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.

[Immobilization of enzymatic inhibitors for the isolation of reversible immunologic sensors]. / Immobilisation d'inhibiteurs enzymatiques pour la réalisation de capteurs immunologiques réversibles.

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.

[Biosensor development in clinical analysis]. / Evolution des capteurs biologiques en analyse clinique.

The use of enzymes immobilized or as markers formed the subject of more than thousand publications in the field of industry or biomedical applications, during the last five years. Recently, some authors published works concerning immobilization of total microorganisms for catalytic purposes, others use the enzymatic activity for marking molecules involved in immunological analysis processes. Together industrial biotechnology and medical analysis laboratory are interested with the evolution of these procedures involving the activity of immobilized enzymes. Enzyme immobilization allowed the lowering of analysis costs for, in this case, the enzyme can be used several times. We take account of the two main cases which are encountered during utilization of immobilized enzymes of analytical purposes. The enzyme is used directly for the catalysed reaction or it is used as enzymatic marker. These both aspects are developed mainly for the elaboration of enzymatic and immunoenzymatic electrodes and the realization of automatic computerized devices allowing continuous estimation of numerous biological blood parameters. From these two precise examples, glucose and antigen determination, the authors show the evolution of these technologies in the field of immobilized enzymes or captors and the analysis of signals given by these electrodes requiring a computerized treatment. This new technology opens to important potentialities in the analytical field. The automatization of these devices allowing the control in real time, will probably make easier the optimization steps of procedures actually used in the biomedical sphere.

A computerised enzyme immunosensor: application for the determination of antigens.

The present report gives preliminary results of a new sensitive method for the amperometric determination of antigens in serum. This method, developed from the biological model 'hepatitis B surface antigen antibodies' is less time-consuming than most immunochemical techniques, and eliminates many inconveniences arising from use of isotopes. Specific antibodies immobilised onto a gelatin membrane are applied in a solid phase 'sandwich' procedure. The antibodies are labelled with glucose oxidase. The measurement consists of an immunological process and an enzymatic reaction. The second part is carried out by fixing the active membrane onto a pO2 electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen due to the enzyme reaction. This response is correlated to the antigen concentration of the sample. It is a function of both the diffusional and the reactional characteristics of the active layer. Under software conditions, the signal is sampled when the stationary state is obtained. The difference between initial signal and the stationary state signal is measured and compared with the pre-set calibration curve. Use of the computerised enzyme immunosensor could easily be extended to assay of other antigens and haptens that are usually determined by radioimmunoassay.

Use of solid phase biochemistry for potentiometric enzyme immunoassay of oestradiol-17 beta - preliminary report.

The present report gives preliminary results of a new sensitive method for potentiometric determination of oestradiol-17 beta in solution, as an application of the competitive enzyme-linked immunoassay technique. Anti-oestradiol-17 beta antibodies are immobilized on a pig skin gelatin membrane, which is incubated with peroxidase-labelled steroid and oestradiol. After fixation of the membrane onto the sensor of an iodide sensitive electrode, the enzymatic activity was evaluated in the presence of hydrogen peroxide and iodides, the electrode potential being a function of hapten concentration in the solution. The purpose of this preliminary work was to determine the optimized conditions for specificity and sensitivity of the antibody-coated membrane in the presence of peroxidase-labelled oestradiol and oestradiol, and to eliminate possible interferences due to adsorption or ionic fixation of enzyme-labelled steroid. The first tests carried out with oestradiol standard solutions gave satisfactory results at levels ranging from 57 pmol/l to 9.2 nmol/l, suggesting that this new procedure should find application in the determination of oestradiol-17 beta in biological fluids.

An "antibody electrode," preliminary report on a new approach in enzyme immunoassay.

We report preliminary experiments on a new, sensitive, and reliable procedure for enzyme immunoassay of various antigens in biological fluids. The method, developed from the biological model "hepatitis B surface-antigen/antibodies," is less time consuming than most immunochemical techniques and eliminates many inconveniences arising from use of isotopes. We use a solid-phase "sandwich" procedure, the antibodies being immobilized on gelatin membranes, and determine antigen concentration with the help of an iodide-sensitive electrode modified by fixing the active membrane onto the crystal sensor. We have established the analytical criteria of the method and compared it with the solid-phase radioimmunoassay for the surface antigen in dilution series. One-tenth microgram of antigen per liter can be reproducibly detected with our method. Use of antibody electrode should easily be extended to assay of other antigens and haptens that usually are determined by radioimmunoassay.

[Determination of alphaxalone in serum and urine by gas-liquid chromatography. Application in anaesthesiological pharmacology (author's transl)]. / Dosage de l'alphaxalone dans le sérum et les urines par chromatographie gaz-liquide. Application en pharmacologie anesthésiologique.

The authors propose a simple and reliable method for determination of alphaxalone [3 alpha-hydroxy(5 alpha)pregnane 11,20 dione] in serum and urine, during long-duration anaesthesia using Althesin, mixture of alphaxalone and alphadolone acetate [21 acetoxy, 3 alpha-hydroxy(5 alpha)pregnane 11,20 dione]. If assays of alphadolone appear difficult and without any precision, because of numerous interferences, the determination of alphaxalone in serum and urine seems interesting for pharmacokinetic and metabolic investigation. We confirm the quick rate of disappearance of alphaxalone from blood, associated with a very active metabolism in the liver. The results show that it exists a relation between the concentration of alphaxalone in blood and the value of anaesthesia judged on clinical and electrical criteria. We cannot however conclude that Althesin presents a cumulative effect, because we used important amounts of anaesthetic agent.

[Potentiometric determination of hepatitis B surface antigen in biological fluids (author's transl)]. / Détermination potentiométrique de l'antigène de surface de l'hépatite B dans les liquides biologiques.

The present report describes a sensitive and reliable method for potentiometric determination of hepatitis B surface antigen in biological fluids, as an application of enzyme-linked immunoassay technique by a "sandwich" procedure. Specific antibodies are immobilized on a gelatine membrane which covers the sensor of an iodide-sensitive electrode. After immersion of the modified electrode in a dilute solution of antigen, the enzymatic activity is evaluated in a solution of peroxidase-labelled antibodies, in the presence of substrate and iodides. The electrode potential is a function of antigen concentration in the solution. This new procedure should find its application in the determination of substances present in very low concentration in biological fluids.

[A simple method for the separation and purification of the surface antigen of hepatitis B from serum (author's transl)]. / Méthodologic simple de séparation et de purification de l'antigène de surface de l'hépatite B à partir du sérum

The present report describes a simple, rapid and reproducible procedure for preparation of hepatitis B surface antigen from human positive sera, with a satisfactory yield. After removal of beta- and alpha-lipoproteins by dextran sulphate precipitation and subsequent removal of IgM and a part of the other serum proteins by dialysis against distilled water, the supernatant obtained presents almost no change in its surface antigen content. After passing this solution through a molecular sieve of Biogel A-5 m, the surface antigen is detected in a well-defined peak. The fraction correspinding to this peak is collected in its totality. After lyophilization, it is passed through a molecular sieve of CPG 10, in phosphate buffer; this allows recovery of a substance which has retained its structural as well as its antigenic properties, and which is intact and practically free of any serum contamination.

[CT 1341 in continuous administration in 75 cases of prolonged general anesthesia in odontostomatology (constant flow assured by a double-lumen venous catheter and electric perfuser)]. / Etude du CT 1341 en administration continue dans 75 cas d'anesthésie générale prolongée en odontostomatologie (débit constant assuré par canule veineuse à double lumière et perfuseur électrique

The authors used during 75 prolonged general anaesthesia in maxillo-facial surgery CT. 1341 (Alfatesine) administered pure at constant flow rate through a double lumen venous cannula and electric perfuser. Four series of patients were thus distinguished depending on their use or not of N2O as the only analgesic and of gallamine. We do not agree with the classical assertion that CT. 1341 has a cumulative effect on some EEG results. The technique is quite inocuous and the authors propose a technique using CT. 1341 at a dose of 0.1 ml/kg for induction of anaesthesia with, later, 1 mg/kg of gallamine. Under a mixture of oxygen and nitrous oxide at 60 p. 100, the maintenance dose advised in 6.17 microliters per kilo, per minute of CT. 1341.