Fucosyltransferase-catalyzed formation of L-galactosylated Lewis structures.

The Lewis (alpha 1-3/4) fucosyltransferase isolated from human milk could be used for preparative fucosylations of the disaccharide acceptors Gal(beta 1-3)GlcNAc(beta 1-O)R (at position OH-4) and Gal(beta 1-4)GlcNAc(beta 1-O)R (at position OH-3) [R = (CH2)8COOMe]. As donors GDP-L-Gal and deoxygenated derivatives were used to lead to a series of novel modified trisaccharides of the Lewis(a) and the Lewis(x) type, respectively.

Bifunctional fusion proteins consisting of a single-chain antibody and an engineered lanthanide-binding protein.

The combination of an antibody fragment with a lanthanide chelating protein has desirable characteristics for fluorescence-based immunoassays and tumor radioimmunotherapy. As a model for this design, a fusion protein consisting of a single-chain antibody linked to an engineered version of oncomodulin, a protein with two Ca(2+)-binding motifs (the CD and EF loops), was produced by secretion from Escherichia coli in good yield. The single-chain antibody was specific for a Salmonella O-polysaccharide. The CD loop of oncomodulin had been redesigned to bind lanthanide ions with high affinity. The fusion protein was shown to have antigen-binding activity that was comparable to that of the unfused single-chain antibody, to bind Tb3+ with very high affinity and to give strong, sensitized Tb3+ luminescence via excitation of the tryptophan residue in the CD loop. A second fusion protein containing a 30-residue helix-loop-helix motif as the lanthanide-binding component was also prepared, but showed considerably lower solubility. Competition for Tb3+ binding by a series of metal chelators indicated that the affinities of the oncomodulin and 30 residue fusions for Tb3+ were approximately 10(11) M-1 and 10(7) M-1, respectively. Time-resolved lanthanide luminescence photography of electrophoresis gels demonstrated that the helix-loop-helix Ca(2+)-binding could be used to specifically visualize the scFv fragment.

Synthesis and expression in Escherichia coli of DNA encoding the murine lambda 1 chain of a monoclonal antibody specific for Salmonella serotype B O-antigen.

A 658 bp DNA sequence corresponding to the murine lambda 1 chain of a monoclonal antibody, Se155-4, specific for the Salmonella serotype B O-antigen, was designed using Escherichia coli preferred codons and chemically synthesized by ligation of synthetic fragments into a linearized plasmid followed by transformation into E. coli. A synthetic signal peptide (ompA) was fused to express the L chain as a free polypeptide into the periplasm of E. coli cells. After isolation and purification, heterologous recombination of the E. coli L chain with mouse H chain gave an active antigen-binding protein. The activity was 15-20% when compared to protein created by an equivalent association of isolated natural mouse L and H chains as measured by a direct EIA assay. In inhibition experiments with the polysaccharide antigen, the two proteins showed identical titration curves and 50% inhibition points, indicating comparable KA values.

Definition of Brucella A and M epitopes by monoclonal typing reagents and synthetic oligosaccharides.

The paradigm that Brucella A and M epitopes are simultaneously expressed on single cells and within one antigen molecule was reinvestigated by using polysaccharide-specific murine monoclonal antibodies. Monoclonal antibodies were generated to the M antigen of Brucella melitensis 16M. Chemically defined lipopolysaccharides and O polysaccharides from Brucella abortus 1119-3, B. melitensis 16M, and Yersinia enterocolitica O:9 were used to dissect the binding profiles of the B. melitensis antibodies and an additional set of antibodies available from a B. abortus fusion experiment. Binding specificities were rationalized in terms of prototype A- and M-antigen structures, an interpretation supported by competitive binding studies with O polysaccharides and synthetic oligosaccharide analogs of the A and M antigens. Three binding patterns were characterized. Antibodies specific for the A antigen required five contiguous alpha 1,2-linked 4,6-dideoxy-4-formamido-D-mannopyranosyl residues, while antibodies with equal affinities for A or M epitopes were effectively inhibited by alpha 1,2-linked tri- or tetrasaccharides. Specificity for the M epitope correlated with binding of a critical disaccharide element alpha-D-Rha4NFo(1----3)alpha-D-Rha4NFo bracketed by alpha 1,2-linked residues. The binding profiles of Brucella monoclonal antibodies were consistent with the concept of simultaneous expression of A and M epitopes within a single molecule. A epitopes were present in the M antigen, and the discovery of isolated alpha 1,3 linkages in the A antigen suggests that M epitopes occur in all A antigens. Three monoclonal antibodies are proposed as standard reagents for the detection and identification of Brucella A and M antigens.

Identification of Escherichia coli serotype O157 strains by using a monoclonal antibody.

The O157 antigenic determinant of Escherichia coli serotype O157:H7, an important bacterial pathogen, resides in the polysaccharide portion of its cellular lipopolysaccharide component which, from structural studies, was identified as a linear polymer of a repeating tetrasaccharide unit composed of D-glucose, L-fucose, 2-acetamido-2-deoxy-D-galactose, and 4-acetamido-4,6-dideoxy-D-mannose residues (1:1:1:1). Hybrid cells producing monoclonal antibodies against the E. coli O157 antigen were obtained by fusion of myeloma cells with lymphocytes from BALB/c mice immunized with killed E. coli O157:117 cells. Clones were selected for binding specificity with purified O polysaccharide. One monoclonal antibody used in direct slide agglutinations or in coagglutination reactions with Staphylococcus aureus Cowan 1 cells sensitized with the affinity column-purified antibody accurately detected all strains of E. coli O157 tested. This selected monoclonal antibody did not agglutinate E. coli strains such as E. coli O7 and E. coli O116 or other bacteria which are known to give positive agglutinations with conventional polyclonal E. coli antisera. These results indicate that the monoclonal antibody is a superior specific-typing reagent.

Development and characterization of monoclonal antibodies to pregnancy-specific beta 1-glycoprotein.

Six monoclonal antibodies were developed to pregnancy-specific beta 1-glycoprotein (PS beta 1G). Studies of ascitic fluid antibodies by a double-antibody radioimmunoassay (RIA) included an evaluation of titers, dose-response parameters, and mass action properties. Four of the antibodies demonstrated moderate to high titers ranging from 1/40 000 to greater than 1/120 000, as determined by the specific binding of 125I-labeled PS beta 1G. In inhibition studies utilizing a standard containing known quantities of placental PS beta 1G, two of the antibodies (AR#11 and B#2) were highly sensitive and only slightly lower in this regard than a high affinity polyclonal antiserum. The binding affinities of AR#11 and B#2 monoclonals were greater than 10(9) mol-1 which underline their importance as potential clinical reagents for the RIA of PS beta 1G. The Scatchard plots, for several of the antibodies, were linear and in full agreement with a single order of binding sites predicted for specific monoclonal reagents. Immunodiffusion results provide preliminary evidence that at least three distinct determinants on PS beta 1G are recognized by a number of the monoclonal antibodies. Further studies on the fine specificities of the antibodies by solid-phase RIA, as well as a detailed evaluation of their clinical applications, are in progress.

Hybridomas specific for carbohydrates; synthetic human blood group antigens for the production, selection, and characterization of monoclonal typing reagents.

A general method for the production of carbohydrate-specific hybridoma antibodies is illustrated by generation of monoclonal antibody to the antigenic determinant of human blood group B. This trisaccharide determinant was chemically synthesized and covalently coupled to bovine serum albumin and human blood group O red cells. Soluble protein antigen and the 'artificial' B red cells were used to immunize BALB/c mice before fusion of spleen cells with the Sp2/0 plasmacytoma cell line. ELISA screening of putative hybrids for B-specific binding activity was facilitated by the availability of a second synthetic conjugate, B-horse hemoglobin. IgM-producing clones were identified by class-specific ELISA reagents and by hemagglutination assay. In this way, clones suitable for blood typing were rapidly identified. The precise antigenic specificity and Ig class of such monoclonal antibodies were defined by inhibition of precipitation and by gel filtration. Hybridoma antibodies were obtained from two separate fusion experiments. One of these, clone 3E-4, was of the IgM class and possessed a binding site that was completely satisfied (100% inhibition) by the trisaccharide determinant of the B blood group. This antibody is shown to be suitable for use in blood typing.

Binding of disaccharides by peanut agglutinin as studied by ultraviolet difference spectroscopy.

The binding of the disaccharides methyl beta-D-lactoside and 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-beta-D-galactopyranose [beta-D-Gal-(l leads to 3)-D-GalNAc] to peanut agglutinin was studied by ultraviolet difference spectroscopy. The magnitude of the difference spectra varied with the concentration of the carbohydrates; association constants and thermodynamic parameters were determined from titration experiments at different temperatures. The enthalpy and entropy changes for binding of methyl beta-D-lactoside were found to be delta H degree = -65 +/- 4 kJ mol-1, delta S degree = -156 +/- 14 J mol-1 K-1. For beta-D-Gal-(1 leads to 3)-D-GalNAc the observed thermodynamic parameters were delta H degree = -78 +/- 5 kJ mol-1,, delta S degree = -177 +/- 16 J mol-1 K-1. For both disaccharides, the enthalpy change upon binding to the lectin is much larger than found for the binding site on peanut agglutinin. The observed parameters are compared with those found for the binding of monosaccharides and oligosaccharides to other lectins and to lysozyme. Molecular models of the minimum energy conformers of beta-D-Gal(1 leads to 3)-D-GalNAc and methyl beta-D-lactoside are used to interpret the interaction of these, and structurally related ligands, with the peanut agglutinin binding site.