A kinetics approach to the characterization of an IgM specific for the glycolipid asialo-GM1.
The unique features of protein recognition of membrane-anchored glycolipids were investigated by surface plasmon resonance (SPR) monitoring of antibody interactions with glycolipids contained in liposomes. Several positive hybridomas belonging to the IgM and IgG classes were identified when tested for binding to the glycosphingolipid asialo-GM1 (Gal beta1-3GalNAcl beta1-4Gal beta1-4Glc beta1-1-Ceramide). Preliminary screening by enzyme immunoassay and thin layer chromatography (TLC) followed by immunostaining indicated that only those of the IgM type showed specificity for this glycosphingolipid. One of the IgMs, H2G10, was purified and further characterized using a SPR technique that involved antibody binding to liposomal asialo-GM1. This method generated kinetic and affinity constants for the interaction and confirmed the specificity of H2G10 for the terminal galactose of asialo-GM1. Interestingly, inhibition of antibody binding to asialo-GM1 liposomes by the asialo-GM1 tetrasaccharide reduced the total amount of bound antibody but increased the affinity of the antigen-antibody interaction due to an inverse relationship between tetrasaccharide concentration and the H2G10 dissociation rate constant. We believe that this effect is due to the selective inhibition of lower valency binding by the tetrasaccharide which, in turn, promotes higher avidity antibody-carbohydrate interactions. The observation that slower dissociation rate constants were also observed at high antigen to antibody ratios supports this interpretation. These results highlight the insight that kinetic data can provide in efforts to promote and inhibit high avidity interactions such as those involving proteins and carbohydrates.
Harrison
B A
BA
Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario.
MacKenzie
R
R
Hirama
T
T
Lee
K K
KK
Altman
E
E
D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D000918:Antibody Specificity; D015374:Biosensing Techniques; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D005677:G(M1) Ganglioside / Q000276:immunology*; D007124:Immunoenzyme Techniques; D007075:Immunoglobulin M / Q000276:immunology*; D007700:Kinetics; D008081:Liposomes; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D011956:Receptors, Cell Surface / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D008081:Liposomes; D011956:Receptors, Cell Surface; D005677:G(M1) Ganglioside; C018835:asialo GM1 ganglioside