Studies on Dermatophytes II. Serological Reactivities of Mannans Prepared from Galactomannans I and II of Microsporum quinckeanum, Trichophyton granulosum, Trichophyton interdigitale, Trichophyton rubrum, and Trichophyton schoenleinii

The contribution of terminal galactofuranose residues to the antigenic specificity and to cross-reactivity of galactomannans isolated from five species of dermatophytes, Microsporum quinckeanum, Trichophyton granulosum, T. interdigitale, T. rubrum, and T. schoenleinii, was investigated. Galactofuranose units were removed from galactomannans I and galactomannans II by mild acid hydrolysis. The resulting mannans were tested for serological reactivity with rabbit antiserum to M. quinckeanum by qualitative precipitation in gel and by quantitative complement-fixation analyses. Our results showed that, with this antiserum, the galactofuranose residues contributed greatly to the antigenic specificity and to cross-reactivity of the galactomannans II, but these residues were less significant as antigenic determinants in the galactomannans I. We have shown that mannans isolated from three Candida species reacted with rabbit antiserum to M. quinckeanum.     

Isolation of allergenically active glycoprotein from Prosopis juliflora pollen

An allergenically active glycoprotein was homogeneously isolated from the aqueous extract of Prosopis juliflora pollen by ConA-Sepharose affinity chromatography. The molecular weight of this glycoprotein was 20,000 dalton, determined by gel filtration and SDS-PAGE. This fraction showed a total carbohydrate concentration of 25%. The purified glycoprotein revealed immunochemically most antigenic or allergenic and demonstrated homogeneous after reaction with P. juliflora pollen antiserum, characterized by gel diffusion, Immunoelectrophoresis and Radioallergosorbent test.     

Immunochemistry of the Cell Walls of Listeria monocytogenes

The antigenic specificity of Listeria monocytogenes types I, II, III, IVa, and IVb was studied by immunochemical techniques. Immunologically active carbohydrates of the various types were extracted from cell walls and were chemically analyzed. Types I and II contained predominantly glucosamine and rhamnose; type III, galactose, rhamnose, and glucosamine; and types IVa and IVb, glucose and galactose. Quantitative precipitin inhibition tests with purified monosaccharides indicated that the major antigenic determinant of types I and II is rhamnose. Precipitin reactions could not be detected with type III carbohydrate and homologous or heterologous antisera. The major determinants of types IVa and IVb were found to be galactose and glucose, respectively. As much as 87% inhibition of the quantitative precipitin test for types I and II was obtained with rhamnose, 72% for type IVa with galactose, and 72% for type IVb with glucose. The immunochemical basis for the antigenic specificity of L. monocytogenes types I, II, IVa, and IVb was further confirmed by using agar gel diffusion. Cross-reactions among the various type-specific carbohydrates and heterologous antisera were also studied. Type II carbohydrate was found to contain galactose and react with type IVa antisera. This reaction could be blocked by galactose. Type I carbohydrate did not contain galactose nor did it react with antiserum prepared from type IVa cells. Therefore, the somatic antigens of type I and type II L. monocytogenes, previously thought to be identical, appeared to differ. The dominant immuno-specific group in the cross-reaction between type IVb carbohydrate and type IVa antisera was found to be galactose. Type IVa absorbed antisera did not produce a significant cross-reaction with type IVb carbohydrate. The results obtained from this investigation indicate a lesser degree of antigenic relationship between type IVa and type IVb L. monocytogenes than was previously believed to exist.     

Production and Purification of Antibody to Bovine White Matter Proteolipid Apoprotein

Circulating antibody to the bovine white matter proteolipid apoprotein was detected in rabbits 1 month after a single injection of the water-soluble form of the apoprotein. By double immunodiffusion, the antiserum reacted specifically with the delipidated proteolipid apoprotein and the crude proteolipid fraction containing complex lipids; after exposure of the proteolipid apoprotein to sodium dodecyl sulfate (SDS), no reactivity was observed. The antiserum did not react with other myelin components, i.e., basic protein, cerebroside or G_M1 ganglioside, nor was there reactivity with non-neural proteolipids. The anti-apoprotein antibody was purified by affinity chromatography. The antibody-antigen interaction is apparently very hydrophobic, since elution of the antibody from the affinity column requires buffer containing 0.5% Triton X-100-4 M-urea.     

Common and form-specific cell wall antigens of Candida albicans as released by chemical and enzymatic treatments

In order to investigate the antigenic properties of the proteins and mannoproteins present in the cell surface of Candida albicans, and to identify individual antigenic moieties and their distribution, a number of polyclonal antisera were obtained by immunizing rabbits with chemical and enzymatic cell wall extracts obtained from intact cells from both growth forms (yeast and mycelium) of the fungus. Prior to injection, wall moieties present in the extracts were subjected to different treatments and/or purification procedures such as adsorption onto polystyrenelatex microbeads or electrophoretic separation. When used as probes in indirect immunofluorescence assays, the different antisera gave rise to different fluorescence patterns varying in intensity and topological localization of the reactivity in C. albicans cells. When the different antisera were used as probes in Western blots of wall proteinaceous materials solubilized from both blastospores and germ tubes, differences in reactivity and specificity were readily discernible, allowing to identify a number of common and form-specific cell wall components. Of special interest was the fact that one of the antisera raised, after adsorption onto heat-killed blastospores, specifically recognized medium to low molecular weight moieties present only in the cell wall extracts from germ tubes. When this antiserum was used as probe in immunofluorescence assays, reactivity was confined to the hyphal extensions. Together, these observations seem to indicate that the different antibody preparations described in this report could represent important tools in the study of different aspects of the cell wall biology in C. albicans, including the identification and study of the distribution of common and form-specific cell wall-bound antigens.Abbreviations IIF Indirect immunofluorescence - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PAb polyclonal antibody     

Further environmental factors affecting the antigenicity of Trichophyton rubrum

Biochemical determinations performed on ammonium sulfate soluble and insoluble fractions of crude mycelial extracts ofTrichophyton rubrum indicated that these antigens were either carbohydrates or carbohydrates with peptide side chains. The antigens contained considerable amounts of galactose and mannose.Gel filtration techniques proved to be effective in separating these antigens. One antigen had a molecular weight greater than or equal to 2.0 × 10⁶. A smaller, more reactive antigen was also found; however, the elution time of this antigen varied with the concentration of dextrose in the medium.Quantitative precipitation tests used to differentiate the serological reactivity of crude antigens, recovered from mycelia grown on media containing variable concentrations of dextrose, indicated that the serological reactivity of the crude antigen was inversely proportional to the concentration of the carbohydrate source, with an optimum reactivity occurring with antigens prepared from mycelia grown in low dextrose concentrations.Nitrogen and carbohydrate concentrations performed on whole mycelia and cell free extracts demonstrated that the total nitrogen, soluble nitrogen, total carbohydrate and soluble carbohydrate concentrations were influenced by the concentration of the carbohydrate source. The optimum carbohydrate concentration necessary for the maximum ratio of protein and carbohydrates per gram of mycelium was 15.0 g/l. This is less than the amount used in most Sabouraud's dextrose media formulations. The effect of these environmental factors on the serological reactivity was discussed.Supported in part by NIH Environmental Health Tranining Grant ES00081-02. The help of Mrs. Gary Swecker is gratefully acknowledged for preparing the graphs.     

Human IgE antibody to the carbohydrate-containing third domain of chicken ovomucoid

Two kinds of the third domain, either with or without a carbohydrate chain, were prepared from chicken ovomucoid. The immunoreactivity of the domain preparations to human IgE antibody directed against ovomucoid was examined by using the sera from patients of egg allergy. About 30% of the serum antibody to ovomucoid reacted with the carbohydrate-containing domain, whereas little or no antibody with reactivity to the carbohydrate-free domain was detected, suggesting that the carbohydrate chain attached to the third domain played an important role in antigenic determinants of the carbohydrate-containing third domain against the human IgE antibody.     

Amino Acid Sequence of αkSubstance,a Mating Pheromone of Saccharomyces kluyveri

A fraction containing IgA (IgA-rich fraction) was prepared from bovine colostrum by anion exchange chromatography using DEAE-Sephadex A-50 and gel filtration on Sephadex G-200. A large amount of IgG1-dimer was found in this fraction, which could not be separated from IgA by repeated gel filtration.

The Fc fragment of bovine colostral IgG (IgG-Fc) was prepared from papain digestion mixtures. IgG-Fc was found to be heterogeneous on DEAE-cellulose column chromatography. Two IgG-Fc fractions were obtained, but no antigenic difference was found between them. Anti-IgG-Fc antibodies raised in rabbits by injection of these Fc preparations reacted only with IgG1 and IgG2. An immunoadsorbent (anti-IgG-Fc-Sepharose) was prepared by coupling these anti-IgG-Fc antibodies to CNBr-activated Sepharose 4B.

IgA was purified from the IgA-rich fraction by affinity chromatography on anti-IgG-Fc-Sepharose adsorbent. IgG1-dimer was effectively removed by this treatment. The purified sample gave only one precipitin arc characteristic of IgA on immunoelectrophoresis with multiple anti-bovine colostral whey antiserum. A small amount of IgA was found to be adsorbed to the affinity column nonspecifically.

When a rabbit was immunized with the purified IgA, besides anti-IgA antibodies, antibodies against the secretory component (SC) were found in the antiserum. This finding leads us to expect that the purified IgA is secretory IgA containing SC.     

Colonic tumor membrane-associated glycoprotein: isolation of antigenically-active peptides after chemical cleavage.

A membrane-associated glycoprotein fraction, referred to a CEA-M was isolated from human colonic tumor tissue by sodium dodecyl sulfate extraction of membrane fragments followed by wheat germ agglutinin affinity chromatography, Bio-Gel A-1.5 gel filtration and preparative slab gel electrophoresis. With a m.w. of approximately 200,000, isoelectric point of about 4.2 and carbohydrate:protein ratio of 2:1, this glycoprotein has physiocochemical and antigenic similarities to carcinoembryonic antigen, CEA. Immunochemical studies have shown that antiserum developed for this glycoprotein possesses relative specificity for human colonic carcinomas. Chemical cleavage of this glycoprotein by 2-nitro-5-thiocyanobenzoic acid resulted in three major Coomassie Blue and two periodic acid Schiff stainable fragments (one of which stains with both). It was found that one of the glycopeptides, labeled as TA, isolated by affinity and covalent chromatography, contained 77% carbohydrates and possessed antigenic determinants recognized by at least 70% of the antibody population raised against the total glycoprotein fraction; purified antibodies to this region of the molecule seem promising for the development of a specific assay for gastrointestinal tumors.     

The carbohydrate moiety of human chorionic gonadotropin: lack of competition with HCG for testicular receptors and anti-HCG-serum.

A glycopeptide fraction has been prepared from human chorionic gonadotropin (HCG) by digesting the reduced, S-carboxymethylated hormone with pronase and fractionating the digest by gel exclusion chromatography. The glycopeptide fraction was estimated to contain (w/w) 29% sialic acid, 31% hexose, 23% hexosamine, and 17% amino acids and/or peptides; thus, the glycopeptide mixture is 83% carbohydrate compared to intact HCG which is about 30% carbohdyrate. There was no cross-reactivity of the glycopeptide fraction with an antiserum directed against HCG. Also, when corrected for minimal non-specific effects, the fraction failed to displace 125I-HCG from a rat testicular preparation even when tested at a 10,000-fold (w/w) excess. Thus, any model involving carbohydrate effects in gonadotropin action must include the protein moiety as a necessary component.     

Conditions of cultivation, composition, and biological activity of mycelium of Flammulina velutipes (Fr.) P. Karst

A study is made on a strain of higher basydiomycete Flammulia velutipes (Fr.) P. Karat. The conditions of maximum biomass production by Flammulia velutipes were studied. Soluble and insoluble fractions were isolated from mycelium. The composition of cultured mycelium and aqueous extracts from mycelium were investigated. These objects mainly contained carbohydrates (65.3 and 84.0% in insoluble and soluble fractions, respectively, and 56% mycelium), proteins (7.5-10.0% in fractions and 17.5% in mycelium), as well as an insignificant amount of mineral substances. The main carbohydrate component of fractions was glucose (53.6-78.8%); galactose and mannose were also present, as well as fucose and xylose in insignificant amounts. The aqueous extracts from mycelium demonstrated immunomodulating activity. They rendered a stimulating effect on the functional activity of macrophages--central cells of the reticluoendothelial system. The soluble fraction had a more pronounced effect than the insoluble fraction.