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Biomedical subjects

C Rivat

Publications and source records attributed to C Rivat.

At least 19 recordsLinked to original sources
PubMed1992

Compared stability of Sepharose-based immunoadsorbents prepared by various activation methods.

During the use of chromatographic supports for the purification of proteins or the selective removal of substances by immunoaffinity, leakage of the antibodies immobilized on the matrix is systematically observed. When the cleansing of blood plasma by extracorporeal circulation is concerned, it is of prime importance that the immunoadsorbents exhibit an extensive chemical stability over the whole range of experimental conditions. To study and minimize this leakage, a matrix, Sepharose CL-4B, was activated by various chemical reagents and coupled to goat anti-apolipoprotein B polyclonal antibodies. Immunoadsorbents thus prepared were compared with those obtained earlier by cyanogen bromide activation. It turns out that divinyl sulphone- and tresyl chloride-activated supports lead to similar results in terms of coupling yield and adsorption capacity, but to a significant reduction in released antibodies.

Animals↗
PubMed1992

Purification of biologically active human plasma transthyretin by dye-affinity chromatography: studies on dye leakage and possibility of heat treatment for virus inactivation.

The application of a purification procedure for the industrial preparation from human plasma of a therapeutic protein may be hindered by several safety concerns. The dye leaching from Remazol Yellow GGL-Sepharose used for the affinity chromatography of human plasma transthyretin was quantitatively studied by a sensitive competitive enzyme immunoassay. The possibility of including a heat treatment step for virus inactivation in the purification process while preserving the biochemical and functional characteristics of the protein is also reported.

Azo Compounds↗
PubMed1992

Single-step method for purification of human transferrin from a by-product of chromatographic fractionation of plasma.

A rapid, simple and convenient method is described for the isolation, on a pilot scale, of pure and functional human transferrin from an unexploited by-product of chromatographic fractionation of plasma. In a single chromatographic step on DEAE-Spherodex, 97% pure transferrin was obtained in 75% yield. A virus inactivation treatment was included in the preparative process in order to guarantee the safety of the final product, which could be used in culture media.

Chromatography, Ion Exchange↗
PubMed1992

Dye-affinity purification of transthyretin from an unexploited by-product of human plasma chromatographic fractionation.

Dye-affinity chromatography of human plasma transthyretin on Remazol Yellow GGL-Sepharose from an unexploited by-product of chromatographic fractionation of plasma was optimized for large-scale preparation of a therapeutic product. With this system, transthyretin is only weakly bound to the gel. The residence time on gel and the transthyretin level in the by-product were observed to have no influence on the binding capacity of gel, and the optimum amount of transthyretin to be applied to the gel was found to be 1 g/l of gel. The adsorbent can be used more than ten times. The procedure resulted in the isolation, with a 30% yield with respect to plasma, of an 80% pure protein, which retained its thyroxine-binding capacity. Although the purity is acceptable for substitutive therapy, it can be improved further with a second chromatography on Cibacron Blue-Sepharose.

Adsorption↗
PubMed1992

A sensitive enzyme immunoassay for the detection of a synthetic affinity ligand, the reactive yellow 13 dye.

Dye-affinity chromatography is widely and increasingly used for the isolation of various proteins. In particular, the purification of transthyretin can be efficiently achieved by affinity chromatography on immobilized Reactive Yellow 13. Measurement of trace-amounts of dye leaching from affinity columns is important because of possible toxicity or side effects linked with the presence of dye in therapeutic transthyretin preparations. A competitive enzyme immunoassay was developed to monitor yellow-dye column leaching. Biotinylated rabbit anti-Reactive Yellow 13 antibodies (immunoglobulin G fraction) were used as principal reagent. The assay is specific sensitive to 1 ng/ml of Reactive Yellow 13, has a good reproducibility and allows the accurate detection of the dye in the presence of transthyretin.

Affinity Labels↗
PubMed1992

[Affinity chromatography for extracorporeal purification or for separation of molecules for biological use: technical and legal restraints].

Affinity and/or immunoaffinity chromatography is one of the less commonly method used for large scale separation purposes. As this technic is greatly selective and gives high yields, it allows in vivo removal, in an extracorporeal circulation, of harmful substances in patient plasma as well as purification of molecules for biological and therapeutic uses. Large development with increased safety of such a method is dependent on some economic, technical and legal requirements. Particular attention must be drawn to the following points. Possibility of multiple uses of affinity supports contributes to lowering the cost of the method. Desorption agents should be chosen according to the best compromise between total desorption and preservation of adsorption capacity over runs. It is likewise essential to store affinity supports in conditions allowing prevention from contamination without loss of adsorption capacity. In developing and optimizing an affinity chromatography procedure, it is important to minimize the amount of ligand released from the affinity support. Besides economic and technical aspects, it is necessary to take into account legal requirements, particularly those related to the preparation of the ligand especially if it is monoclonal or polyclonal antibodies. An important consideration for the therapeutic use of antibodies (either in the preparation of biological products or in an extracorporeal circulation procedure) is the possible presence of viruses and/or potentially oncogenic macromolecules. Source material should be shown to be free of viruses, the cell banks (for monoclonal antibodies) and the production of antibodies must be closely controlled, viral inactivation of antibodies should be performed and the inactivation process should be shown to be effective.

Chromatography, Affinity↗
PubMed1992

Specific removal of transthyretin from plasma of patients with familial amyloidotic polyneuropathy: optimization of an immunoadsorption procedure.

Familial amyloidotic polyneuropathy is characterized by the presence in patients plasma of a genetic variant of transthyretin. No specific treatment has been found and extracorporeal immunoadsorption on immobilized anti-transthyretin antibodies appears as a potentially attractive procedure. Parameters involved in specific immunoadsorption of transthyretin were studied and optimized. Several monoclonal anti-TTR antibodies were compared as affinity ligands and one of them was found to be suitable for such purposes. Optimum quantities of antibodies to be immobilized on the gel were determined. Three desorption agents were tested for regenerating immunoadsorbents and best results were obtained with basic variation of pH, allowing total desorption of TTR and possibility of multiple use without loss of adsorption capacity. Simulation of an immunoadsorption procedure in well-defined conditions showed efficiency and specificity of adsorption to remove TTR and the system thus should be subjected to clinical trials.

Amyloidosis↗
PubMed1991

Monoclonal antibodies against human plasma protein C and their uses for immunoaffinity chromatography.

Human protein C, isolated by conventional multistep methods, was used for immunization of mice. Monoclonal antibodies were prepared and screening of antibodies to human protein C was achieved using an immunoblotting technique. Five monoclonal anti-protein C antibodies were compared as affinity ligands. Different parameters were studied (adsorption capacity, specificity of adsorption, possibility of desorption under mild conditions) and two antibodies were selected. One antibody allows preparation of highly purified protein C in a single-step procedure from a fraction of plasma containing high levels of coagulation factors whereas the other can be used for preparation of protein C deficient plasma.

Antibodies, Monoclonal↗
PubMed1991

[Immunohistochemical localization of transforming growth factor-beta (TGF-beta) in smooth muscle tissue].

Anti-TGF-beta antibodies were raised in rabbits against a C-terminal synthetic peptide whose amino acid sequence was chosen after human TGF-beta 1 primary sequence antigenic study by continuous epitope prediction. Immunopurified antibodies were used for an immunohistochemical localization in normal and pathological human tissues. Smooth muscular type tissue seems to be an elective target.

Animals↗
PubMed1990

[Human plasma fibronectin. Comparison of methods for preparation of a concentrate for therapeutic use from different sources].

Three methods, successive precipitations, affinity chromatography on immobilized gelatin and immunoaffinity chromatography with monoclonal anti-fibronectin antibodies were optimized and compared in order to be used for large scale preparation of human plasma fibronectin (Fn). The functional properties of the various Fn preparations were investigated by means of two assays: quantitation of the gelatin-binding activity by ELISA and quantitation of the Fn-mediated attachment of fibroblasts on plastic. Functional alterations of the purified Fn were observed when it was isolated by successive precipitations. Both chromatographic methods provide a rapid and convenient way for isolation of pure and functional Fn. Mass production of monoclonal antibodies is too expensive and legislative requirements for the therapeutic use of monoclonal antibodies are limiting factors for the choice of immunopurification as large scale isolation procedure. Plasma Fn can be isolated from different sources: fresh frozen plasma, cryoprecipitate supernatant or by-products from factor VIII preparation. When gelatin-Sepharose chromatography is performed under optimized conditions, fibronectins isolated from these sources show similar properties. Large scale purifications of Fn from a by-product of factor VIII preparation were performed either by gelatin affinity chromatography or by successive precipitations. These two purification methods can be easily scaled-up since the data obtained closely correlate with analytical results. The chromatographic method supplies a higher purified (98 vs 75%) and functional (95 vs 50%) material when compared with successive precipitations. Yield is also higher (50 vs 26%). The starting material undergoes viral inactivation and the affinity purified Fn, sterile, atoxic, apyrogen, which can be freeze-dried without additives fulfils all requirements for an injectable product.

Antibodies, Monoclonal↗
PubMed1990

[Development of a technique of immunoadsorption of LDL-cholesterol].

An immunoadsorption system for lowering plasma cholesterol was optimized. Several polyclonal and monoclonal antibodies were compared and the best results were obtained with goat polyclonal antibodies. The optimum quantity of antibodies to be immobilized on the gel was 5 mg/ml. Taking into account two variables, i.e., 1) that the regeneration must be as complete as possible and, 2) that immunoadsorbents must be used several times without a loss of adsorption capacity, desorption was achieved with 0.3 M glycine adjusted to pH 2.8. Antibody release from the immunoadsorbent was determined and can be minimized by glutaraldehyde treatment of the immunoadsorbent. Each phase, adsorption and desorption, respectively, was well-defined and synchronized, so that two columns could be used in parallel in an automated procedure. The kinetics of plasma protein removal demonstrated the efficiency and the specificity of the procedure.

Animals↗
PubMed1990

Study of parameters involved in specific immunoadsorption of apolipoprotein B.

A therapeutic immunoadsorption system on immobilized and anti-apolipoprotein B as a plasma cholesterol lowering procedure was optimized. Several antibodies were compared and highest adsorption capacity was obtained with goat polyclonal antibodies. Optimum quantities of antibodies to be immobilized on the gel and quantities of apo-B to be applied to columns were determined. The amount of antibodies released from immunoadsorbents can be minimized by treatment with a 0.005% glutaraldehyde solution with an acceptable reduction rate of adsorption capacity. Each phase, adsorption and desorption respectively, were well-defined and synchronized so two columns could be used in parallel in an automated procedure. In these conditions, the immunoadsorption system can efficiently, specifically and safely remove cholesterol and has to be subjected to clinical trials.

Animals↗
PubMed1989

Biotechnology of plasma proteins. Fractionation and applications.

Without discussing all of the current problems of industrial plasma fractionation, this INSERM International Symposium emphasized the importance of the technological progress of the last ten years. Although Cohn's classical technique of alcohol precipitation is still the most widely used, new techniques are rapidly being developed based on more selective methods with high extraction yields. However, it is certain that, given the growth in biotechnologies with the industrial applications of genetic engineering and cellular engineering, various problems will eventually be raised concerning plasma fractionation. It is probable that in the relatively near future certain fractions such as F VIII will no longer be prepared from plasma; but can the other fractions be reasonably replaced in the foreseeable future? Furthermore, the problems of purification will always be the crux of the development of biotechnologies. The technical committee of the OECD has recently drawn the attention of European governments to the considerable importance for the future of bioindustries of 'downstream processings' and the need to develop research on methods of extraction and purification. Human plasma is certainly the ideal model for the development of new methods of protein purification which could subsequently be applied to other industrial fields such as cellular engineering or genetic engineering. European countries definitely have an edge in this field as a result of their industries and fractionation centers.

Biotechnology↗
PubMed1988

Affinity purification of human plasma fibronectin on immobilized gelatin.

Several problems are associated with the biospecific affinity purification of plasma fibronectin on gelatin-Sepharose. Large-scale development of this purification procedure requires optimization of adsorption and elution conditions. The adsorption capacity depends on the amount of gelatin coupled to the Sepharose, the residence time, the temperature and the amount of fibronectin loaded on the adsorbent. Elution of adsorbed fibronectin with 3 M urea leads to incomplete recovery. The elution yield was found to vary with both the gelatin concentration and the amount of adsorbed fibronectin. Despite the incomplete elution, the adsorption capacity did not decrease after twelve consecutive isolation procedures. Under optimized conditions, the method described here provides a rapid, single-step and convenient way for the isolation of pure and functional fibronectin, either for analytical or large-scale preparative purposes.

Adsorption↗
PubMed1988

[Highly purified, functionally active human fibronectin preparation].

Fibronectin has been purified by gelatin-Sepharose affinity chromatography from fresh frozen human plasma. The bound fibronectin was eluted with 3 M urea. The purity of the fibronectin obtained has been checked on (immunoelectrophoresis, polyacrylamide gel electrophoresis, FPLC). Biological activity of the purified molecule has been monitored by means of three assays: quantitation of the gelatin-binding activity by ELISA, quantitation of the fibronectin-mediated attachment of fibroblasts on plastic and evaluation of the opsonic activity (uptake of gelatin latex particles by a murine macrophage line). When deep-frozen, fibronectin retains all of its properties. This highly purified and functional fibronectin fulfills the basic requirements for a standard reagent. It will allow to investigate physicochemical and functional alterations of various fibronectins.

Cell Adhesion↗
PubMed1988

[Immunosorption of apolipoproteins B. Optimization of the method in an in vitro bench test].

The aim of the present study was to determine optimum conditions for preparation and use of immunoadsorbents for removal of apolipoprotein B from plasma. An in vitro study was carried out to optimize different chromatography parameters: linear flow rate, apo B quantity loaded, gel regeneration conditions. Specificity of removal of apo B, stability of antibody covalent linkage and possible multiple use of immunoadsorbents were assessed. No significant loss of adsorption capacity was observed after sterilization and storage of immunoadsorbents.

Apolipoproteins B↗
PubMed1987

A potential new procedure for removing anti-factor VIII antibodies from hemophilic plasma.

A new approach for removing the anti-factor VIII antibodies in hemophilic patients by immunoadsorption is proposed. The method is based on the fact that the anti-factor VIII antibodies were predominantly of the IgG4 subclass; anti-human IgG4 antibodies were covalently linked to agarose and large amounts of anti-factor VIII antibodies can be eliminated. A study of 21 blood samples from hemophilic patients with anti-factor VIII antibodies allows us to confirm the large predominance of IgG4 in the anti-factor VIII population. In some samples, the presence of IgG3 related anti-VIII:C was checked by adsorption on an anti-IgG3 column. In a majority of cases, after IgG4 (or IgG4 + IgG3) immunoadsorption, the substitution therapy becomes possible or easier.

Antibody Specificity↗