Anti-glycosyl antibodies. Two sets of isoantibodies with specificity for different carbohydrate moieties of the same glycosyl antigen.

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.     

Isolation and characterization of sets of anti-l -fucose and anti-bovine serum albumin antibodies directed against a glycoconjugate ofl -fucose and bovine serum albumin

A set of anti-carbohydrate antibodies and a set of anti-protein antibodies were isolated from the serum of rabbits immunized with a glycoconjugate ofl-fucose and bovine serum albumin. The sets were separated by affinity chromatography by a two-column method on adsorbents withl-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.     

Isolation and characterization of sets of anti-l-fucose and anti-bovine serum albumin antibodies directed against a glycoconjugate ofl-fucose and bovine serum albumin

A set of anti-carbohydrate antibodies and a set of anti-protein antibodies were isolated from the serum of rabbits immunized with a glycoconjugate ofl-fucose and bovine serum albumin. The sets were separated by affinity chromatography by a two-column method on adsorbents withl-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.     

Ascaris suum: specific antibodies in isolated intestinal loop washings from immunized swine.

Six pigs had been immunized with multiple dose of embryonated eggs and an isolated intestinal loop was prepared in each animal. Specific antibodies to Ascaris suum were detected in the soluble protein fraction of washings from the intestinal loops using an indirect fluorescent antibody test. The specific antibodies belonged to the IgA, IgG and IgE classes of immunoglobulins. In contrast, specific antibodies were not detected in the soluble protein fraction from the accumulated fluid from the intestinal loop of one pig. Soluble proteins from the washings of intestinal loops consisted of serum albumin, a large molecular size glycoprotein, and variable amounts of several α-globulins, transferrin, and immunoglobulins. The individual soluble protein solutions were efficiently fractionated using DEAE-cellulose, Sephadex G-200, and Sepharose 6B Chromatographic columns.     

Antibodies against non-immunizing antigens derived from a large immune scFv library

Target-specific antibodies can be rapidly enriched and identified from an antibody library using phage display. Large, naïve antibody libraries derived from synthetic or unimmunized sources can yield antibodies against virtually any antigen, whereas libraries from immunized sources tend to be smaller and are used exclusively against the antigen of immunization. In this study, 25 scFv libraries made from the spleens of immunized rabbits, each with a size ranging from 10⁸ to higher than 10⁹, were combined into a single large library with > 10¹⁰ individual clones. Panning of this combined library yielded target-specific rabbit scFv clones against many non-immunizing antigens, including proteins, peptides, and a small molecule. Notably, specific scFv clones against a rabbit self-antigen (rabbit serum albumin) and a phosphorylated protein (epidermal growth factor receptor pTyr1173) could be isolated from the library. These results suggest that the immune library contained a significant number of unimmunized clones and that a sufficiently large immune library can be utilized similarly to a naïe library, i.e., against various non-immunizing antigens to yield specific antibodies.     

An improved ELISA for the determination of sialyl Lewisx structures on purified glycoconjugates

The membrane carbohydrate antigen, sialyl Lewis x (sLe^x), is involved in cellular adhesive interactions in many diseases, such as cancer, inflammation and thrombosis. This antigen is also found on soluble macromolecules, such as serum glycoproteins, but the precise role of soluble sLe^x in modifying disease processes, or reflecting the pathological changes is still unclear. Although methods were previously reported for the measurement of soluble sLe^x, many of these were not well characterised, measurements were mainly made on mixtures of molecules, and the anti-sLe^x antibodies were used at concentrations that made the assay expensive. In this study an ELISA has been devised that detects sLe^x in purified soluble glycoconjugates using the anti-sLe^x antibody, CSLEX 1. Commercially-available haptoglobin (Hp) and synthetic complexes of Lewis antigens with polyacrylamide were used as model substances in developing the procedure. Key steps were washing the antibody/antigen complex with ten times diluted salt solution to prevent dissociation of the complex and the use of bovine serum albumin for blocking non-specific interactions. The assay was shown to be very specific, its precision was in the range 6–12%, and it could detect less than a pmol of sLe^x. It could also distinguish between different densities of sLe^x on the same amount of glycoconjugate. Determination of sLe^x in Hp isolated from small groups of healthy individuals, cancer patients, and rheumatoid arthritis sufferers suggested that the antigen expression is increased in disease. This method, which is an improvement on those previously described, will be useful for determining sLe^x in many different types of soluble glycoconjugate, and used in combination with synthetic carbohydrate polyacrylamide complexes, will help to standardize measurements of soluble sLe^x in the future.     

Antibodies directed against the thiomannose moiety of a glycoconjugate of 2-imino-2-methoxyethyl 1-thio-alpha-D-mannopyranoside and bovine serum albumin

Anti-thiomannose antibodies were induced in rabbits immunized with a glycoconjugate of 2-imino-2-methoxyethyl 1-thio--d-mannopyranoside (Man-S) and bovine serum albumin (BSA). Also anti-BSA antibodies directed against the BSA moiety of the glycoconjugate were detected in low concentrations in the immune serum. However, antibodies against the combinatorial epitope of the hapten group and the carrier protein were not detected. The anti-thiomannose and the anti-BSA antibodies were isolated in pure forms by affinity chromatography on Sepharose 4B-bearing thiomannosyl-BSA ligands or BSA ligands. The anti-thiomannose antibodies constituted the major fraction of the antibodies, and these antibodies were isolated in pure form for the first time. The specificity of the thiomannose antibodies was established from data of experiments of periodate oxidation, perpropionic acid oxidation, hapten inhibition, and agar diffusion. Isoelectrofocusing showed that the anti-thiomannose antibody preparation consisted of at least six isomeric proteins, all of which exhibited antibody activity against the glycoconjugate of thiomannose and BSA.     

The expression of Lewisx on carcinoembryonic antigen (CEA)-related glycoproteins of normal and inflamed oesophageal squamous mucosa

Carcinoembryonic antigen (CEA)-related antigens were detected histologically in normal and inflamed oesophageal squamous mucosa using polyclonal anti-CEA antisera and monoclonal antibodies recognizing CEA or NCAs (non-specific cross-reacting antigens). Expression was limited to the surface of more mature squames. Immunoblotting of detergent extracts of oesophageal mucosa separated on polyacrylamide gels using polyclonal anti-CEA antisera showed a number of CEA-related proteins, of 195, 145, and 80 kDa. CEA-specific monoclonal antibodies recognized only the 195-kDa glycoprotein. The lower molecular weight species were recognized by anti-NCA antibody DD9 and a CD66 antibody. The carboyhydrate antigen Lewis^x (Le^x, CD15), previously shown to be a marker of mature squames, was present predominantly on a subpopulation of the 195-kDa antigen and was demonstrable on the higher molecular weight component of a doublet recognized by the CEA antibodies. Expression of Le^x carbohydrate antigens in inflamed oesophageal squamous mucosa was shown to be significantly reduced relative to the expression seen in normal tissue. A suprabasal layer of CEA-positive, Le^x-negative cells became apparent in inflamed tissue showing altered glycosylation of the CEA under these conditions. It is postulated that CEA plays a role in maintaining the integrity of the squamous mucosa.     

Quantitative label-free screening for antibodies using scattering biophotonic microarray imaging

A biophotonic array based on gold nanoparticles functionalized with antigen proteins has been used to determine the concentrations of the respective antibodies in solution. Four proteins—fibrinogen, bovine serum albumin, transferrin, and C-reactive protein—were used to construct a test array with the assay repeated a number of times. The antibody-antigen association and dissociation rate constants were determined for the antibody assays from a series of calibration experiments. The label-free determination of the unknown antibody concentrations was performed using two related kinetic analyses. From these results, the current array assay sensitivity is 250 ng ml^-1 with an accuracy of 15% using an 8-min kinetic measurement and a 16-spot averaged assay.     

Prediction of carbohydrate binding sites on protein surfaces with 3-dimensional probability density distributions of interacting atoms

Non-covalent protein-carbohydrate interactions mediate molecular targeting in many biological processes. Prediction of non-covalent carbohydrate binding sites on protein surfaces not only provides insights into the functions of the query proteins; information on key carbohydrate-binding residues could suggest site-directed mutagenesis experiments, design therapeutics targeting carbohydrate-binding proteins, and provide guidance in engineering protein-carbohydrate interactions. In this work, we show that non-covalent carbohydrate binding sites on protein surfaces can be predicted with relatively high accuracy when the query protein structures are known. The prediction capabilities were based on a novel encoding scheme of the three-dimensional probability density maps describing the distributions of 36 non-covalent interacting atom types around protein surfaces. One machine learning model was trained for each of the 30 protein atom types. The machine learning algorithms predicted tentative carbohydrate binding sites on query proteins by recognizing the characteristic interacting atom distribution patterns specific for carbohydrate binding sites from known protein structures. The prediction results for all protein atom types were integrated into surface patches as tentative carbohydrate binding sites based on normalized prediction confidence level. The prediction capabilities of the predictors were benchmarked by a 10-fold cross validation on 497 non-redundant proteins with known carbohydrate binding sites. The predictors were further tested on an independent test set with 108 proteins. The residue-based Matthews correlation coefficient (MCC) for the independent test was 0.45, with prediction precision and sensitivity (or recall) of 0.45 and 0.49 respectively. In addition, 111 unbound carbohydrate-binding protein structures for which the structures were determined in the absence of the carbohydrate ligands were predicted with the trained predictors. The overall prediction MCC was 0.49. Independent tests on anti-carbohydrate antibodies showed that the carbohydrate antigen binding sites were predicted with comparable accuracy. These results demonstrate that the predictors are among the best in carbohydrate binding site predictions to date.     

Serologic demonstration of the albuminoid nature of the B700 murine melanoma antigen.

Limited available evidence indicates that the B700 murine melanoma antigen is related to serum albumin, but potential relationships to other members of the serum albumin protein family have not yet been established. Using specific antibodies raised against each of the members of the albumin family, we have studied cross-reactivity by solid phase enzyme-linked immunosorbent assay and Western immunoblotting. We demonstrate that B700 is serologically cross-reactive to members of the serum albumin family, which includes alpha-fetoprotein and vitamin D binding protein. Therefore, B700 is part of the serum albumin family of proteins, although the mechanism underlying its specific expression by transformed melanocytes remains unknown.     

Immunocytochemical identification of phenylalanine hydroxylase and albumin in cultured hepatoma cells and isolated rat hepatocytes

Rhodamine-conjugated antibodies specific for phenylalanine hydroxylase and serum albumin were employed as cytochemical probes to identify these two proteins in H4 hepatoma cells and in isolated rat hepatocytes. Each fluorescent antibody stained the cells specifically and in a distinctive manner. In both cell types, albumin staining was discretely localized in cytoplasmic and in H4 cultures varied somewhat from cell to cell. Evidence from cultures of REB15 cells, a strain derived by cloning H4 cells in tyrosine-free medium, suggested that the staining variability of H4 cells could reflect a variability in phenylalanine hydroxylase content. Hydrocortisone-treated H4 cells and REB15 cultures contain increased amounts of phenylalanine hydroxylase; and all cells in the culture appear to be induced by the hormone. Evidence was presented to show that the albumin visualized within the isolated hepatocytes had been synthesized by these cells, and, furthermore, that quantitatively nearly all intracellular albumin in the isolated rat hepatocytes appeared to be entrained in the secretion pathway (analogous data already exist for H4 cells [Baker, R.E., and R. Shiman. 1979. J. Biol. Chem. 254:9633-9639]). By scoring specific fluorescence, 86 and 98% of the H4 cells and 89 and 98% of the isolated hepatocytes were found to contain phenylalanine hydroxylase and albumin, respectively. Therefore, almost all cells in each population appeared to synthesize both proteins. An implication of these findings is that in rat virtually all liver parenchymal cells must synthesize both phenylalanine hydroxylase and albumin.     

Characterization of a human granulocyte differentiation antigen (CDw15) commonly recognized by monoclonal antibodies

Many different anti-human granulocyte monoclonal antibodies recognize the same carbohydrate antigen which contains the trisaccharide 3-fucosyl-N-acetyllactosamine. The antigen is expressed mainly on two cell surface glycoproteins of molecular weights around 105 K and 160 K which are apparently not members of the LFA-1 family of proteins. Although specific for granulocytes in blood, the antigen is expressed on a wide range of non-haemopoietic cell types.     

Association of an albumin antigen with phosphatidylcholine liposomes alters the nature of immunoglobulins produced during the immune response against the antigen

Human serum albumin has been injected intravenously in rabbits either free in solution or associated with liposomes. Blood samples were obtained from the rabbits at various time intervals after injection, and two different antibody determinations were performed in each sample. Whereas a haemagglutination technique was applied for determination of predominantly IgM anti-human serum albumin antibodies, a second technique, using antigen-coated Sepharose beads and horseradish peroxidase-conjugated anti-rabbit IgG, was used to detect the IgG anti-human serum albumin antibodies. Liposomes appeared to enhance strongly the IgM response against human serum albumin. No such marked differences were found, however, between the IgG responses against liposome-associated or free human serum albumin. The conclusion is drawn that the immunoadjuvant effect of liposomes during the primary immune response against an albumin antigen is mainly due to an enhanced IgM antibody production.     

Rat sulfite oxidase antibodies cross-react with two gene family-related proteins: albumin and vitamin D-binding protein.

Screening lambda cDNA libraries from rat liver with antibody to native rat liver sulfite oxidase (RLSO) showed cross-reaction with two proteins that belong to the same gene family: serum albumin and vitamin D-binding protein. Antibodies raised against native RLSO or sodium dodecyl sulfate-denatured protein cross-reacted with these proteins by Western blot analysis. The relative effectiveness of RLSO antibody binding was estimated to be 1/5 for rat serum albumin and 1/10 for rat vitamin D-binding protein. This result was not caused by contaminating proteins in the RLSO used for immunization as the RLSO preparation did not react with rat serum albumin antibody. RLSO antibodies, selected for their ability to bind rat serum albumin immobilized on nitrocellulose, recognized both rat serum albumin and RLSO. RLSO antibody, with albumin-reactive antibody removed, still recognized vitamin D-binding protein, suggesting that multiple determinants specific to each protein are involved in the cross-reaction. Comparison of RLSO antibody binding to the rat and human proteins indicated that the determinants were species-specific. cDNA clones identified by screening cDNA libraries with RLSO antibody demonstrated that these determinants reside in the C-terminal domain of these proteins. These results suggest that these proteins contain some common immunological features and may be evolutionarily related.     

Schistosoma mansoni: reactivity with infected human sera and monoclonal antibody characterization of a glycoprotein in different developmental stages

Cercarial glycoproteins of Schistosoma mansoni were purified by concanavalin A affinity chromatography. The purified fraction consisted of at least 15 polypeptides when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sera of infected humans specifically immunoprecipitated all of these polypeptides. These purified glycoproteins were used as antigen for preparing monoclonal antibodies. One of these monoclonal antibodies immunoprecipitated cercarial polypeptides that were identical to polypeptides immunoprecipitated with sera of infected humans as analyzed by two-dimensional gel electrophoresis. Direct binding assays with ¹²⁵I-labeled monoclonal antibody showed that proteins sharing antigenic determinants recognized by this monoclonal antibody were present not only in cercariae (the source of the immunogen) but also in adult male and female worms and in eggs. The protein molecules expressing these antigenic determinants were glycosylated in each of the developmental stages of the larvae, but differed with respect to molecular weight. These findings indicate a role for this monoclonal antibody in serodiagnosis and immunoprophylaxis.     

Isomeric, anti-rhamnose antibodies having specificity for rhamnose-containing, streptococcal heteroglycans

L-Rhamnose (6-deoxy-L-mannose) is a constituent carbohydrate unit of microbial, immunogenic heteroglycans and lipopolysaccharides, and often functions as the immunodeterminant group of such immunogens. Two types of anti-rhamnose antibody have now been isolated by affinity chromatography of immune sera obtained from rabbits immunized with vaccines of Streptococcus mutans, strain KI-R, and Streptococcus pneumoniae, type 32. The antibodies of one type were directed at a glycan of L-rhamnose, D-glucose, and D-galactose in the cell wall of S. mutans, and those of the other type, against a capsular glycan of L-rhamnose and D-glucose from S. pneumoniae. The two types of anti-rhamnose antibody were immunologically distinct, and showed no reciprocal cross-reactivity. Additional properties of the two types of antibody were determined; thus, both types of antibody were of the IgG class of immunoglobulins, both possessed molecular weights of 1.45 X 10(5), and both consisted of multiple or isomeric forms.     

Characterization of monoclonal antibodies specific for the Lewis a human blood group determinant

Four hybridoma cell lines were derived from the spleen cells of mice immunized with the neutral glycolipids of human meconium. The antibodies secreted by these lines were specific for the Lewis a antigen of the human Lewis blood group system as determined by solid phase immunoassay using synthetic carbohydrate antigens and by plate binding assay and thin layer chromatography-autoradiography using natural glycolipid antigens. Coating protein A-bearing Staphylococcus aureus with one of the antibodies yielded a stable reagent that produced rapid agglutination of Lewis a positive human erythrocytes. The fine structural specificity of these antibodies was assessed by competition radioimmunoassay using synthetic structural analogs of Lewis a conjugated to bovine serum albumin. One antibody was specific for the Lewis a trisaccharide (Gal beta 1 leads to 3(Fuc alpha 1 leads to 4) beta GlcNAc), while a second recognized the entire Lea (1 leads to 3) beta Gal tetrasaccharide. The third and fourth were directed at topography largely provided by only the alpha Fuc and beta GlcNAc units. These monoclonal antibodies not only represent potentially useful reagents for detecting the Lewis a antigen but also provide a system for studying precise relationships between anticarbohydrate antibody structure and binding specificity.     

Immunochemical studies on methyl α- and methyl β-glucosaminide-azoprotein conjugates

Conjugates consisting of methyl 2(p-azobenzamido)2-deoxy-α-d-glucopyranoside and methyl 2(p-azobenzamido)-2-β-d-glucopyranoside coupled to bovine serum albumin were used as synthetic antigens. Rabbits immunized with artificial antigen provided specific anti-methyl α-glucosaminide or anti-methyl β-glucosaminide anti-hapten sera. Quantitative hapten inhibition was employed to elucidate the specificity of anti-hapten combining sites and to evaluate some structural and configurational features contributing to the immunochemical interaction with the antibody. It is shown that the configuration at the anomeric carbon atom makes a major contribution to the immunochemical specificity of the anti-hapten antibodies against α- and β-methyl glycosides.     

A Modified IF-test to Demonstrate IgM Antibodies to Babesia Divergens of Cattle

An IF–test modified to increase sensitivity was used to detect the presence of Babesia divergens specific IgM antibodies. It consisted of three steps: (1) the incubation of Babesia antigen with test serum, followed by; (2) its incubation with protein A; and (3) the addition of rabbit anti-bovine IgM bound to protein A, which was bound with FITC. Compared to the conventional IF–technique, this modified IF-technique detected bovine anti-Babesia-IgM in test serum at a two–fold lower dilution and with a diminished background staining. This modified IF-technique improves the possibilities for studying class Μ antibody in the serum of cattle which have been infected with B. divergens. It is possible to determine whether a calf s antibody response is due to maternally transmitted antibodies or actively acquired antibodies by determining the presence of each of the classes of immunoglobulin against B. divergens present in the calf s serum.