Dissociation of immunocomplexes by ionic shock for the development of immunosensors: application to measurement of alpha 1-fetoprotein.

We report here our experimental results on the reaction rate of immunological complexes and on potential repeated use of membranes as a result of dissociation of the complexes between alpha 1-fetoprotein and catalase-labeled antibodies by different buffers with various ionic strengths. The measurement consists of an immunological process and an enzymatic reaction. The protein membrane activated by glutaraldehyde for the immobilization of antibodies is fixed over an oxygen electrode. After incubation with antibody/alpha 1-fetoprotein and antibody/alpha 1-fetoprotein antibodies coupled to catalase, the reaction medium is introduced into a continuous-flow cell. Oxygen production by the catalase is measured on-line, with the electrode in contact with hydrogen peroxide. This response is correlated to the alpha 1-fetoprotein concentration of the sample. We show a typical calibration curve between 0.5 and 120 micrograms/L. Replicate (n = 20) equilibrium measurement with the same membrane gave a CV of 2.2%. The reversible immunochemical sensor has been tested for 30 measurements without significant loss of activity.

Development of a reversible bienzymatic system in immunoenzymatic technique.

The use of specific captors solubilized with a ligand on to proteic membranes together with an automatic-computerized system is proposed for the determination of various haptens or antigens in biological and industrial fluids by an enzyme-linked immunoassay test. Two enzymes are used in this technique: the first enzyme for linking reversibly the immunocomplex to the insoluble matrix, the substrate of this enzyme being immobilized on the matrix; the second (beta-d-glucose oxidase) for labelling the antigen. Its activity is measured by fixing the immunocomplex gelatin membrane on to a pO(2) sensor. After incubation of the antigen labelled with glucose oxidase and the free antigen with specific antibodies linked with the first enzyme in a pre-established concentration, the reaction medium is introduced inside the continuous flow cell. O(2) consumption due to the enzyme reaction is measured in actual time when the electrode is in contact with the glucose standard solution. The signal is correlated by an injection of urea solution. The signal is processed with an automated microcomputer system.