A heterogeneous immunoassay performed on a rotating carbon disk electrode with electrocatalytic detection. Mass transfer control of the capture of an enterotoxin.
An ELISA procedure for the determination of enterotoxin A from Staphylococcus aureus conducted on the surface of a glassy carbon electrode is described. The electrocatalytic detection of the immobilized labelled second antibody is based upon the electrochemical reaction and the enzymatic catalysis occurring on the same surface. The indirect quantification of the bound antigen is, therefore, very sensitive (10(-15) mol cm-2). This heterogeneous technique was used to study the kinetics of antigen binding to the immunological solid phase, the mass transfer of the antigen being controlled under well-defined hydrodynamic conditions. The experiments were performed with a rotating solid phase disk in such a way that thickness of the diffusion layer was known. We found that the capture of the antigen by the immobilized monoclonal antibody was solely limited by diffusion. A simple theoretical model permitted the amount of bound antigen and the sensitivity of the method to be predicted as a function of the incubation time, the rotational speed of the solid phase and the volume of the sample. Both the theory and the experimental results indicate that the assay may be performed with the sample volume undefined.
Huet
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D
Labortoire de Technologie Enzymatique, URA 523 du CNRS, Université de Compiègne, France.
Gyss
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C
Bourdillon
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C
D000327:Adsorption; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D004563:Electrochemistry; D004566:Electrodes; D004768:Enterotoxins / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007700:Kinetics; D008433:Mathematics; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D013211:Staphylococcus aureus
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D004768:Enterotoxins; C026066:enterotoxin A, Staphylococcal