Biomedical subjects

F E Regnier

Publications and source records attributed to F E Regnier.

At least 55 records · Page 3Linked to original sources

PubMed1994

Process monitoring of the production of gamma-interferon in recombinant Chinese hamster ovary cells.

The production of recombinant gamma-interferon was monitored using high-performance liquid chromatographic methods. These methods were able to distinguish between glycosylated and non-glycosylated forms of gamma-interferon by complexing the carbohydrate with borate. Sufficient quantities of standard glycosylated gamma-interferon were not available for peak identification so immunological techniques were used to identify gamma-interferon variants. These techniques were validated with the non-glycosylated form. The non-glycosylated form was then shown to be retained only on a cation-exchange column, while the glycosylated form, complexed with borate, was retained only on an anion-exchange column. Samples were drawn at 2-h intervals over a 60-h production cycle and analyzed by both anion- and cation-exchange chromatography. Results indicated that the production of each form was coincidental and that the glycosylated form of gamma-interferon is produced in greater abundance than non-glycosylated.

Animals↗

PubMed1993

Electrophoretically mediated microanalysis of ethanol.

Capillary electrophoresis was used to determine ethanol by the methodology of electrophoretically mediated microanalysis (EMMA). In EMMA, spatially distinct analyte and analytical reagent zones of differing electrophoretic mobility are merged under the influence of an electric field, and the resulting product is transported to the detector. The enzymatic oxidation of ethanol to acetaldehyde by alcohol dehydrogenase was utilized, and the concurrent reduction of NAD+ to NADH was monitored at 340 nm as a measure of the quantity of ethanol injected. Quantitation using an internal standard and normalization for peak migration time yielded a R.S.D. of 2.7%, and the linear range extended to that quantity of ethanol which could be reacted prior to passing by the detection window. Comparison of the EMMA technique to the Sigma spectrophotometric procedure revealed that the two methods do not yield significantly different values for the determination of ethanol. The EMMA method offered the advantages of electrophoretic mixing and miniaturization.

Acetaldehyde↗

PubMed1993

Automated process monitoring of monoclonal antibody production.

Antifibronectin, monoclonal antibody was monitored through 52 h of production. Samples were automatically drawn from a bioreactor into the injection valve of an HPLC system without prior sample preparation. The hybridoma cell line was nonadherent, so whole cells were injected directly onto the perfusable protein A affinity column. There was only a modest column back pressure (ca. 1700 psi at a linear flow rate of 1.5 cm/s) after over 75 injections over the 52-h experiment. These experiments demonstrate the utility of high-speed chromatography for rapid process monitoring.

Animals↗

PubMed1993

Electrophoretically mediated microanalysis of leucine aminopeptidase in complex matrices using time-resolved laser-induced fluorescence detection.

Leucine aminopeptidase, a clinically significant enzyme, was assayed in complex biological samples using a new technique termed electrophoretically mediated microanalysis. The assay was performed in capillary electrophoresis columns using time-resolved laser-induced fluorescence detection. Human serum, human urine, and Escherichia coli supernatant samples were assayed using this method. Results for serum and urine were within the ranges of expected values found in the literature. A low concentration of 6 x 10(-13) M enzyme in buffer was detected using this method. A detection limit (3 sigma) of 400 enzyme molecules in buffer was determined.

Buffers↗

PubMed1993

Mathematical treatment of electrophoretically mediated microanalysis.

A new concept in reaction-based chemical analysis is introduced and theoretically described. By utilization of the variability in electrophoretic mobilities among charged species, spatially distinct zones of chemical reagents can be electrophoretically merged under the influence of an applied electric field. Electrophoretically mediated microanalysis (EMMA) exploits this phenomenon as a basis for chemical analysis utilizing capillary electrophoretic systems. EMMA is described in terms of the four stages required for reaction-based analysis: (1) analyte and analytical reagent metering; (2) initiation of reaction; (3) control of reaction conditions and product formation; (4) detection of species whose production or depletion is indicative of the concentration or quantity of the analyte of interest. The method is illustrated by the enzymatic oxidation of ethanol to acetaldehyde by alcohol dehydrogenase with the concurrent reduction of NAD+ to NADH monitored at 340 nm. Experimental results for both substrate and enzyme determinations are shown to agree with the presented theory.

Electrophoresis↗

PubMed1993

Native protein separations and enzyme microassays by capillary zone and gel electrophoresis.

Native protein separations by capillary gel electrophoresis are achieved using linear acrylamide gel matrices. Polyacrylamide gels with a concentration range of 3.5-5% did not exhibit size separations for native proteins with molecular weights from 20,000 to 47,000. The separation of native proteins in gel-filled capillaries is based solely on the charge of the protein as in normal zonal electrophoresis. Retention of protein activity in the acrylamide matrix was demonstrated by performing enzymatic assays in the gel matrix. Alkaline phosphatase (ALP) and beta-galactosidase assays were conducted in both C18-PF108-modified and polyacrylamide gel-filled capillaries. Enzyme assays were achieved by filling the capillary with an appropriate substrate dissolved in the electrophoresis buffer. The product formed by the reaction of enzyme with substrate was monitored using a standard UV-visible detector. Both constant potential and zero potential modes of analysis were demonstrated. The polyacrylamide gel columns provide the advantages of minimized diffusion and limited band spreading due to the high viscosity of the gel matrix. The lowest detection limit achieved was 5.2 x 10(-20) mol (7.6 x 10(-12) M sample injected) of ALP. The dual enzyme assay of ALP and beta-galactosidase was achieved in gel-filled capillaries simultaneously.

Electrophoresis↗

PubMed1993

Liquid chromatography based enzyme-amplified immunological assays in fused-silica capillaries at the zeptomole level.

Through the use of fused-silica capillaries it was shown that reducing the liquid volume of an enzyme-amplified immunological assay increases the rate of amplification and sensitivity of the assay by several orders of magnitude. Human immunoglobulin G (hIgG) captured on protein G-coated 100-microns-i.d. columns was saturated with F(ab) anti-hIgG conjugated to alkaline phosphatase (ALP). Conjugated enzyme captured by the antigen was subsequently assayed in a stop-flow incubation with p-nitrophenyl phosphate. p-Nitrophenol produced in the stop-flow incubation was then swept to the detector and quantitated at 405 nm. The detection limit in the stop-flow mode was approximately 3 fmol. Three problems were identified in this flow-through, capillary assay format. The first was that the rate of immunological complex formation within the capillary was too slow. Preforming the immunological complex before application to the column increased the sensitivity by 2 orders of magnitude. Another problem was that the rate of mass transfer within the capillary limited capture of the preformed immunological complex. This problem was solved by stop-flow incubation of the complex in the column. The combination of preformation of the immunological complex and stop-flow binding within the column reduced the detection limit to approximately 3 amol. Finally, reducing the amount of F(ab)-ALP used in the assay minimized nonspecific binding of the conjugated enzyme and reduced the detection limit further to 333 zmol.

Alkaline Phosphatase↗

PubMed1993

Manipulation of electroosmotic flow in capillary electrophoresis.

This paper reports the use of surfactant and polymer-C18 coated capillaries that allow manipulation of electroosmotic flow (EOF). Although this approach to the control of EOF involves the preparation and use of multiple capillaries, all the coatings were prepared by a single procedure. It is shown that the ability to control EOF allows optimization of both separation time and resolution. In the case of proteins, low EOF maximizes resolution whereas high flow gives the shortest analysis time. It should be noted that proteins are a special case and this conclusion may not be true with other molecular species. Through selection of a specific coating, it is possible to complete a separation in the shortest time while maintaining sufficient resolution to give baseline resolution of proteins. The various coated capillaries were examined in capillary zone electrophoresis (CZE) and capillary isoelectric focusing (cIEF) separations of native protein standards and hemoglobin variants. Separation of glycosylated hemoglobin A1 variants was achieved by cIEF within 10 min, including the focusing time. Good run-to-run reproducibility was obtained by flushing the capillary with the coating solution between analyses.

Electrophoresis↗

PubMed1993

Immunochromatographic analysis of proteins. Identification, characterization and purity determination.

Antibodies specific to a protein and its structural variants were immobilized on a high-performance Protein G column. This column recognized and selectively subtracted specific molecules from a sample. When a size-exclusion column was coupled with this high-performance affinity column, a comparison between the elution profile before and after the antibody immobilization was used to study antigen components present in the sample. Various human growth hormone structural variants and aggregates were studied using this approach. The technique is simple, fast and does not involve the usage of radioactive material.

Antibodies, Monoclonal↗

PubMed1993

Polymer- and surfactant-coated capillaries for isoelectric focusing.

This paper reports a method for deactivation of fused-silica capillaries to be used in capillary isoelectric focusing (cIEF). Deactivation was achieved by adsorbing either a surfactant or hydrophilic polymer to alkylsilane-derivatized capillaries. The surfactant PF-108 and methyl cellulose reduced electro-osmotic flow (EOF) 20 to 30 fold in comparison to underivatized capillaries. Although EOF was reduced sufficiently to allow focusing to permit separations to be completed before proteins were swept through the capillary, there was adequate flow to obviate the need for a separate mobilization step. This reduces the complexity of cIEF and increases reproducibility. Based on resolution of hemoglobin variants, proteins that varied 0.03 pH units in isoelectric point were resolvable. This is equivalent to the highest resolution achieved in conventional slab and tube gel isoelectric focusing.

Buffers↗

PubMed1993

Fimbriated stationary phases for proteins.

This paper describes synthetic procedures for preparing fimbriated stationary phases on poly(styrene-divinylbenzene) (PS-DVB) packing materials. The synthesis consists of a five-step procedure in which the order in which the steps are carried out may be varied. These steps are (i) polymerization of monomers to form an amphiphilic copolymer or oligomer, (ii) adsorption of either monomers, or polymer onto the PS-DVB surface, (iii) solvent induction of functional group orientation at the PS-DVB-polymer interface and polymer-water interface, (iv) a cross-linking reaction that forms a hydrophilic surface layer, and (v) derivatization of the surface layer with stationary phase.

Chromatography, Affinity↗

PubMed1993

Tandem chromatographic-immunological analyses.

The function of conventional immuno-logical assays is to determine the presence and amount of known substances. Cross-reactivity of antibodies with unknown species of similar structure is a problem that can be circumvented by using chromatography in a second level of discrimination. Tandem LC-IA systems increase the selectivity and the sensitivity of assays by removing interfering species. For cases in which the goal is to prove that similar species are absent from a sample or to identify antigen variants, conventional assays fail. LC-IA systems are adept at dealing with these tasks. The necessary steps of metering reagents, separating Ag:Ab complex from other species, and enhancing detection in an immunological assay are easily accommodated by the LC system. In fact, the LC system provides a wide variety of new, high-sensitivity, high-speed methods for carrying out immunological assays. On the basis of the enormous versatility, selectivity, and sensitivity that LC-IA brings to analytical chemistry, we believe this technique will become increasingly important for monitoring analytes in complex biological matrices.

Amino Acids↗

PubMed1993

Rapid process monitoring in biotechnology.

Detection of protein variants in the production of recombinant DNA products is an important and complex task. Rapid acquisition of this information permits feedback control of the production process and continuous validation of the product. Much of the technology required for rapid process monitoring is currently available or under development.

Biotechnology↗

PubMed1992

Ultramicro enzyme assays in a capillary electrophoretic system.

This paper describes an ultramicro method for achieving enzyme assays. Enzyme saturating concentrations of substrate, coenzyme when appropriate, and running buffer were mixed and used to fill a deactivated fused-silica capillary in a capillary zone electrophoresis apparatus. The enzyme glucose-6-phosphate dehydrogenase was injected by either electrophoresis or siphoning and mixed with the reagents in the capillary by electrophoretic mixing. Enzyme activity was assayed by electrophoresing the product, reduced nicotinamide adenine dinucleotide phosphate, to the detector where it was detected at 340 nm. Under constant potential, the transport velocity of enzyme and the product was generally different. This caused product to be separated from the enzyme after it was formed. Because product formation was much faster than the rate of enzyme-product separation, product accumulated. The amount of accumulated product was inversely related to operating potential. In the extreme case, the operating potential was zero. Zero potential assays were generally carried out by electrophoresing the enzyme partially through the capillary and then switching to zero potential. This capillary was left at zero potential for several minutes to allow additional product to accumulate. After this additional amplification step, potential was again applied and the product transported to the detector. Product formed under constant potential appears as a broad peak with a flat plateau. When the voltage is switched to zero at intermediate migration distance, a peak will be observed on top of this plateau. Either the eight of the plateau or the area of the peak may be used to determine enzyme concentration. The lower limit of detection was 4.6.10(-17) mol of glucose-6-phosphate dehydrogenase.

Electrophoresis↗

PubMed1992

Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide.

Proteins with relative molecular masses of 14,000 to 205,000 were separated by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using non-cross-linked linear polyacrylamide gels on both coated and uncoated fused-silica capillaries. It was determined that viscosity of the acrylamide solution was a major factor affecting column stability with linear acrylamide gels. When the viscosity of the acrylamide solution reaches 100 cP, electro-osmotically driven displacement of the gels is insignificant. Uncoated capillaries provided better resolution, stability, and reproducibility than surface coated capillaries when the concentration of linear polyacrylamide was greater than 4%. At lower gel concentrations, non-cross-linked polyacrylamide is easily displaced from the columns. A calibration plot of log molecular mass vs. mobility with non-linear polyacrylamide was linear, which indicated that resolution was equivalent to that obtained with cross-linked acrylamide. Separations with model proteins indicated that baseline resolution between protein species that vary 10% in molecular mass can be achieved.

Acrylic Resins↗

PubMed1992

Kinetic chromatographic sequential addition immunoassays using protein A affinity chromatography.

A new type of chromatographic immunoassay based on sequential addition is described. On a protein A column, the antibody, the sample containing the antigen, and then a known amount of antigen are sequentially injected. This assay is designed to shorten analysis times and reduce complexity of dual-column chromatographic immunoassays, circumvent desorption buffer interferences common to affinity chromatography, and eliminate the need for tagged molecules. This new technique is named kinetic immunochromatography sequential addition (KICQA). Because of its kinetic nature, flow rate will have a large effect on KICQA, and the impact of changing flow rate is studied extensively. By use of various amounts of antibody, the dynamic range of KICQA is shown to be selectable over 2.5 orders of magnitude. Finally, KICQA was used to determine transferrin and albumin in human serum. Both analytes show good agreement with their respective reference methods, and an albumin assay was performed in under 1 min.

Antigen-Antibody Complex↗

PubMed1992

Automated real-time immunoassay of biomolecules.

ImmunoDetection is a novel technique combining perfusion chromatography technology with antibodies to perform the steps of an immunoassay in a flow-through column format. Sensitive and precise measurements are performed in seconds to minutes using automated liquid chromatography instrumentation.

Antibodies↗

PubMed1992

Contribution of soluble aluminium species to absorption of aluminium from the rat gut in situ.

1. Rat gut perfusion studies in vivo at pH 4, 6 or 8 using aluminium chloride or equimolar aluminium chloride and sodium citrate showed that elevated plasma aluminium concentrations were associated with aluminium solubility in the perfusion. Elevated plasma aluminium levels and soluble aluminium in the perfusate occurred with perfusion of equimolar aluminium chloride and sodium citrate at all three pH values. 2. Partitioning studies in vitro, utilizing water and ethyl acetate, revealed that uncomplexed aluminium exhibited maximum partitioning into the ethyl acetate phase at pH 2.5. When complexed with citrate, aluminium exhibited partitioning over a much broader pH range, pH 2.5-8.0. 3. A direct linear relationship was observed between the soluble aluminium concentration of the perfusate and the increase in the plasma aluminium level, suggesting that soluble aluminium is absorbed by a passive diffusion mechanism.

Aluminum↗