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Properties and applications of anti-xanthan antibodies
Abstract
Xanthan, an exocellular bacterial polysaccharide, was used to immunize rabbits to induce the synthesis of anti-xanthan antibodies. The antibodies were isolated by affinity chromatography on an appropriate adsorbent. The antibodies are of the IgG type and possess a molecular weight of 1·55 × 105. On immunization with xanthan, eight isomers of anti-xanthan antibodies are produced in the immune response to the polysaccharide and all isomers exhibit anti-xanthan activity. Several lines of evidence from inhibition tests and antibody reactivity with chemically modified xanthan indicate that the immunodominant group of the antigen is 4,6-pyr-β-d-Man-(1 → 4)-β-d-GlcA. The antibodies were used to identify xanthan in gum blends of commercial origin and in several food items.
... Antibodies to specific sugar alcohols can be used as analytical reagents for detection and quantification of the respective sugar alcohol in foods, biological fluids, and biological samples. IgG antibodies specific to xylitol, for example, should prove useful in developing immunoassays for D-xylitol or D-xylitol epitopes on modified proteins in biological samples, natural and processed foods [Pazur & Li, 2004;Pazur et al., 1995], and pharmaceuticals. It is interesting to note in this context that xylosyl-lysyl linkages (aldimine) and xylosyl-seryl linkages are present on core proteins of dermatan sulfate of connective tissues [Longas & Meyer, 1982], and reducible lysine-carbohydrate condensation products have been identified in collagen as a result of age-related changes [Robins & Bailey, 1972]. ...
Sugar alcohols such as sorbitol, mannitol, xylitol, erythritol, maltitol and lactitol are widely used as additives (alternative sweeteners) in foods and as excipients in pharmaceuticals. Intravenous infusion of mannitol is the therapeutic osmotic agent of choice in various clinical situations. Xylitol is used as a therapeutic agent for the treatment of osteoporosis and ear infections. Although these sugar alcohols have enjoyed an enviable record of safety, there have been some reports of adverse reactions since 1966. In recent years, a rare case of anaphylaxis to mannitol present in pomegranate, cultivated mushroom, and a chewable drug has been described in an atopic subject. Mannitol was identified as a haptenic allergen based on positive skin prick test, mannitol-specific IgE by ELISA, and double-blind placebo-controlled food challenge. Since mannitol is a small molecule and does not contain reactive groups, a hypothesis was proposed to explain the mechanism of sensitization and hypersensitivity. The hypothesis was validated by the use of mannitol-protein conjugates prepared by reductive amination of D-mannose with proteins. Three cases of allergic reactions to erythritol in foods and one case of oral erosive eczema to xylitol in chewing gum have been reported since 2000 from the U.S. and Japan. The mechanism of sensitization and hypersensitivity to any monosaccharitol can easily be explained on the basis of the hypothesis proposed for the allergenicity of mannitol. Nonenzymic glycation of proteins with sugars in vivo leads to the production of glycated sugars which appear to be responsible for the immunogenicity and allergenicity of sugar alcohols. The proposed hypothesis for the allergenicity of sugar alcohols has also been validated to prove the immunogenicity of sugar alcohols in experimental animals. Polyclonal antiserum was generated using sugar alcohol-protein (bovine serum albumin) conjugates in rabbits. Antibodies specific to sugar alcohol was purified from the immune serum by hapten affinity chromatography on sugar alcohol-protein (keyhole limpet hemocyanin)-solid phase matrix. The specificity of the purified antibodies towards sugar alcohols was demonstrated by hapten-inhibition in ELISA; most sugars and other sugar alcohols showed minimal cross-reactivity. Further, the inhibition studies provided evidence for the haptenic and mono-epitopic nature of monosaccharitols. IgG antibodies specific to mannitol, erythritol, and xylitol have been generated in rabbits; in addition, IgE antibodies specific to xylitol have been generated in BALB/c mice by repeated intradermal administration of xylitol-protein conjugate. Antibodies specific to erythritol and xylitol have been utilized for the analysis of the respective sugar alcohols in natural and processed foods by polyclonal antibody-based indirect competitive ELISA.
... Alternatively, mannitol conjugates or corresponding Schiff bases formed under certain food processing conditions may also act as sensitizing agents (Venkatesh and Hegde, 2003). Antibodies specific to saccharides or their derivatives have found wide applications as analytical reagents in immunoassays for the identification and quantification of corresponding analytes in biological samples, natural foods, and processed foods (Pazur et al., 1995(Pazur et al., , 2001Pazur and Li, 2004). IgG antibodies specific to mannitol described here should prove useful in developing immunoassays for D-mannitol or D-mannitol epitopes on proteins in biological samples, natural and processed foods, and pharmaceuticals containing mannitol as an excipient. ...
D-mannitol is commonly used as a food additive and excipient due to its sweetness, non-toxicity, and low calorific value. However, several cases of hypersensitivity reactions to mannitol have been reported. Owing to its inert nature, mannitol cannot produce an immunological response. In order to explain the mechanism of immunogenicity of mannitol, a method to obtain mannitol epitopes on a carrier protein, which serves as an immunogen to generate antibodies against mannitol, is described. In the present investigation, D-mannitol-specific polyclonal antibodies were generated by immunizing rabbits with reductively aminated mannose-bovine serum albumin (BSA) (33 haptens/molecule) as the hapten-carrier conjugate. Anti-mannitol IgG antibodies were purified from the immune serum by hapten-affinity chromatography on a D-mannitol-keyhole limpet hemocyanin (KLH)-Sepharose CL-6B affinity matrix. The yield of mannitol-specific antibodies was approximately 40 microg per mL of rabbit antiserum. The specificity of the purified antibodies towards D-mannitol was demonstrated by hapten-inhibition in enzyme-linked immunosorbent assay (ELISA). The affinity-purified antibodies were found to be very specific to D-mannitol showing less than 5% cross-reactivity with other sugars and sugar alcohols, with the exception of its epimer, sorbitol, which showed 8.8% cross-reactivity. Importantly, the antibodies showed <1% cross-reactivity with L-mannitol epitope, thus exhibiting configurational specificity. The inhibition studies provided evidence for the haptenic nature of mannitol and confirmed that the mannitoyl group is a single epitope. The reaction scheme utilized here for the generation of mannitol epitopes provides the basis for the immunogenicity of mannitol.
The chapter discusses procedures for the isolation of polysaccharides, methods for the structural analysis, and the types of assays for antigenicity. The pioneering studies of Avery and Heidelberg that established for the first time an immunological function for microbial cell wall polysaccharides are included along with some appropriate contributions of other notable scientists. Some applications of polysaccharide antigens and the anti-carbohydrate antibodies are also considered in the chapter. The antigenic polysaccharides from gums have useful properties such as high viscosity, gel forming ability and stabilizing of suspensions. Included in this class are gum arable, gum mesquite, gum xanthan, gum guaran and gum linseed. The polysaccharides have been used to prepare many anticarbohydrate antibodies and these have specificity for glucose, galactose, glucuronic acid, rhamnose, glucose-1-phosphate, N-acetyl- glucosamine-1-phosphate, and oligosaccharides of glucose, galactose, glucuronic acid 4,6-pyruvyl-mannose, arabinose, N-acetyl-glucosamine and N-acetyl-galactosamine. Such anti-carbohydrate antibodies are likely to have value for the detection and identification of glycoproteins and glycolipids in normal and diseased tissues.
An immunoassay is described to detect galactomannans in foods. The assay uses a monoclonal antibody that specifically binds the (1→4)-β-linked mannan backbone of galactomannans. Carob and guar galactomannan additives in commercial food samples can be detected at concentrations as low as 5 ng/ml without prior sample preparation other than dilution.
Antibodies are an important group of bioactive natural products and are of the globulin class of proteins. Anti-caibohydrate antibodies are produced by immunization of animals with carbohydrate containing antigens. The antigens may be a polysaccharide, glycoprotein, glycolipid or a synthetic conjugate of carbohydrate and protein. The antibody that is produced is polyclonal and has specificity for the carbohydrate residue of the antigen. Many types of anti-carbohydrate antibodies have been isolated in pure form by affinity chromatography. These are specific for mono- or disaccharide units of antigens (e.g. glucose, galactose, mannose, rhamnose, fucose, glucuronic acid, N-acetyl glucosamine, lactose, isomaltose, lactosamine, neuraminic-galactose, glucuronic-galactose, mannose-glucuronic, rhamnose-glucuronic, arabinose-glucuronic, me glucuronic-galactose). The chemical and biological properties of these antibodies have been determined. To illustrate, molecular weight, light and heavy chain type, immunoglobulin class, inhibition, ultracentrifiigation, electrophoresis, isoelectrofocusing and specificity are recorded. Many of these anti-carbohydrate antibodies have been useful in applications. Some have been used in medicine for the identification of pathogenic microorganisms, some in studies to determine the structure of carbohydrate polymers, some for the analysis of additives to processed food and beverages and some in pharmaceutical uses to prepare vaccines for treatment of diseases.
Anti-carbohydrate antibodies with specificities for polysaccharide gums were isolated from the serum of rabbits that were immunized with a solution of the gums and Freund's complete adjuvant. The primary objective was to test an immunological method for the detection of the polysaccharide gums as additives to processed foods. Analysis involved the extraction of food with phosphate buffer and the testing of the extract for a reaction with anti-gum antibodies by the agar diffusion method. Reaction by a specific gum with the homologous antibodies establishes the presence of the gum in the food. The method is a novel application of antibodies. The antibody method is highly specific for a gum and thus possesses advantages over other methods of analysis for polysaccharide gums as additives in processed foods.
Flow properties of aqueous deacetylated xanthan solutions could be approximated to pseudoplastic behavior at concentrations below 0.1% but to plastic behavior above 0.3%. The flow indices in the power law for the deacetylated xanthan were somewhat different at various concentrations. The apparent viscosity of deacetylated xanthan decreased with increasing temperature at relatively low concentrations from 0.1 to 0.5%, however, it increased with increasing temperature, showed a maximum value at 40°C and decreased gradually at 1.0%. Compared with native xanthan, deacetylated material showed higher dynamic viscoelasticity at high concentrations. The dynamic viscoelasticity of deacetylated xanthan decreased with increasing temperature at various concentrations. The dynamic viscoelasticity of deacetylated xanthan was decreased by addition of urea (4.0 M).This suggests that acetate residues, which are attached to the inner mannose residues of the side chains, contribute to the intramolecular association, and tha...
Gellan gum, a microbial polysaccharide from Pseudomonas elodea, is sold now for use in microbiological media as a gelling agent to replace agar. Gellan gum's unique properties also appear to have great potential in structured food products. S-130, a biopolymer from a strain of Alcaligenes, has excellent heat stability, suspending capabilities, and rheological properties which are useful in a variety of oil field applications. S-194, another biopolymer, has unusual compatibility with high levels of salts, is stable to shear, and has excellent suspending properties. S-194 is particularly useful in agricultural applications, especially suspension (liquid) fertilizers and flowable pesticides. New applications for xanthan gum, including its use in retort pouch foods, candy, farinaceous foods, jet and foam printing are briefly reviewed also.
Two sets of anti-glycosyl antibodies have been isolated by affinity chromatography methods from the antisera of rabbits immunized with a vaccine of nonviable cells of Streptococcus faecalis, strain N. Both types of antibodies are directed against a dineteroglycan of glucose and galactose present in the cell wall of this organism. The members of one set, anti-galactose antibodies, combine with the terminal lactose residues of the glycan and the member of the other set, anti-lactose antibodies, combine with terminal lactose residues of the same glycan. Each set of antibodies is composed of multiprotein components. The electrofocusing method had been used to isolate the individual antibody proteins in homogeneous states as shown by both electrophoresis and ultracentrifugation techniques. Since the components of each set combine with the same structural unit of the antigen, they have been designated as isoantibodies. The sedimentation constants, electrophoretic properties, carbohydrate constituents, and amino acid compositions of the two sets of antibodies are recorded.
Some properties of the extracellular polysaccharide produced by the bacterium Xanthomonas campestris NRRL B-1459 were measured in partially purified culture fluids and in liquid systems prepared from thoroughly purified and lyophilized material. High intrinsic viscosities resulted from dispersions and solutions of polysaccharide B-1459 in 0.01m ammonium acetate and 4m urea. Heating a dispersion of B-1459 in 4m urea yielded a true macromolecular solution in which the polysaccharide had an apparent molecular weight of approximately 2 × 106. Lightscattering measurements on culture fluids of B-1459 made 4m in urea gave molecular weights of 13 × 106 and 50 × 106. Various solution-properties and factors that affect the viscosity of dispersions prepared from lyophilized polysaccharide are presented.
Antibodies directed against terminal carbohydrate units of gum arabic and gum mesquite were deteced in sera of rabbits immunized intramuscularly with solutions of the gums and Freund's complete adjuvant. The antibodies were isolated by affinity chromatography on adsorbents of AH-Sepharose 4B containing ligands of the appropriate gum. From the sera of animals immunized with gum arabic, two sets of anti-carbohydrate antibodies were isolated and these were shown to have specificity for different disaccharide units at the non-reducing ends of the gum molecule. From the sera of animals immunized with gum mesquite only one set of anti-carbohydrate antibodies with specificity for a terminal disaccharide unit was obtained. Isoelectric focusing coupled with agar diffusion of the purified antibodies showed that all of the sets of antibodies were composed of isomeric proteins with each isomer exhibiting antibody activity. The antibodies of a set are appropriately termed isoantibodies and it is likely that each isomer is synthesized by a different immunocyte of the host. Hapten inhibition studies with oligosaccharides isolated from the gums showed that gum arabic possesses two different immunodeterminants with the structure α-l-arabinofuranosyl-(1→4)-d-glucuronic acid and β-d-glucuronosyl-(1→6)-d-galactose while gum mesquite possessed only one determinant with the structure 4-methyl-β-d-glucuronosyl-(1→6)-d-galactose.
Xanthan with various pyruvic acid and acetate contents has been prepared from a single commercial polysaccharide sample using optimised chemical conditions (acid and alkali hydrolysis, respectively) for removal of acetal and acyl groups. The only significant change found on analysis of the modified xanthans was loss of pyruvic acid and/or acetate; no low moleculur weight carbohydrate-containing material was released. Contrary to some previous reports, evidence is presented to show that the pyruvic acid acetal and o-acetyl contents of xanthan do not affect solution viscosity. The viscosities of native, pyruvate-free and pyruvate/acetate-free xanthan solutions (0·3% w/v) were similar at shear rates 8·8–88·3 s−1 in both distilled water and 1% KCl. Over the concentration range 0·2-1·5%, the viscosities of native and pyruvate-free xanthan at 10 s−1 were similar. The viscosity increase on addition of 1% KCl to salt-free xanthan solutions was independent of pyruvic acid acetal substitution. Our results suggest that xanthan samples with various pyruvic acid acetal and o-acetal contents, prepared under different fermentation conditions of Xanthomonas campestri should not normally be used for assessing the contribution of these groups to solution viscosity.
Xanthan gum, the extracellular polysaccharide from Xanthomonas comperstris, provides aqueous solutions with properties that are extremely useful in a large number of industrial applications, both food and non-food. These properties, a consequence of xanthan gum's particular molecular shape, are: high at-rest or low-shear viscosity even at low gum concentrations, yield value, high pseudoplasticity, increased viscosity in the presence of added salt depending on gum concentration, stable viscosity over a wide range of temperature and pH in the presence of added salt, synergistic increase in viscosity in the presence of guar gum and locust bean gum, thermoreversible gelation with locust bean gum at appropriate gum concentratins, and gelation with di-and trivalent metal ions and borates under specific conditions. Xanthan gum is thus an excellent stabiliser for a wide variety of suspensions, emulsions, and foams and is highly effective over a range of temperature, pH and ionic strength. In addition, its pseudoplasticity allows the formulation of products which require not only high at-rest viscosity but also low viscosity under highshear application conditions.
The exocellular polysaccharide fermented from glucose in good yield by Xanthomonas campestrìs NRRL B-1459, has been characterized. The general aspects of chemical constitution have been established, as well as the physical properties related to practical applicability. This macromolecular polysaccharide is composed of D-mannose, D-glucose, D-glucuronic acid (as the potassium salt), and a small proportion of acetyl groups. It can be produced on an industrial scale and is stable is storage. Analytical fractionation indicates fairly sharp molecular distribution for the native polysaccharide. The polysaccharide forms homogeneous dispersions in water which show plastic rheological properties and viscosity comparable with that of high-grade plant gums. Outstanding characteristics of practical significance are the atypical insensitivity of solution viscosity to salt effects and to heat, especially when salt is present. Solutions of low concentration show a restricted viscosity decrease upon salt addition; those of higher concentrations show substantial increases. Viscosity is enhanced still further by monovalent cations at basic pH and by divalent cations at neutral or slightly basic pH. Salt moderates or eliminates any viscosity decrease due to heat and, in somewhat higher concentrations, it increases the viscosity of heated solutions. Heating or deacetylating Polysaccharide B-1459 causes no impairment of its properties, but actual improvement. The constitutional basis for these unusual properties is discussed.
The location of inter-residue, cyclic hemiacetals formed following the periodate oxidation of four representative heteroglycans has been determined by methylation analysis of the periodate-oxidized glycans. The cyclic hemiacetals led to the protection of hydroxyl groups during methylation in methyl sulfoxide, and their positions were located by analysis of the resulting di- and mono-methyl ethers. Such derivatives were not observed upon methylation analysis of the native and the periodate-oxidized-borohydride-reduced glycans. Inter-residue hemiacetals were thus identified in all oxidized glycans, between aldehydic groups at C-2 or C-3 of oxidized residues and hydroxyl groups at C-3 or C-2 of adjacent, unoxidized residues. Selective removal of 6-O-substituents from oxidized residues resulted in a decreased ability of the latter to form the inter-residue hemiacetals. Analysis of the types and proportions of the methyl ethers resulting from inter-residue hemiacetal formation may also yield structural information on the glycan.
Xanthan gum, the extracellular polysaccharide from Xanthomonas campestris, has been reinvestigated by methylation analysis, and by uronic acid degradation followed by oxidation and elimination of the oxidized residue. The polysaccharide is composed of pentasaccharide repeating-units with the following structure:
Partial, acid hydrolysis of the extracellular polysaccharide from Xanthomonas campestris gave products that were identified as cellobiose, 2-O-(β-d-glucopyranosyluronic acid)-d-mannose, O(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-d-glucose, O-(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→4)]-d-glucose, and O-(β-d-glucopyranosyluronic acid)-(1→2)-O-α-d-mannopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-d-glucose. This and other evidence supports the following polysaccharide structure (1) which has been proposed independently by Jansson, Kenne, and Lindberg:
Some properties of the glucoamylase from Rhizopus niveus have been determined and compared with the comparable properties of the glucoamylase from Aspergillus niger. The enzymes from these organisms possess the following common properties: quantitative conversion of starch to glucose, molecular weights in the range 95,500 to 97,500, and glycoprotein structures with many oligosaccharide side chains attached to the protein moieties of the enzymes. Differences in the glucoamylases exist in electrophoretic mobility, amino acid composition, nature of carbohydrate units, and types of glycosidic linkages. Lysine, threonine, serine, glutamic acid, tyrosine, and phenylalanine differ in the two glucoamylases by 25 to 50%. Whereas the enzyme from R. niveus contains mannose and glucosamine, in the N-acetyl form, as the carbohydrate constituents, the enzyme from A. niger contains mannose, glucose, and galactose. The carbohydrate chains of the R. niveus enzyme are linked by O-glycosidic and N-glycosidic linkages to the protein, while those of the A. niger enzyme are linked by O-glycosidic linkages only. Antibodies directed against the two glucosamylases have been isolated by affinity chromatography and found to be specific for the carbohydrate units of the glucoamylases. Cross reactions did not occur between the glucoamylases and the purified antibodies.
We have prepared murine hybridomas secreting monoclonal antibodies against the exopopolysaccharide xanthan from Xanthomonas campestris pv. campestris 646 after fusing NSO myeloma cells and spleen cells from BALB/c mice immunized with xanthan. Four hybridomas, secreting antibodies designated A6 (IgM kappa), B3 (IgM kappa), D1 (IgM kappa), and D3 (IgG2A kappa), were selected for further studies. All antibodies reacted with a range of different xanthans. Competition studies using variants of the exopopolysaccharide as competitors suggested that specificity was mainly against the side-chain. One of the antibodies (B3) appeared to require the fully acylated side-chain with the pyruvylated terminal mannose as the immunodominant part. The three others were assumed to be directed against the nonsubstituted trisaccharide with the inner mannose-glucuronic acid being immunodominant. None of the antibodies reacted with cellulose (the xanthan backbone). Using immunoblotting techniques on nitrocellulose paper both a mixture of monoclonal antibodies, and also polyclonal ascitic fluid, could detect xanthan quantities of approximately 0.1 microgram.
Reaction of hydroxy acids with water-soluble carbodiimides leads to the formation of lactones which can be reduced with sodium borohydride to the corresponding alcohols. In a similar manner polyuronides and glycosaminoglycuronans react with water-soluble carbodiimides. The exact nature of the products formed from the polymers has not been established but they appear to be lactones or intramolecular esters. These products can be reduced with sodium borohydride to convert the uronic acid residues in the polymers to the corresponding neutral sugars. Reduction facilitates acid hydrolysis of the acid-resistant glycosyluronic acid bonds in the polymer by replacing them with the more acid-labile glycosyl bonds. When hyaluronic acid, chondroitin sulfate, and heparin are subjected to this reduction and acid hydrolysis reaction sequence, more than 90% of the original uronic acid glycosidic bonds are cleaved. The remaining acid-resistant amino sugar glycosidic linkages are cleaved with nitrous acid to complete the essentially quantitative, nondestructive depolymerization of these polymers.
Conditions for isoelectric focusing in polyacrylamide gel of IgG globulins have been refined. Using undenatured antibodies against estradiol, preliminary information about structural and functional properties of antibodies was obtained.
The determination of the chemical nature of immunodeterminant groups of carbohydrate antigens has been achieved by a micro method coupling inhibition and double diffusion in agar. This method has been tested with antigens which react with anti-lactose, anti-galactose, anti-N-acetyl-glucosamine and antirhamnose antibodies. The analysis can be performed with as little as 10 micrograms of inhibitor, 0.2 microgram of antigen and 10 micrograms of antibody. The procedure has also been used for the identification of the determinant groups of 2 antigens with a phosphoglycan structure. The determinants of these antigens have been found to be N-acetyl-beta-glucosamine 1-phosphate and beta-glucose 1-phosphate. The glycosyl 1-phosphate units are novel types of antigenic determinants and antigens with such determinants should be useful for investigating the interactions of antigens with homologous antibodies. The specificity of monoclonal antibodies directed at an O-antigen has been determined by use of the coupled method.
A set of anti-carbohydrate antibodies and a set of anti-protein antibodies were isolated from the serum of rabbits immunized with a glycoconjugate of L-fucose and bovine serum albumin. The sets were separated by affinity chromatography by a two-column method on adsorbents with L-fucose or bovine serum albumin ligands. Isoelectrofocusing results showed that the anti-carbohydrate antibodies consisted of 11 molecular species and the anti-bovine serum albumin antibodies consisted of seven molecular species. The anti-carbohydrate antibodies are all of the IgG type while the anti-protein antibodies contain three types of globulin molecules, IgA, IgG, and IgM. The former antibodies should be useful as markers for unique glycoproteins of diseased cells and the latter antibodies may be useful for investigating the mechanism of simultaneous synthesis of three types of immunoglobulins.
PYRUVIC acid, a compound widely distributed in Nature as a metabolic intermediate, is a building unit for many compounds of biological origin. For example, in the biosynthesis of sialic acid - a carbohydrate associated with mucoproteins in animals - pyruvic acid is combined enzymatically with N-acetyl mannosamine by an aldol-type condensation1,2. Muramic acid - a carbohydrate constituent of bacterial cell walls - is thought to arise from a condensation of pyruvic acid, as phosphoenol pyruvate, with N-acetyl glucosamine3. Also, pyruvic acid has been isolated and identified as a component of a commercial red seaweed polysaccharide. Structural studies indicate the presence of one acid residue for each 51 hexose units4,5.
Antibodies specific for the polysaccharide xanthan
- Pazur