2-DIMENSIONAL PARACRYSTALLINE GLYCOPROTEIN S-LAYERS AS A NOVEL MATRIXFOR THE IMMOBILIZATION OF HUMAN-IGG AND THEIR USE AS MICROPARTICLES IN IMMUNOASSAYS

In the present study, cup-shaped 1-3 mu m large cell wall fragments fr om Thermoanaerobacter thermohydrosulfuricus L111-69 covered with a hex agonal S-layer lattice composed of glycoprotein subunits were shown to act as a matrix for tile immobilization of human IgG. After cross-lin king the S-layer glycoprotein lattice with glutaraldehyde (S-layer mic roparticles), IgG was either bound to carbodiimide activated carboxyl groups from acidic amino acids from the protein moiety or to the carbo hydrate chains activated with cyanogen bromide or oxidized with period ate. After determining the binding capacity of the S-layer lattice for human IgG, the orientation of the immobilized antibody molecules was investigated using anti-human IgG peroxidase conjugates with different specificity. Attachment of S-layer microparticles with covalently bou nd human IgG to microplates precoated with anti-human IgG of different specificity led to clear correlations between the amount of applied h uman IgG and the absorption values in the immunoassays. The steepest a bsorption curves were obtained when human IgG was bound to the carbohy drate chains exposed on the surface of the S-layer lattice. This confi rmed that the location and the accessibility of the immobilized antibo dies on S-layer microparticles is of major importance for the response in immunoassays. In addition to the high reproducibility of the amoun t of IgG which could be bound to the S-layer lattice and the high repr oducibility. of the absorption curves in the immunoassays, one major a dvantage of using cup-shaped S-layer microparticles can be seen in the considerable increase of the actual surface available for binding pro cesses and immunological reactions.