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Biomedical subjects

K Mosbach

Publications and source records attributed to K Mosbach.

At least 73 records · Page 4Linked to original sources
PubMed1987

The design of a simple competitive ELISA using human proinsulin-alkaline phosphatase conjugates prepared by gene fusion.

The gene encoding human proinsulin has been fused in-frame with the E. coli alkaline phosphatase gene (pho A) (EC 3.1.3.1). Two constructions are described. One construction consists of the entire proinsulin gene fused to the 5'-terminal end of pho A. In the other construction a 42 base pair DNA fragment has been deleted from the 3'-terminal end of the proinsulin gene. The two purified fusion proteins are enzymatically active showing a specific activity of 10-15 U/mg and 18-25 U/mg, respectively. The first construction exhibited insulin antigenicity and was used to design a simple competitive ELISA for insulin. The lower detection limit was found to be at least 2.5 ng/ml. Both fusion proteins were also shown to have potential for use in a competitive ELISA for proinsulin.

Alkaline Phosphatase↗
PubMed1987

The expression in E. coli of a polymeric gene coding for an esterase mimic catalyzing the hydrolysis of p-nitrophenyl esters.

We have prepared a hybrid protein consisting of seven esterase units, Glu-Ala-His-Ala-Ser-Phe-Phe-Phe, fused to the N-terminal of galactokinase (E. coli). The structural gene for this bifunctional protein was obtained by cloning a polymer made up of three chemically synthesized oligonucleotides to which the galactokinase gene was fused in frame. The hybrid protein was purified to homogeneity with the aid of the galactokinase moiety and showed an Mr of 51,000-53,000. The preparation could catalyze the hydrolysis of p-nitrophenyl esters and, due to the inbuilt hydrophobic spacers, Phe-Phe-Phe, improved catalysis of more hydrophobic substrates was obtained.

4-Nitrophenylphosphatase↗
PubMed1987

A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions.

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.

Antibodies, Monoclonal↗
PubMed1987

Poly-N-acryloyl-Tris gels as anticonvection media for electrophoresis and isoelectric focusing.

We describe in detail the synthesis of an acrylic monomer, N-acryloyl-tris(hydroxy-methyl)aminomethane (NAT), which was successfully used for the preparation of gels for electrophoresis and isoelectric focusing. The polymerization kinetics and transparency of the poly(NAT) gels crosslinked by N,N'-methylenebisacrylamide (Bis) are also shown. Poly(NAT)-Bis gradient (4-24%) gel resolves proteins according to their size. The exclusion limit of this gel is slightly over 3 X 10(6), which is more than threefold higher than the exclusion limit of the polyacrylamide gradient gel of the same concentration. The gel made of 6% NAT and 3% Bis represents a suitable matrix for isoelectric focusing. These results demonstrate that poly(NAT)-Bis gels could be advantageously used in those applications where the extensive sieving by the polyacrylamide matrix is not desir desirable.

Acrylates↗
PubMed1986

Galactosyltransferase-dependent sialylation of complex and endo-N-acetylglucosaminidase H-treated core N-glycans in vitro.

Purified beta-N-acetylglucosaminide beta(1-4)galactosyltransferase and partially purified beta-galactoside alpha(2-6)-sialyltransferase were used to elongate and terminate glycan chains of agalacto-ovalbumin and endo-beta-N-acetylglucosaminidase H-treated yeast invertase in vitro. In the presence of both transferases, 0.1 mol sialic acid was incorporated per mol agalacto-ovalbumin within 24 h. Evidence is presented to show that purification of the galactosylated intermediate increases the efficiency of sialylation. Incorporation of sialic acid into endo-beta-N-acetylglucosaminidase H-treated oligomannose glycoproteins may be useful for in vivo stabilization of these glycoproteins by preventing uptake in liver or reticuloendothelial cells.

Animals↗
PubMed1986

Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli.

We have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 micrograms/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 degrees C in the enzyme thermistor unit. Thus, immediate assay start up was possible.

Enzyme-Linked Immunosorbent Assay↗
PubMed1986

Reversible and specific interaction of dehydrogenases with a coenzyme-coated surface continuously monitored with a reflectometer.

Reversible affinity binding of NAD-dependent dehydrogenase to an NAD-coated silicon surface ("NAD biochip") has been accomplished. The silicon surface, which is favorable for use with optical techniques because of its excellent reflection properties, was precoated with a polymer to prevent nonspecific and irreversible adsorption. Using a new reflectometry technique based on measurement of the polarization change of light reflected upon the biochip, continuous monitoring of the affinity binding and subsequent desorption of alcohol dehydrogenase and lactate dehydrogenase from the NAD surface were possible; allowing repeated use of the same NAD chip--an advantage when the assay was carried out in a continuous reflectometer. With a flow rate of 0.5 ml/min, response times on the order of 30 s were obtained.

Adsorption↗
PubMed1985

Immobilized enzymes in organic synthesis.

The immobilization of enzymes and cells by different methods and the possible stabilization of immobilized preparations are discussed. An outlook on 'second generation enzyme technology', which involves immobilized multi-enzyme systems and coenzymes, is given with examples: the immobilization of dehydrogenases with their active sites facing one another, and systems containing NAD(H) coenzymes immobilized by coupling to dextran (in an enzyme electrode), to polyethylene glycol (in a membrane reactor), or to enzymes themselves. The use of immobilized enzymes to synthesize peptides and disaccharides is described.

Chemical Phenomena↗