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Biomedical subjects

F Rüker

Publications and source records attributed to F Rüker.

29 records · Page 2Linked to original sources
PubMed1991

Expression of a human monoclonal anti-HIV-1 antibody in CHO cells.

The cDNA coding for the light and heavy chains, respectively, of the human monoclonal antibody 3D6 (IgG1, kappa), which binds specifically to human immunodeficiency virus-1 (HIV-1) gp41, was inserted into three different mammalian expression vectors and transfected into Chinese hamster ovary (CHO) cells. Transcription was under the control of Rous sarcoma virus long terminal repeat (RSV LTR), human cytomegalovirus major immediate early (CMV IE) promoter, and mouse mammary tumor virus long terminal repeat (MMTV LTR), respectively. Antibody productivity was monitored in the supernatants of selected clones. The binding characteristics of the CHO-derived antibody to HIV-1 gp41 were found to be identical to that of the original antibody produced by hybridoma cells.

Animals↗
PubMed1990

Detection of the trans activity of the plum pox virus NIa-like protease in infected plants.

The NIa-like protein of plum pox virus is a protease with high sequence specificity that is autocatalytically released from the viral polyprotein. In order to determine whether the protease is active in trans we constructed a fusion protein consisting of the C-terminal region of the plum pox virus polyprotein and the staphylococcal Protein A. The authentic protease recognition sequence Asn-Val-Val-Val-His-Gln-Ala occurs in the centre of this protein fusion. This protein was cleaved specifically by extracts of plum pox virus-infected plants due to the strong activity of the viral protease making it a useful tool for diagnostic purposes.

Amino Acid Sequence↗
PubMed1987

Combination of Zetaprep mass ion-exchange media and high-performance cation-exchange chromatography for the purification of high-purity monoclonal antibodies.

A procedure involving diafiltration, mass ion exchange on a QAE Zetaprep disk, gel chromatography and cation-exchange chromatography was used for the purification of mouse monoclonal antibodies from hybridoma culture supernatant. Prior to the separation steps, the starting solution was adjusted to the desired pH and conductivity. Diafiltration was used for this purpose in order to keep the volume constant or even to reduce the volume of sample. A QAE Zetaprep disk was used to remove the main protein contaminants from the culture supernatant. After washing unbound proteins out of the Zetaprep disk, slightly bound protein was eluted with a buffer solution containing 50 mM sodium chloride. The monoclonal antibody was eluted with a solution containing 150 mM sodium chloride. The purity of the eluted antibody was 50%, and was increased to 99% by subsequent high-performance cat-ion-exchange chromatography. The purity was determined by means of sodium-dodecyl sulphate polyacrylamide gel electrophoresis and silver staining. The advantage of the two high-performance techniques, mass ion exchange and high-performance cation-exchange chromatography, are the high-flow-rates and the high resolution that can be obtained. These techniques are suitable for the production of injectable therapeutic preparations.

Animals↗
PubMed1987

Screening for monoclonal antibodies with covalently linked antigen.

Microtitre plates prepared for ELISA are treated for the maximum adsorption of gamma-globulins (at pH 9). The adsorption of other soluble proteins (e.g. antigens) is sometimes less effective and therefore requires a higher amount of protein for the primary coat of wells. In order to reduce the amount of pure antigen required for the screening of mAb-producing hybridomas, we improved the sensitivity of our screening ELISA technique by coupling the antigen covalently to the surface of the microtiter wells. The antigen (urokinase) was coupled by the glutaraldehyde or carbodiimide procedures respectively using "aminoplates" (Nissho Iwai, Japan). The glutaraldehyde method led to at least a five fold increase of the sensitivity compared to coventional adsorption. Reduced requirement of antigen for the assay is thus achieved by a simple procedure.

Animals↗
PubMed1987

Cross flow diafiltration of serum with basal medium suitable for growth of hybridomas, sterilization and protein reduction.

Fetal Calf Serum (FCS) was extensively extracted by cross flow diafiltration (Pellicon, Millipore) and sterilized using the basal growth medium (DMEM) for the extraction. Ultrafiltration membranes of 10(5) and 3 X 10(5) Dalton cut off were used respectively. The diafiltrates were used for hybridoma cultivation and the results of growth and mAb-production were compared with standard medium (DMEM + 5% FCS). Slightly reduced mAb-titers were achieved. These were, however, compensated by decreased concentration of contaminating protein and higher specific mAb/protein ratio as examined by SDS-PAGE and enzyme linked immuno electro transfer blot (EITB).

Animals↗
PubMed1985

A laboratory fermentor for agarose immobilized hybridomas to produce monoclonal antibodies.

Mass culture of immobilized cells in airlift-fermenters usually ends up with the beads accumulating in the foamy layer on the surface of the reactor fluid or, in stirred tankreactors, with partial destruction of the beads. We tried to use an airlift fermenter vessel for growing cells, immobilized in agarose beads. Instead of using the gas for driving, we mounted a slowly turning marine type impeller within the drought tube. Oxygen was supplied on occasional demands by the original sparger. This set up leads to sufficient operational characteristics of the reactor without accumulation of the beads in the foamy layer and without mechanical destruction. Different productivities of either immobilized cells or cells in free suspension culture are reported.

Animals↗