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Biomedical subjects

T Scheper

Publications and source records attributed to T Scheper.

At least 37 records · Page 2Linked to original sources
PubMed1999

Immunoaffinity layering of enzymes. Stabilization and use in flow injection analysis of glucose and hydrogen peroxide.

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae.

Animals↗
PubMed1999

Fluorescence monitoring during cultivation of Enterobacter aerogenes at different oxygen levels.

On-line monitoring of NAD(P)H fluorescence and 2D fluorescence spectroscopy was performed with Enterobacter aerogenes, a bacterium sensitive to oxygen availability. The organism was grown in a reactor under low and high dissolved oxygen concentrations and circulated through a bypass attached to the reactor. Under low dissolved oxygen concentration in the reactor, NAD(P)H fluorescence in the reactor and the bypass showed a deviation, but not when the dissolved oxygen level in the reactor was high. The pattern of growth curves was identical under low and high oxygen levels. This indicates a difference in the metabolic activity of E. aerogenes in response to oxygen. The difference spectrum of the 2D fluorescence shows that growing E. aerogenes under high dissolved oxygen levels increases the NAD(P)H content of the cells.

Bioreactors↗
PubMed1998

Flow injection analysis system for the supervision of industrial chromatographic downstream processing in biotechnology.

Sugar beet molasses is a natural resource for various products used in daily life, ranging from sucrose to amino acids for pharmaceutical industry. The separation of molasses into these high value components is performed on a large scale by ion exchange/exclusion chromatography. A biosensor system was set up for the "in time" analysis of serine and sucrose during molasses desugarisation. D-Serine was analysed with the multi-enzyme system D-serine dehydratase/lactic dehydrogenase and photometric detection of the NADH consumed. Sucrose was determined with invertase/mutarotase/glucose oxidase and the oxygen consumed was monitored amperometrically. An analysis could be performed within 2-5 min by directly injecting samples from the chromatographic process into the flow injection analysis system. The determination range for the sucrose analysis was 0-2.5 gl-1 and for the analysis of D-serine 0-0.5 gl-1. The standard deviation for the measurement of D-serine was 1.7%.

Biosensing Techniques↗
PubMed1998

In situ microscopy for on-line determination of biomass.

A sensor is presented, which allows on-line microscopic observation of microorganisms during fermentations in bioreactors. This sensor, an In Situ Microscope (ISM) consists of a direct-light microscope with a measuring chamber, integrated in a 25 mm stainless steel tube, two CCD-cameras, and two frame-grabbers. The data obtained are processed by an automatic image analysis system. The ISM is connected with the bioreactor via a standard port, and it is immersed directly in the culture liquid-in our case Saccharomyces cerevisiae in a synthetic medium. The microscopic examination of the liquid is performed in the measuring chamber, which is situated near the front end of the sensor head. The measuring chamber is opened and closed periodically. In the open state, the liquid in the bioreactor flows unrestricted through the chamber. In closing, a defined volume of 2,2. 10(-8) mL of the liquid becomes enclosed. After a few seconds, when the movement of the cells in the enclosed culture has stopped, they are examined with the microscope. The microscopic images of the cells are registered with the CCD-cameras and are visualized on a monitor, allowing a direct view of the cell population. After detection, the measuring chamber reopens, and the enclosed liquid is released. The images obtained are evaluated as to cell concentration, cell size, cell volume, biomass, and other relevant parameters simultaneously by automatic image analysis. With a PC (486/33 MHz), image processing takes about 15 s per image. The detection range tested when measuring cells of S. cerevisiae is about 10(6) to 10(9) cells/mL (equivalent to a biomass of 0.01 g/L to 12 g/L). The calculated biomass values correlate very well with those obtained using dry weight analysis. Furthermore, histograms can be calculated, which are comparable to those obtained by flow cytometry.

Automation↗
PubMed1998

Determination of the minor whey protein bovine lactoferrin in cheese whey concentrates with capillary electrophoresis.

In our present work we present the determination of bovine lactoferrin in whey concentrates as they are typically produced by milk and cheese industry after production of cheese. Due to the high total protein content the analysis of whey concentrate samples is difficult and even not possible by using capillary zone electrophoresis with UV detection. To enhance the detection sensitivity we applied a more promising approach by using affinity interactions in combination with laser-induced fluorescence detection. By mixing fluoresceine isothiocyanate (FITC)-conjugated polyanionic lipopolysaccharide with the mostly positively charged lactoferrin we found a significant migration time shift which is clearly dependent on the concentration of the added protein. In the second approach we developed an immunoassay using FITC-conjugated specific antibody against bovine lactoferrin. The results of the immunoassay measurements were compared with data obtained by standard enzyme-linked immunosorbent assay analysis.

Animals↗
PubMed1997

Fast on-line flow injection analysis system for IgG monitoring in bioprocesses.

An automated immunoassay, with one affinity component immobilized on a solid surface, has been developed to monitor the production of different immunoglobulins during mammalian cell cultivation processes. The whole analysis device is based on the principle of flow injection analysis (FIA) and a cartridge with the immobilized affinity component is implemented into the FIA system. This cartridge is filled with a carrier material to which protein G is covalently bound. After sample injection, binding of the IgG on the protein G within the cartridge takes place while after a washing step, the IgGs are eluted by a pH shift, and the IgG concentration is monitored via fluorescence. In the automated immunoassay, undiluted cell free samples from the reactor or from down-stream processing can be analyzed directly. Due to the separation the IgG can be detected without interference from other sample components by protein fluorescence. The results are obtained with analysis times below 6 min. Sample volumes of less than 100 microliters may be used. The assay is sensitive to concentrations from 5 up to 500 micrograms ml-1. Using this FIA-System, immunoglobulins G, produced in different media, were successfully monitored. The results of the assay were validated by ELISA.

Biochemistry↗
PubMed1997

Bioaffinity layering: a novel strategy for the immobilization of large quantities of glycoenzymes.

A simple strategy for increasing considerably the quantities of glycoenzymes immobilized on insoluble supports is described. The strategy that we call bioaffinity layering makes use of the multivalent nature of concanavalin A (Con A) and the multiple oligosaccharide chains of most glycoenzymes to build alternating lectin and glycoenzyme layers on a Sepharose matrix with precoupled Con A. Using this procedure, it was possible to increase the amounts of several glycoenzymes immobilized on Sepharose and 19.0 mg glucose oxidase could be associated with one ml Sepharose matrix after seven Con A/glucose oxidase incubation cycles. Bioaffinity layered preparations of glycoenzymes exhibited high activities as indicated by very high effectiveness factor (eta) values and those of glucose oxidase and invertase exhibited a layer-by-layer increase in thermostability. The sensitivity of a flow-through glucose monitoring cartridge integrated into a flow injection analysis (FIA) system was enhanced significantly by increasing the amount of immobilized glucose oxidase via bioaffinity layering. A cartridge bearing six layers of glucose oxidase on Sepharose support was used effectively and repeatedly for analysis of medium glucose concentration during a fed-batch cultivation of the yeast Saccharomyces cerevisiae.

Biosensing Techniques↗
PubMed1997

Fermentation monitoring and process control.

The use of modern analytical online methods such as two-dimensional fluorescence measurements gives new insights into bioprocesses. With the resulting data, it is not only possible to better understand and document, for example, biotransformations, but also to develop efficient control strategies that lead to better productivity and lower costs.

Journal Article↗
PubMed1995

Analysis of immunoglobulin G using a capillary electrophoretic affinity assay with protein A and laser-induced fluorescence detection.

A method for the rapid and sensitive determination of immunoglobulin G (IgG) in cultivation media by an affinity assay using capillary electrophoresis is presented. For that purpose we evaluated protein A conjugated with a fluorescent dye such as fluorescein diisocyanate or dichlorotriazinyl-aminofluorescein as an affinity ligand. The ligand formed a fluorescing complex with immunoglobulin G in the sample and rapid separation from excess protein A was performed by capillary zone electrophoresis. However, only partial resolution of the zones was achieved when protein A as a whole molecule was utilized. In contrast, baseline resolution of the zones was obtained when recombinant fragments of protein A were used as affinity ligands. Immunoglobulin concentrations in the range of two orders of magnitude were determined. Due to the specificity of protein A for immunoglobulin G, analysis can be carried out even in the presence of high concentrations of other components and in cultivation media. Thus, the capillary electrophoretic affinity assay was successfully applied to monitor monoclonal antibodies in a cultivation process.

Affinity Labels↗
PubMed1994

Fluorescein isothiocyanate-labeled protein G as an affinity ligand in affinity/immunocapillary electrophoresis with fluorescence detection.

Antibodies from human sera (h-IgG) were tagged with a fluorescent dye (fluorescein isothiocyanate, FITC) through the affinity reaction of FITC-labeled protein G with the Fc fragment of the antibodies. The complexes were quantified by capillary zone electrophoresis (CZE) within 1 min, i.e., fast enough to prevent their dissociation during the measurement. Conditions for the affinity reaction and the CZE analysis could thus be optimized independently. When an FITC-labeled protein G concentration of 10(-6) mol/L was used, h-IgG concentrations between 10(-6) and 10(-9) mol/L were reproducibly quantified (STD < 2%), using an LIF detector. A correlation coefficient, r2, of 0.9988 was established between the peak height and the IgG concentration. Alternatively, h-IgG containing serum samples and the FITC-labeled protein G were simply injected into the CE capillary in consecutive zones, followed by the application of the electrical field. Within 2 min, the affinity complexes were resolved and the IgG content of the serum quantified (r2 = 0.9986). The injection sequence was of no consequence. The measurements agreed well with those found in a single radial immunodiffusion (SRID) assay. In addition FITC-labeled protein G-tagged anti-h-IgG1 antibodies were used to detect the specific antigen of the involved antibody, namely, h-IgG1, in human sera.

Electrophoresis↗
PubMed1994

Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization.

Commercial lipase (triacylglycerol lipase, EC 3.1.1.3) of Pseudomonas cepacia (Amano) has been purified to homogeneity by a single chromatography on phenyl Sepharose. The eluted lipase crystallized spontaneously at 4 degrees C in the eluent, containing 58-69% 2-propanol. The yield of the lipase was 87-100% and the specific activity during the hydrolysis of triolein 5800 U/mg protein. This protein has a molecular weight of 34.1 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Its purity was determined by SDS-PAGE and capillary zone electrophoresis to be > or = 99%. Immobilization on Sepharose increased its stability in organic solvents. This lipase of P. cepacia differs from that of other Pseudomonas strains in respect to substrate specificity and during crystallization. It exhibits a high stability in organic solvents and supercritical carbon dioxide.

Amino Acid Sequence↗
PubMed1994

Automated imunoanalysis systems for monitoring mammalian cell cultivation processes.

Two different automated immunoanalysis systems are presented. Both are based on the principles of flow-injection analysis and were developed to provide reliable, rapid monitoring of relevant proteins in animal cell cultivation processes. One system uses a turbidimetric analysis, and the other employs a heterogeneous chemistry with immobilized immunocomponents. For both systems, the analysis time is in the range of a few minutes, and a complete analysis cycle, including triplicate analyses and various washing steps, is in the range of 20-30 minutes. Samples from cultivation processes can be analyzed directly without dilution. Quantitation of proteins such as rt-PA or monoclonal antibodies can be performed over an analyte concentration range of 1-1000 mg/L. Both systems were compared to conventional ELISA assays on microtiter plates. The turbidimetric analysis system also included a biosensor for simultaneous glucose determination.

Animals↗
PubMed1994

Application of a flow injection fibre optic biosensor for the analysis of different amino acids.

The analysis of different amino acids is described using a fibre optic biosensor previously described in its principles. This biosensor works according to the measurement principle of flow injection analysis (FIA) and consists of a fluorosensor and a measurement cell operating under pressure. In the measurement cell, enzymes and molecular weight enlarged coenzyme PEG (MW 20000)-N6-(2-aminoethyl)-NAD(H) are confined behind a solid ultrafiltration membrane. Assays for the analysis of L-phenylalanine and L-alanine were developed. The analysis frequency is in the range of 1 to 2 samples per hour, and the sensor stability was found to be sensitive to the stability of each enzyme system used. This effect was studied in detail. The L-alanine assay was found to be especially reliable, sensitive, specific, and highly selective for the L-enantiomer.

Alanine↗
PubMed1993

Specific flow injection sandwich binding assay for IgG using protein A and a fusion protein.

A sandwich-type flow-injection binding assay for quantitation of various IgG's was developed. The assay is based on the pseudoimmunological reaction between protein A from Staphylococcus aureus and immunoglobulin G from different species. Protein A immobilized on a solid support and a fusion protein of protein A and beta-galactosidase from Escherichia coli are used for detection. The fusion protein is produced with a temperature-inducible recombinant E. coli strain. A sandwich is formed by subsequent injection of IgG and fusion protein into the buffer stream flowing through the immobilized protein A column. The amount of enzyme activity bound is proportional to the amount of IgG bound and is measured by pumping a lactose solution as substrate for beta-galactosidase through the protein A column. Lactose is converted to glucose and galactose. The detector is an enzyme thermistor that measures the heat evolved in the enzymatic conversion of glucose by coimmobilized glucose oxidase and catalase. The assay takes 16 min at a flow rate of 0.6 mL min-1 with a lower detection limit of 33 pmol per injection of rabbit IgG. The precision of replicate measurements has a standard deviation of 4-5%, and the column can be used for more than 50 cycles.

Animals↗
PubMed1993

Rapid capillary gel electrophoresis of proteins.

The rapid separation of sodium dodecyl sulphate-protein complexes according to their molecular masses (M(r)) by capillary gel electrophoresis is described. Using commercial equipment, standard proteins with M(r) in the range 29,000-97,400 were resolved to the baseline in less than 2 min by utilizing a separation distance of 7 cm. A linear relationship between migration time and log M(r) was found and rapid determination of the molecular mass of light and heavy chains of human immunoglobulin G is reported. The results are compared with applications using longer separation distances, showing that rapid and efficient analysis and adequate resolution can be obtained by using short separation distances.

Animals↗
PubMed1993

Two FIA-based biosensor systems studied for bioprocess monitoring.

In this paper, two different FIA-based biosensor systems are described for application to different biotechnologically relevant purposes. In the first system, single fiber optodes were used to determine the pH, urea and penicillin V concentrations. A two-channel system was developed for the simultaneous monitoring of different variables to increase the analysis accuracy. This system was used for monitoring the penicillin V concentration during a cultivation of Penicillium chrysogenum. The second system described is a calorimetric immunoassay based on the use of an enzyme thermistor. A sandwich assay with protein A immobilized on a solid support for the determination of various IgGs was established. A fusion protein of protein A and beta-galactosidase obtained from a recombinant E. coli strain was used in the labelling and detection reaction. This system is designed for future application in bioprocess monitoring.

Biosensing Techniques↗