Activity in saline of phthalylated or succinylated derivatives of mycobacterial water-soluble adjuvant. (1/2830)

A water-soluble fraction (WSA) of the cell wall can substitute for mycobacterial cells in Freund complete adjuvant. However, when WSA is administered in saline instead of in a water-in-oil emulsion, its adjuvant activity is very weak, and under certain experimental conditions it can even inhibit the humoral immune response. The data reported in the present study show that after treatment by phthalic or succinic anhydride the adjuvant activity of WSA was markedly changed, since high levels of circulating antibodies were produced when these derivatives were administered with an antigen in an aqueous medium. Moreover, the antigenic determinants of WSA were modified and acylated WSA had no tuberculin-like activity.  (+info)

Variable domain-linked oligosaccharides of a human monoclonal IgG: structure and influence on antigen binding. (2/2830)

The variable-domain-attached oligosaccharide side chains of a human IgG produced by a human-human-mouse heterohybridoma were analysed. In addition to the conserved N-glycosylation site at Asn-297, an N-glycosylation consensus sequence (Asn-Asn-Ser) is located at position 75 in the variable region of its heavy chain. The antibody was cleaved into its antigen-binding (Fab) and crystallizing fragments. The oligosaccharides of the Fab fragment were released by digestion with various endo- and exoglycosidases and analysed by anion-exchange chromatography and fluorophore-assisted carbohydrate electrophoresis. The predominant components were disialyl- bi-antennary and tetra-sialyl tetra-antennary complex carbohydrates. Of note is the presence in this human IgG of oligosaccharides containing N-glycolylneuraminic acid and N-acetylneuraminic acid in the ratio of 94:6. Furthermore, we determined N-acetylgalactosamine in the Fab fragment of this antibody, suggesting the presence of O-linked carbohydrates. A three-dimensional structure of the glycosylated variable (Fv) fragment was suggested using computer-assisted modelling. In addition, the influence of the Fv-associated oligosaccharides of the CBGA1 antibody on antigen binding was tested in several ELISA systems. Deglycosylation resulted in a decreased antigen-binding activity.  (+info)

The role of homophilic binding in anti-tumor antibody R24 recognition of molecular surfaces. Demonstration of an intermolecular beta-sheet interaction between vh domains. (3/2830)

The murine antibody R24 and mouse-human Fv-IgG1(kappa) chimeric antibody chR24 are specific for the cell-surface tumor antigen disialoganglioside GD3. X-ray diffraction and surface plasmon resonance experiments have been employed to study the mechanism of "homophilic binding," in which molecules of R24 recognize and bind to other molecules of R24 though their heavy chain variable domains. R24 exhibits strong binding to liposomes containing disialoganglioside GD3; however, the kinetics are unusual in that saturation of binding is not observed. The binding of chR24 to GD3-bearing liposomes is significantly weaker, suggesting that cooperative interactions involving antibody constant regions contribute to R24 binding of membrane-bound GD3. The crystal structures of the Fabs from R24 and chR24 reveal the mechanism for homophilic binding and confirm that the homophilic and antigen-binding idiotopes are distinct. The homophilic binding idiotope is formed largely by an anti-parallel beta-sheet dimerization between the H2 complementarity determining region (CDR) loops of two Fabs, while the antigen-binding idiotope is a pocket formed by the three CDR loops on the heavy chain. The formation of homophilic dimers requires the presence of a canonical conformation for the H2 CDR in conjunction with participation of side chains. The relative positions of the homophilic and antigen-binding sites allows for a lattice of GD3-specific antibodies to be constructed, which is stabilized by the presence of the cell membrane. This model provides for the selective recognition by R24 of cells that overexpress GD3 on the cell surface.  (+info)

Ganglioside GM2-activator protein and vesicular transport in collecting duct intercalated cells. (4/2830)

This study describes the molecular characterization of an antigen defined by an autoantibody from a woman with habitual abortion as GM2-activator protein. The patient showed no disorder of renal function. Accidentally with routine serum screening for autoantibodies, an immunoreactivity was found in kidney collecting duct intercalated cells. Three distinct patterns of immunostaining of intercalated cells were observed: staining of the apical pole, basolateral pole, and diffuse cytoplasmic labeling. Ultrastructurally, the immunoreactivity was associated with "studs," which represent the cytoplasmic domain of the vacuolar proton pump in intercalated cells. This pump is subjected to a shuttling mechanism from cytoplasmic stores to the cell membrane, which exclusively occurs in intercalated cells. Peptide sequences of a 23-kD protein purified from rat kidney cortex showed complete identity with corresponding sequences of GM2-activator protein. In the brain, GM2-activator protein is required for hexosaminidase A to split a sugar from ganglioside GM2. Because neither ganglioside GM2 nor GM1 (its precursor) is present in significant amounts in the kidney, the previous finding that this tissue contains the highest level of activator protein in the body was confusing. In this study, a novel role for GM2-activator protein in intercalated cells is proposed, and possible roles in the shuttling mechanism are discussed.  (+info)

In vitro comparison of the antigen-binding and stability properties of the various molecular forms of IgA antibodies assembled and produced in CHO cells. (5/2830)

The hallmark of a mucosal immune response is the production of antigen-specific secretory IgA (S-IgA) antibodies in external secretions. S-IgA consists of ten polypeptides produced in two different cell lineages. The heavy and light chains in plasma cells assemble into IgA, which on association with J chain become polymerized, whereas secretory component (SC) is added during transport across the epithelium. Recombinant chimeric mouse-human monomeric, dimeric, and S-IgA antibodies have been produced in a single CHO cell sequentially transfected with expression vectors carrying three independent selective markers for chimeric heavy and light chains, human J chain, and human SC, respectively. Biochemical characterization of the various molecular forms indicates that the assembly of the various polypeptides resulted in species of the expected size and covalence. All chimeric IgA antibodies retained the antigen-binding capacity of the parent mouse IgA antibody. The resistance of S-IgA to protease-rich intestinal washes was enhanced when compared with dimeric IgA lacking associated SC. Up to 20 micrograms of recombinant S-IgA per 1 x 10(6) cells were recovered in 24 h with the best producing clones. We conclude that CHO cells programmed de novo with four different genetic elements can assemble functional chimeric S-IgA.  (+info)

Antifactor VIII antibody inhibiting allogeneic but not autologous factor VIII in patients with mild hemophilia A. (6/2830)

Two unrelated patients with the same Arg2150His mutation in the factor VIII (FVIII) C1 domain, a residual FVIII activity of 0.09 IU/mL, and inhibitor titres of 300 and 6 Bethesda Units, respectively, were studied. Further analysis of patient LE, with the highest inhibitor titer, showed that (1) plasma or polyclonal IgG antibodies prepared from LE plasma inhibited the activity of allogeneic (wild-type) but not of self FVIII; (2) the presence of von Willebrand factor (vWF) increased by over 10-fold the inhibitory activity on wild-type FVIII; (3) the kinetics of FVIII inhibition followed a type II pattern, but in contrast to previously described type II inhibitors, LE IgG was potentiated by the presence of vWF instead of being in competition with it; (4) polyclonal LE IgG recognized the FVIII light chain in enzyme-linked immunosorbent assay and the recombinant A3-C1 domains in an immunoprecipitation assay, indicating that at least part of LE antibodies reacted with the FVIII domain encompassing the mutation site; and (5) LE IgG inhibited FVIII activity by decreasing the rate of FVIIIa release from vWF, but LE IgG recognized an epitope distinct from ESH8, a murine monoclonal antibody exhibiting the same property. We conclude that the present inhibitors are unique in that they clearly distinguish wild-type from self, mutated FVIII. The inhibition of wild-type FVIII by LE antibody is enhanced by vWF and is associated with an antibody-dependent reduced rate of FVIIIa release from vWF.  (+info)

Antibody response to antigens distinct from smooth lipopolysaccharide complex in Brucella infection. (7/2830)

The smooth lipopolysaccharide complex of the outer surface of smooth Brucella abortus cells is believed to be the antigenic component involved in serological tests routinely used for the diagnosis of brucellosis. Sera from cattle vaccinated or infected with B. abortus generally contain antibody directed toward the smooth lipopolysaccharide complex. The brucella organism contains a large number of other antigenically distinct components. The biological significance of some of these antigens has been demonstrated by showing that sera from infected cattle have precipitins to these components. These sera revealed up to seven distinct lines in immunoelectrophoresis with a protein-rich antigen mixture prepared from rough strain B. abortus 45/20, whereas sera from strain 19-vaccinated cattle did not reveal these lines at 4 or more months after vaccination. Monospecific antisera were prepared against six antigens in this mixture, and the purification of two of them by antibody affinity chromatography is described.  (+info)

Cyanobacterial phycobilisomes. Characterization of the phycobilisomes of Synechococcus sp. 6301. (8/2830)

A procedure is described for the preparation of stable phycobilisomes from the unicellular cyanobacterium Synechococcus sp. 6301 (also known as Anacystis nidulans). Excitation of the phycocyanin in these particles at 580 nm leads to maximum fluorescence emission, from allophycocyanin and allophycocyanin B, at 673 nm. Electron microscopy shows that the phycobilisomes are clusters of rods. The rods are made up of stacks of discs which exhibit the dimensions of short stacks made up primarily of phycocyanin (Eiserling, F. A., and Glazer, A. N. (1974) J. Ultrastruct. Res. 47, 16-25). Loss of the clusters, by dissociation into rods under suitable conditions, is associated with loss of energy transfer as shown by a shift in fluorescence emission maximum to 652 nm. Synechococcus sp. 6301 phycobilisomes were shown to contain five nonpigmented polypeptides in addition to the colored subunits (which carry the covalently bound tetrapyrrole prosthetic groups) of the phycobiliproteins. Evidence is presented to demonstrate that these colorless polypeptides are genuine components of the phycobilisome. The nonpigmented polypeptides represent approximately 12% of the protein of the phycobilisomes; phycocyanin, approximately 75%, and allophycocyanin, approximately 12%. Spectroscopic studies that phycocyanin is in the hexamer form, (alpha beta)6, in intact phycobilisomes, and that the circular dichroism and absorbance of this aggregate are little affected by incorporation into the phycobilisome structure.  (+info)