Two-dimensional paracrystalline glycoprotein S-layers as a novel matrix for the immobilization of human IgG and their use as microparticles in immunoassays.
In the present study, cup-shaped 1-3 microns large cell wall fragments from Thermoanaerobacter thermohydrosulfuricus L111-69 covered with a hexagonal S-layer lattice composed of glycoprotein subunits were shown to act as a matrix for the immobilization of human IgG. After cross-linking the S-layer glycoprotein lattice with glutaraldehyde (S-layer microparticles), IgG was either bound to carbodiimide activated carboxyl groups from acidic amino acids from the protein moiety or to the carbohydrate chains activated with cyanogen bromide or oxidized with periodate. 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 bound human IgG to microplates precoated with anti-human IgG of different specificity led to clear correlations between the amount of applied human IgG and the absorption values in the immunoassays. The steepest absorption curves were obtained when human IgG was bound to the carbohydrate chains exposed on the surface of the S-layer lattice. This confirmed that the location and the accessibility of the immobilized antibodies on S-layer microparticles is of major importance for the response in immunoassays. In addition to the high reproducibility of the amount of IgG which could be bound to the S-layer lattice and the high reproducibility of the absorption curves in the immunoassays, one major advantage of using cup-shaped S-layer microparticles can be seen in the considerable increase of the actual surface available for binding processes and immunological reactions.
Küpcü
S
S
Zentrum für Ultrastrukturforschung, Universität für Bodenkultur, Vienna, Austria.
Sleytr
U B
UB
Sára
M
M
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D002241:Carbohydrates / Q000737:chemistry / Q000276:immunology; D002473:Cell Wall / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D003460:Crystallization; D006023:Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D016988:Gram-Positive Asporogenous Rods, Irregular / Q000737:chemistry / Q000276:immunology / Q000648:ultrastructure; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D010316:Particle Size
D001425:Bacterial Outer Membrane Proteins; D002241:Carbohydrates; D006023:Glycoproteins; D007074:Immunoglobulin G