Quantitation of monomeric and oligomeric forms of membrane-bound staphylococcal alpha-toxin by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody.
A murine monoclonal antibody generated against staphylococcal alpha-toxin was shown to react only with the monomeric (native), 3S form of the toxin. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) constructed with this antibody permitted detection of 0.25 to 0.5 ng of native toxin per ml. Toxin oligomers formed either by heat aggregation in solution, on target erythrocyte membranes, or on phosphatidylcholine-cholesterol liposomes were unreactive in the ELISA when membranes were solubilized with the nondenaturing detergent Triton X-100. After dissociation of the oligomers by boiling in sodium dodecyl sulfate, however, the ELISA reactivity of the liberated 3S toxin was fully restored. Parallel determinations of membrane-bound toxin with sodium dodecyl sulfate and Triton X-100 solubilization thus permitted direct quantitation of total and monomeric toxin, respectively; the difference between these two values was represented by toxin oligomers. The detection limits for membrane-bound oligomeric and monomeric toxin on erythrocyte membranes are in the order of 100 molecules and 1 molecule per cell, respectively. Using this ELISA, we show that over 90% of alpha-toxin molecules bound to target membranes at 37 degrees C are in oligomeric form. Evidence is given that the monoclonal antibody neutralizes alpha-toxin by inhibiting its binding to both rabbit and human erythrocytes. This ELISA is the first assay that quantitatively discriminates between mono- and oligomeric forms of a pore-forming protein on target cell membranes.
Hugo
F
F
Institute of Medical Microbiology, University of Giessen, Federal Republic of Germany.
Sinner
A
A
Reichwein
J
J
Bhakdi
S
S
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001427:Bacterial Toxins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004912:Erythrocytes / Q000378:metabolism; D006460:Hemolysin Proteins; D008081:Liposomes; D046911:Macromolecular Substances; D008970:Molecular Weight; D009500:Neutralization Tests
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008081:Liposomes; D046911:Macromolecular Substances; C021091:staphylococcal alpha-toxin