Recognize Carbohydrate Epitopes Research Articles

Changes of glycoconjugate expression profiles during early development.

A variety of glycoconjugates, including glycosphingolipids (GSLs), expressed in mammalian tissues and cells were isolated and characterized in early biochemical studies. Later studies of virus-transformed fibroblasts demonstrated the association of GSL expression profiles with cell phenotypes. Changes of GSL expression profile were observed during mammalian embryogenesis. Cell surface molecules expressed on embryos in a stage-specific manner appeared to play key roles in regulation of cell-cell interaction and cell sorting during early development. Many mAbs showing stage-specific reactivity with mouse embryos were shown to recognize carbohydrate epitopes. Among various stage-specific embryonic antigens (SSEAs), SSEA-1 was found to react with neolacto-series GSL Lex, while SSEA-3 and SSEA-4 reacted with globo-series Gb5 and monosialyl-Gb5, respectively. GSL expression during mouse early development was shown to shift rapidly from globo-series to neolacto/lacto-series, and then to ganglio-series. We found that multivalent Lex caused decompaction of mouse embryos, indicating a functional role of Lex epitope in the compaction process. Autoaggregation of mouse embryonal carcinoma (EC) F9 cells provided a useful model of the compaction process. We showed that Lex-Lex interaction, a novel type of molecular interavction termed carbohydrate-carbohydrate interaction (CCI), was involved in cell aggregation. Similar shifting of GSL expression profiles from globo-series and neolacto/lacto-series to ganglio-series was observed during differentiation of human EC cells and embryonic stem (ES) cells, reflecting the essential role of cell surface glycoconjugates in early development.

Adaptive humoral immune responses to type III capsular polysaccharide of Streptococcus pneumoniae.

Abstract Capsular polysaccharides (CPSs) are important vaccine candidates given that they are located on the outermost surface of bacteria and have distinct structures. To induce CPS specific adaptive immune responses (i.e., T cell-mediated B cell responses), CPSs are conjugated with carrier proteins, to make glycoconjugate vaccines. The production of the current generation of glycoconjugate vaccines does not make use of specific scientific knowledge to maximize stimulation of critical immune cells (i.e., helper T cells) involved in producing protective IgG antibodies. Our previous discovery demonstrated that the mammalian CD4+ T cell repertoire contains a population of carbohydrate-specific T cells (i.e., Tcarbs) that recognize carbohydrate epitopes. Here, we investigate the interactions of carbohydrate epitopes with the key molecules of the adaptive immune system, MHCII proteins and T cell receptors (TCRs). This knowledge will have fundamental biological implications in our comprehension of how the adaptive immune system functions, and in future knowledge-based vaccine design. We employed a model bacterial polysaccharide antigen (capsular polysaccharide of type 3 Streptococcus pneumoniae, Pn3P) and its protein or peptide conjugates. We have collected evidence for the presence of a Tcarb repertoire that recognizes MHCII-presented Pn3P epitopes and drives Pn3P-specific IgG production by B cells. We have also generated Pn3 oligosaccharides through controlled depolymerization of Pn3P to be used in interation studies. Building on our expertise in these systems, we will define the MHCII and TCR interactions of processed Pn3P epitopes, and characterize immune responses that result from these interactions.

Differential glycosylation of -dystroglycan and proteins other than -dystroglycan by like-glycosyltransferase

Genetic defects in like-glycosyltransferase (LARGE) cause congenital muscular dystrophy with central nervous system manifestations. The underlying molecular pathomechanism is the hypoglycosylation of α-dystroglycan (α-DG), which is evidenced by diminished immunoreactivity to IIH6C4 and VIA4-1, antibodies that recognize carbohydrate epitopes. Previous studies indicate that LARGE participates in the formation of a phosphoryl glycan branch on O-linked mannose or it modifies complex N- and mucin O-glycans. In this study, we overexpressed LARGE in neural stem cells deficient in protein O-mannosyltransferase 2 (POMT2), an enzyme required for O-mannosyl glycosylation. The results showed that overexpressing LARGE did not lead to hyperglycosylation of α-DG in POMT2 knockout (KO) cells but did generate IIH6C4 and VIA4-1 immunoreactivity and laminin-binding activity. Additionally, overexpressing LARGE in cells deficient in both POMT2 and α-DG generated laminin-binding IIH6C4 immunoreactivity. These results indicate that LARGE expression resulted in the glycosylation of proteins other than α-DG in the absence of O-mannosyl glycosylation. The IIH6C4 immunoreactivity generated in double-KO cells was largely removed by treatment either with peptide N-glycosidase F or with cold aqueous hydrofluoric acid, suggesting that LARGE expression caused phosphoryl glycosylation of N-glycans. However, the glycosylation of α-DG by LARGE is dependent on POMT2, indicating that LARGE expression only modifies O-linked mannosyl glycans of α-DG. Thus, LARGE expression mediates the phosphoryl glycosylation of not only O-mannosyl glycans including those on α-DG but also N-glycans on proteins other than α-DG.

Carbohydrate epitopes are immunodominant at the surface of infectious Neoparamoeba spp.

Amoebic gill disease, the main disease of concern to the salmon industry in Tasmania, is caused by the amoeba, Neoparamoeba spp. Experimental infection can only be induced by exposure to wild-type (WT) parasites isolated from the gills of infected fish, as cultured amoebae are non-infective. To characterize the surface antigens of WT parasites, we produced monoclonal antibodies (mAbs) using subtractive immunization. Mice inoculated with non-infective parasites were treated with cyclophosphamide, to deplete reactive lymphocytes, and then immunized with different antigen preparations from infective parasites. When whole parasites were used for boosting, the percentage of WT unique mAbs was very high (86%) as was the percentage of mAbs specific for carbohydrate epitopes (89%). When deglycosylated membranes were used, the numbers of mAbs specific for non-carbohydrate epitopes did not increase, but the total number of WT unique mAbs was reduced (86-40%). Using an untreated membrane preparation, the total number of mAbs to surface molecules was very high, but all recognized carbohydrate epitopes. The total number of mAbs recognizing carbohydrate epitopes on the surface of the WT parasites was 97%, suggesting that the dominant epitopes on the surface molecules unique to WT parasites are carbohydrate in nature.

Comparison between the native glycosylated and the recombinant Cup a1 allergen: role of carbohydrates in the histamine release from basophils

Cypress pollinosis is an important cause of respiratory allergies. Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed. The native counterpart of this allergen has been purified and characterized by our group. It has been suggested that sugar moieties play a role in the in vitro IgE binding on Cupressus arizonica pollen extract. To characterize the immunoreactivity of the recombinant major allergen in comparison with its native counterpart. To evaluate the role of carbohydrate moieties in the IgE-mediated in vitro histamine release from basophils by using the native glycosylated Cup a1 as compared with the recombinant one. Recombinant Cup a1 was expressed in E. coli. IgE reactivity of Cupressaceae-allergic patients on the native as well as the recombinant molecule was investigated by immunoblotting, ELISA experiments and histamine release test from passively sensitized basophils. Fourteen out of 17 Cup a1-positive sera had IgE antibodies reactive with the native molecule only and lost their reactivity-after periodate deglycosylation of the allergen. Moreover, only native molecule was capable of inducing histamine release by this group of sera. Both the recombinant and the native molecules were recognized by three out of the 17 sera and were equally capable of triggering degranulation. A large number of sera reactive with the major allergen recognize carbohydrate epitopes only. IgE from these sera are able to induce histamine release from basophils and they might play a functional role in the clinical symptoms of allergy.

Chondroitin sulfate proteoglycans in the rat thalamus: expression during postnatal development and correlation with calcium-binding proteins in adults.

Postnatal expression of chondroitin sulfate proteoglycans was studied in the rat thalamus by immunocytochemistry and Western immunoblotting techniques with monoclonal antibodies that recognize carbohydrate epitopes (clones CS-56, 1-B-5, 2-B-6). The complex of the results shows that these antibodies recognize mostly nonoverlapping molecules whose expression is regulated during postnatal development. Chondroitin sulfate proteoglycans, recognized by antibody CS-56, and hyaluronan, identified by antibody 1-B-5 after hyaluronidase digestion, are abundant in the neuropil of most thalamic nuclei at the perinatal stage and progressively decrease during the second week of life, attaining levels barely detectable by immunocytochemistry at the end of the third week. In adult thalamus, chondroitin sulfate proteoglycans of high molecular mass, bearing glycosaminoglycans unsulfated in the linking region, and recognized by antibody 1-B-5 are confined to perineuronal nets around neurons chiefly localized in thalamic reticular nucleus. The immunoreactvity for antibody 2-B-6, specific for chondroitin-4-sulfate, is low at the perinatal stage and is not detectable in adult thalamus. Double-immunolabeling has shown that, along the rostrocaudal extension of reticular nucleus, the most developed perineuronal nets are associated with a subset of neurons expressing calretinin, and not with parvalbumin-positive neurons, which represent the largest neuronal population of the nucleus. The distribution of perineuronal nets supports the presence, in thalamic reticular nucleus, of neuronal subpopulations with different morphological and physiological features.

Tick-bite-induced anaphylaxis in Spain

Although there are very few reports of human anaphylaxis induced by tick bites, two such cases have recently been seen in Salamanca, Spain. To identify the tick species responsible, salivary-gland extracts from six species of hard tick and two of soft tick were prepared and used as allergens/antigens in skin-prick tests and serological analyses. For each case, the results of the skin tests were positive for several species of hard tick but negative for the soft ticks. ELISA and western blots revealed high titres of IgG against hard ticks (but not soft ticks) in the sera from both cases. However, serum from only one of the cases was found to be ELISA- and western-blot-positive for tick-specific IgE. Accordingly, the anaphylaxis seen in one case was IgE-mediated whereas that in the other case appeared to be IgE-independent. In both cases, most of the tick-specific antibodies only recognized carbohydrate epitopes. High levels of cross-reactivity between the salivary-gland extracts from several species of hard tick made it impossible to identify which species was responsible for each anaphylactic reaction, although the immunological results seem to point to Ixodes ricinus.

Monoclonal Antibodies Specific to Carbohydrates of <i>Echinococcus multilocularis</i>

To investigate the complexity of epitopes presented on Echinococcus multilocularis (E.m.) metacestode carbohydrates, a panel of monoclonal antibodies (MoAbs) was generated and characterized. Thirty of the clones were obtained and classified into three types (types I to III) based on Western blotting (WB) and dot-ELISA. One MoAb (type I) appeared to react with one of the carbohydrate antigens (C-antigens) located at 30-35 kDa, and was the most effective diagnostic antigen for human alveolar hydatid disease (AHD) in Hokkaido, Japan. The second group (15 clones) of MoAb (type II) reacted with another C-antigen; one which also induced antibody response in AHD patients. The third group (14 clones) of MoAb (type III) reacted with other C-antigens both in ELISA and dot-ELISA, but did not react in WB. Cross-reaction to the antigens of Echinococcus granulosus was faintly observed in only the type I-MoAb by dot-ELISA. In the immunohistological studies, all of the MoAbs reacted strongly with the laminated layer though not with protoscoleces in metacestode tissue prepared from experimentally infected cotton rats. Tissue sections treated with sodium periodate lost their immunoreactivity, suggesting that these MoAbs recognized carbohydrate epitopes of the E.m. metacestode.

Production and characterization of monoclonal antibodies against human airway mucins.

The objective of this study was to generate and characterize monoclonal antibodies (MAbs) against human airway mucins, and therefore, should serve as a useful tool in studying the regulation of airway mucins in various physiological or pathological situations of human airway. As an antigen, we used a high molecular mass mucin preparation purified from the sputum of normal human subjects. Two monoclonal hybridomas, namely MAbs HM02 and HM03 were obtained and they showed strong immunoreactivity against purified or crude mucin in sputum or bronchial washing of normal human subject. With the high immunoreactivity of these MAbs, mucin contents could be analyzed with more than 100-fold dilution of human airway secretion. The antibodies recognized carbohydrate epitopes because their immunoreactivity was completely abolished by treatment of the mucin with 5 mM periodate. Further characterization of MAbs HM02 and HM03 showed that: (1) they belong to the IgM type; (2) they bind to high molecular mass mucins based on Western blot; (3) they could indirectly immunoprecipitate human airway mucin and as we know, this is the first to demonstrate immunoprecipitation of human airway mucin with anti-human mucin antibodies; and (4) they bind to the goblet cell in airway epithelium as well as some submucosal glands based on immunohistochemistry. Therefore, MAbs HM02 and HM03 should be able to serve as an invaluable tool in studying the regulation of airway mucins in various physiological and pathological situations of human airway.

Identification and characterization of excreted-secreted products and surface coat antigens of animal and plant-parasitic nematodes.

Nematode surface coat (SC) proteins and excreted-secreted products (E-S) are likely to play important roles in the host-parasite interaction and considerable similarities can be found in SC proteins and E-S products from certain plant and animal parasitic nematodes. Monoclonal antibodies raised to E-S products of plant-parasitic nematodes were shown to cross-react with E-S products and the surface coats of the animal parasites Trichinella spiralis and Haemonchus contortus. Most of the antibodies recognized carbohydrate epitopes but the activity of 2 MAbs (IACR-CCNj.2a.15 and IACR-Misec.8D.3) which recognized proteic epitopes in these nematodes were further characterized. Antibody 2a.15 recognized the SC and oral exudate of Meloidogyne incognita, T. spiralis and H. contortus. This antigen was also immunolocalized in the lining of the oesophagus and gut and in the exudate present during ecdysis of H. contortus L3. Antibody 8D.3 reacted with the SC of these nematodes on cryosections but on live nematodes the immunofluorescence was very patchy and was shed from the nematode SC.

Immunocytochemical studies of the infection mechanisms of Botrytis fabae I. The fungal extracellular matrix in penetration and post‐penetration processes

Extracellular matrices associated with conidia and germ tubes of Botrytis fabae (Sard.) sporelings grown on Vicia faba L. leaves were clearly visualized by epi‐fluorescence microscopy following immunolabelling with the monoclonal antibodies, BC‐KH4 and BC‐FD7‐G9. These antibodies were raised against surface washings of B. cinerea, are directed against B. cinerea and B. fabae, and are known to recognize carbohydrate epitopes on a glycoprotein. Both BC‐KH4 and BC‐FD7‐G9 also labelled matrix material located at the surface of penetration and infection hyphae inside the leaf tissue by epi‐fluorescence microscopy. Such matrix material was not visible by DIC microscopy.Immunoelectron microscopy of B. fabae‐infected leaf tissue, prepared by progressive low‐temperature dehydration and embedding in acrylic resin, allowed further investigation of the spatial distribution of the antibody‐binding sites. An abundance of BC‐KH4 and BC‐FD7‐G9 antigenic sites were observed throughout the fibrillar‐like matrix material surrounding the germ tubes on the leaf surface and the infection hyphae inside the host cells. However, close examination of the V. faba–B. fabae interface inside the host tissue showed that this fibrillar material extended some distance from the surface of the infection hyphae and through the swollen epidermal and mesophyll cell walls. Such fibrillar matrix material is thought to be of fungal origin. The possible role(s) of this matrix material in the infection process are discussed.Double‐immunolabelling studies using the BC‐KH4 MAb and a polyclonal antiserum directed against oligosaccharides containing β‐(1→3)‐glucose were carried out in order to localize and distinguish between the fungal extracellular matrix material and translucent cell wall respectively. This technique allowed a closer examination of the interactions of the fungal matrix components with the host walls and degenerate host cytoplasm. Finally, inward curling of the leaf cuticle suggested that mechanical pressure is involved in the penetration process.

Production and characterization of monoclonal antibodies against rat tracheal mucins.

The objective of this study was to generate and characterize monoclonal antibodies against rat airway mucins. Therefore, it should serve as a useful tool in studying the regulation of airway mucins using various in vivo rat models that are currently available. As an antigen, we used a high molecular mass mucin preparation purified from the spent media of rat tracheal surface epithelial cells in primary culture. Seven monoclonal hybridomas were obtained, among which mAbRT03 showed the highest immunoreactivity against the mucin. All of the antibodies secreted by these hybridomas recognized carbohydrate epitopes but not sialic acid residues, since their immunoreactivity was completely abolished by treatment of the mucin with 20 mM periodate but not with neuraminidase. Further characterization of mAbRT03 showed that (1) it belongs to the IgM type, (2) it binds to high molecular mass mucins based on Western blot, (3) it could indirectly immunoprecipitate rat airway mucin--and, as we know, this is the first demonstration of immunoprecipitation of airway mucin with anti-mucin antibodies--(4) it binds to the luminal side of tracheal epithelium as well as some goblet cells based on immunohistochemistry, and (5) it also recognizes in vivo airway mucins from rats, but not from human nor hamsters, which have been purified from the airway lavage fluids. This is the first monoclonal antibody against rat airway mucin, which has been developed against purified rat airway mucins. Therefore, mAbRT03 should be able to serve as an invaluable tool in studying the regulation of airway mucins using various intact rat models that are currently available.

Haemonchus contortus Glycoproteins Contain N-Linked Oligosaccharides with Novel Highly Fucosylated Core Structures

Structural studies on the N-linked oligosaccharides of Haemonchus contortus, an economically important nematode that parasitizes domestic ruminants, have revealed core fucosylation of a type not previously observed in any eukaryotic glycoprotein. Mass spectrometric analyses were performed on detergent extracts of homogenized adult H. contortus and on purified H11, a glycoprotein isolated from intestinal brush borders which has been previously shown to be an effective vaccine antigen. The major N-linked glycans identified in the present study have up to three fucose residues attached to their chitobiose cores. The fucoses are found at the 3- and/or 6-positions of the proximal GlcNAc and at the 3-position of the distal GlcNAc. The latter substitution is unique in N-glycans. Most anti-H11 monoclonal antibodies are known to recognize carbohydrate epitopes, and it is possible that the newly discovered multifucosylated core structures are highly immunogenic in this glycoprotein.

Lacto-N-fucopentaose III (Lewis x), a target of the antibody response in mice vaccinated with irradiated cercariae of Schistosoma mansoni

Carbohydrates on soluble egg antigens are major epitopes for the antibody responses of patients and mice infected with Schistosoma mansoni. Recently, protective sera of mice vaccinated with irradiated cercariae were shown to recognize carbohydrate epitopes on schistosomal glutathione S-transferase. The present study demonstrates that carbohydrate epitopes are major targets of sera from C57BL/6J and CBA/J mice vaccinated with 15- or 50-kilorad-irradiated cercariae of S. mansoni. Antibody titers to carbohydrate epitopes increased with the number of vaccinations and were considerably higher in C57BL/6J mice than in CBA/J mice. The specificity of this anticarbohydrate response was determined by measuring antibody binding to defined oligosaccharide residues known to be present on the parasite. A predominant target of the humoral anticarbohydrate response of vaccinated mice was lacto-N-fucopentaose III, a molecule relevant for cell trafficking. We observed no binding to its nonfucosylated homolog, lacto-N-neotetraose, or to oligosaccharides present on keyhole limpet hemocyanin. The strongest antibody response to lacto-N-fucopentaose III was observed for C57BL/6J and CBA/J mice repeatedly vaccinated with 15-kilorad-irradiated cercariae, which also achieve the highest levels of protection. Immunoglobulin M was the predominant antibody class binding to lacto-N-fucopentaose III. We conclude that in the irradiated-cercariae vaccine model, C57BL/6J and CBA/J mice produce anticarbohydrate antibodies against various stages of S. mansoni and that the oligosaccharide lacto-N-fucopentaose III is one target of this response. Lacto-N-fucopentaose III and its specific antibodies may profoundly affect host resistance and parasite homing.

Monoclonal antibodies as tools to quantify mycelium of aquatic hyphomycetes.

Quantitative ELISAs have been developed to determine the biomass of two aquatic hyphomycetes in natural mixed assemblages. These assays employ a species specific rat monoclonal antibody (MAb) raised to Alatospora acuminata and a genus-specific murine MAb raised to Tetracladium marchalianum. The respective antigens are produced constitutively and their production is not affected by a range- of culture conditions. The MAbs can also be used to study the spatial distribution of these fungi using Immunofluorescence. Both antibodies recognize carbohydrate epitopes and belong to the immunoglobulin class IgM. The potential applications of these immunoassays are discussed.

Human thymic epithelial cells express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells.

Thymic epithelial cells play a crucial role in the selection of developing thymocytes. Thymocyte-epithelial cell interactions involve a number of adhesion molecules, including members of the integrin and immunoglobulin superfamilies. We found that human thymic epithelial cells synthesize an endogenous lectin, galectin-1, which binds to oligosaccharide ligands on the surface of thymocytes and T lymphoblastoid cells. Binding of T lymphoblastoid cells to thymic epithelial cells was inhibited by antibody to galectin-1 on the epithelial cells, and by two antibodies, T305 and 2B11, that recognize carbohydrate epitopes on the T cell surface glycoproteins CD43 and CD45, respectively. T lymphoblastoid cells and thymocytes bound recombinant galectin-1, as demonstrated by flow cytometric analysis, and lectin binding was completely inhibited in the presence of lactose. The degree of galectin-1 binding to thymocytes correlated with the maturation stage of the cells, as immature thymocytes bound more galectin-1 than did mature thymocytes. Preferential binding of galectin-1 to immature thymocytes may result from regulated expression of preferred oligosaccharide ligands on those cells, since we found that the epitope recognized by the T305 antibody, the core 2 O-glycan structure on CD43, was expressed on cortical, but not medullary cells. The level of expression of the UDP-GlcNAc:Gal beta 1,3GalNAc-R beta 1, 6GlcNAc transferase (core 2 beta 1, 6 GlcNAc transferase, or C2GnT), which creates the core 2 O-glycan structure, correlated with the glycosylation change between cortical and medullary cells. Expression of mRNA encoding the C2GnT was high in subcapsular and cortical thymocytes and low in medullary thymocytes, as demonstrated by in situ hybridization. These results suggest that galectin-1 participates in thymocyte-thymic epithelial cell interactions, and that this interaction may be regulated by expression of relevant oligosaccharide ligands on the thymocyte cell surface.

Production and characterization of monoclonal antibodies against guinea pig tracheal mucins.

Thirty-five hybridomas that secrete mouse monoclonal antibodies (MAb) against guinea pig (G.P.) tracheal mucins were established. The MAbs were characterized immunologically, biochemically, and immunohistochemically at both light and electron microscopic levels. Isotyping of the MAbs revealed 14 to be IgM, 13 IgG1, 3 IgG2, and 5 IgG3. The MAbs demonstrated various patterns of binding in immunoblots against mucins derived from G.P. tracheal explants. This suggested the presence of "subpopulations" of G.P. tracheal mucins with specific MAbs binding to different epitopes on the mucin molecules. Periodate oxidation indicated that 33 of the 35 MAbs recognized carbohydrate epitopes on the mucin molecules. Ten of the MAbs also reacted with both bovine and ferret tracheal mucins, while 7 and 6 MAbs bound only to bovine and ferret tracheal mucins, respectively. The generated MAbs should be useful for immunomeasurement of mucin secretion in vivo (e.g., in bronchoalveolar or airway lavage fluid) and in vitro (e.g., cell and organ cultures) from cells of guinea pig and (with certain MAbs) bovine and ferret origin.

Molecular analysis of antigens targeted by protective antibodies in rapid expulsion of Trichinella spiralis

Rapid expulsion is a protective immune mechanism which eliminates as much as 99% of a challenge infection of Trichinella spiralis muscle larvae from the gastrointestinal tract of suckling rats. Protective monoclonal antibodies (mAbs) generated against larval excretory-secretory antigens (ESA) specifically recognize a 45-kDa glycoprotein, gp45, in addition to a distinct profile of other cross-reactive antigens that are also recognized by non-protective mAbs. Recent data indicate that protective mAbs recognize carbohydrate epitopes. To complement biochemical studies on the target(s) of rapid expulsion, we describe here the cloning and characterization of the cDNA, TspE1, which belongs to a multigene family and encodes several larval proteins in the 40–50 kDa range. A second cDNA, TspM6 encodes a 45-kDa antigen and is homologous to the published sequence of gp45. Anti-TspE1 antibodies detected antigens within β and γ-stichocytes while anti-TspM6 antibodies detected antigens within α-stichocytes of the secretory organs of muscle larvae. Sequence analysis has provided no functional information on the encoded gene products. Neither recombinant antigen is recognized by the mAbs but native parasite molecules with peptide homology to both the TspE1 and TspM6 recombinant antigens bear the glycan recognized by the protective mAbs. These molecules are candidate targets in rapid expulsion.

Cloning, expression and characterization of the Fv fragments of the anti-carbohydrate mAbs B1 and B5 as single-chain immunotoxins.

The mAbs B1 (IgG1 kappa) and B5 (IgM kappa) recognize carbohydrate epitopes on human carcinoma cells. The Fv regions of these antibodies were separately cloned from hybridoma RNA using reverse transcription and PCR with oligonucleotide primers designed according to the amino acid sequences of the N-termini. The Fv regions also provide sequences for translation initiation in Escherichia coli (Fr1 oligos) and sequences of the constant region of the heavy and light domains (CH1 or C-kappa oligos). Following the determination of the DNA sequence of the Fvs, primers were designed according to the 3' ends of the VH and VL domains. These also provided for a peptide linker at the C-terminus of the VH and a short connector at the C-terminus of the VL (Fr4 oligos). The VH and VL were then each PCR-amplified using their corresponding Fr1 and phosphorylated Fr4 oligos. The resulting PCR products were annealed as 'mutagenic primers' to a uracil-containing single-stranded template obtained from an expression plasmid encoding a single-chain immunotoxin in which the B3 single-chain Fv is fused to a truncated form of Pseudomonas exotoxin (PE). Thus, the B1 and B5 variable domains replaced their corresponding B3 domains in the expression plasmid by 'variable domain shuffling' without subcloning. The resulting B1(Fv)-PE38 and B5(Fv)-PE38 were expressed in E. coli and purified to near homogeneity. Both show specific cytotoxicities to human carcinoma cell lines, but B1(Fv)-PE38 is much more active, reflecting its higher affinity to the target cells.

Monoclonal antibodies raised against membrane glycoproteins from mouse brain recognize N-linked oligomannosidic glycans.

Monoclonal L3 and L4 antibodies have been shown to recognize carbohydrate epitopes on several neural cell adhesion molecules; these epitopes can be released by treatment with endoglycosidase H. In the present study, we have identified the oligosaccharides released by endoglycosidase H from the cell adhesion molecules AMOG and L1 by fast-atom bombardment mass spectrometry as being solely of the oligomannosidic type. Using neoglycolipids of oligomannosidic glycans, we also report that both antibodies show the highest reactivity with the alpha-manno-pentaose Man alpha 1-3-[Man alpha 1-6(Man alpha 1-3)Man alpha 1-6]-Man, but decreasing reactivity with the alpha-manno-hexaose, heptaose, octaose and nonaose glycans. Thus, to our knowledge, we describe here for the first time monoclonal antibodies recognizing N-glycosidically linked oligomannosidic glycans.