[Immobilization of enzymatic inhibitors for the isolation of reversible immunologic sensors].
The authors propose the use of specific sensors immobilized by ligands onto artificial supports, and the elaboration of a computerized system for the determination of various antigens, haptens or antibodies in biological fluids according to enzyme-linked immunosorbent assay techniques. Two enzymes are applied in this technique: the first (ribonuclease) for reversibly linking the immunocomplex to the insoluble support via disulphur bridges; the second (beta-D-glucose oxidase) for labelling the antigen. Enzyme activity is measured in the presence of glucose oxidase by fixing the immunocomplex onto a pO2 electrode. After incubation of the antigen labelled with glucose oxidase and the free antigen with specific antibodies linked with ribonuclease, to reduce the pre-established concentration, the reaction medium is introduced into the continuous flow cell. O2 consumption due to the enzyme reaction is measured by the actual time that the electrode is in contact with a glucose standard solution. Cleavage of the disulphur bridges is caused by an injection of dithiothreitol solution. Treatment of the signal obtained is realized with an automatic microcomputer system. The preliminary results show that reproducibility with the same membrane for ten measurements is less than 5%. Elution performed using dithiothreitol for example, shows that cleavage between the immunocomplex and the thiol-containing support is obtained after a few minutes, and 98% of the immunocomplex is eluted.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D016227:Benzoquinones; D002234:Carbodiimides; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D005780:Gelatin; D005947:Glucose / Q000032:analysis; D005949:Glucose Oxidase / Q000302:isolation & purification; D005976:Glutaral / Q000032:analysis; D006041:Goats / Q000276:immunology; D007074:Immunoglobulin G / Q000302:isolation & purification; D008567:Membranes, Artificial; D011809:Quinones / Q000032:analysis; D011817:Rabbits; D012260:Ribonucleases / Q000302:isolation & purification
D000906:Antibodies; D016227:Benzoquinones; D002234:Carbodiimides; D004791:Enzyme Inhibitors; D007074:Immunoglobulin G; D008567:Membranes, Artificial; D011809:Quinones; C004532:quinone; D005780:Gelatin; D005949:Glucose Oxidase; D012260:Ribonucleases; D005947:Glucose; D005976:Glutaral
Immobilisation d'inhibiteurs enzymatiques pour la réalisation de capteurs immunologiques réversibles.