Observations on the Binding of Four Anti-carbohydrate Monoclonal Antibodies to Their Homologous Ligands

Abstract

The binding of four monoclonal immunoglobulins, two with specificity for beta(1-->6)-linked D-galactopyranans (IgA X24 and IgA J539) and two with specificity for the chain terminus of alpha(1-->6)-linked d-glucopyranans (IgA W3129 and IgA 16.4.12E), was measured with a number of their homologous oligosaccharide ligands at different temperatures. The results show a linear relationship between lnKa and 1/T, where Ka is the affinity constant and T is the absolute temperature. The unitary free energy of binding, DeltaGu, is virtually independent of T, and the DeltaSu is small when compared with DeltaGu. The enthalpy changes derived from van't Hoff plots are large and negative, indicating an exothermic binding effect, whereas the entropy changes are small and negative, indicating minor overall hydrophobic contributions. Measurements of the free energies of binding, in low and high salt buffers, of methyl beta-d-galactopyranoside and the methyl glycoside of beta(1-->6)-D-galactopyranotetraose with anti-galactan IgA X24 indicate that the monosaccharide has no hydrophobic interaction with the highest affinity subsite of IgA, whereas the tetraoside might have a modest hydrophobic interaction with the three other hapten-binding subsites of IgA. The standard entropy change of binding of the two groups (galactosyl and glucosyl) of oligosaccharides to the two respective sets (anti-galactan and anti-dextran) of antibodies shows a distinct, differing correlation with the hapten chain length within each set. This correlation agrees with the type of association previously established between the antibodies and either the interior determinants of the antigen (in the case of the anti-galactans) or the chain terminus (in the case of the anti-dextrans).

High-Efficiency Intracellular Magnetic Labeling with Novel Superparamagnetic-Tat Peptide Conjugates

“…Although the fluorescence signal was clearly intracellular, we performed additional experiments to exclude the possibility of peptide dissociation. Briefly, cells were stained for dextran using a monoclonal antibody (24). As shown in Figure 5, dextran was concentrated in and distributed through the nucleus thus confirming internalization of the entire construct IV.…”

“…Cells were incubated with IV for 1 h at 20 µg of Fe, washed extensively with HBSS and fixed with MeOH:acetone (1:1) mixture for 10 min at -80°C. After fixation cells were incubated with antidextran IgA antibody W1329 (generous gift from Dr. Glaudemans) (24), followed by incubation with secondary biotinylated goat anti-mouse IgA antibody (Sigma). Biotin was probed for with avidin peroxidase (Bio-Rad, Hercules, CA).…”

Thermodynamics and Density of Binding of a Panel of Antibodies to High-Molecular-Weight Capsular Polysaccharides

“…However, the lower size limit for oligosaccharides needed to demonstrate detectable binding to antibodies is not clearly defined, and very low affinity interactions of smaller oligosaccharides and monosaccharides with antibodies can be detected by using sufficiently sensitive methodologies. Microcalorimetric methods have been used to demonstrate the low-affinity binding of di-, tri-, and tetrasaccharide haptens to the antidextran IgM antibody MOPC-104E (47); methylglycosides and di-and trisaccharides to IgA antidextran and antigalactan antibodies (27); and a Salmonella serogroup B trisaccharide epitope to an IgG MAb (38,39). The binding of two IgA antigalactans to tri-and tetrasaccharides (18) and the binding of methylgalactosides and di-and trisaccharides to an IgM anti-Shigella dysenteria type 1 O antigen (28) were also quantified by fluorescence spectroscopy.…”

Partial Analysis of the Flagellar Antigenic Determinant Recognized by a Monoclonal Antibody to Clostridium tyrobutyricum

“…Indeed, according to the key-lock model, parts of a ligand could compete with this full ligand for the binding site of a receptor. Thus, polysaccharide-associated lectins are eluted by monosaccharides (11) and interactions between antibodies and polysaccharides could be specifically disrupted by monosaccharides (16) and oligosaccharides (14,20 L. BEDOUET ET AL affinity increase with similitude to the natural ligand and the degree of polymerization of glycosidic ligands (13). The general combining area of an antibody is made up of N-terminal ends of the heavy and light chains together.…”