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Biomedical subjects

H Katinger

Publications and source records attributed to H Katinger.

At least 109 records · Page 6Linked to original sources
PubMed1989

Comparison of protein A, protein G and copolymerized hydroxyapatite for the purification of human monoclonal antibodies.

Protein A Superose, protein G Sepharose fast flow and copolymerized hydroxyapatite were used for the purification of human monoclonal antibodies against HIV 1. Both desalted culture supernatant and a prepurified protein solution were used as starting materials. The different runs were compared with respect to yield and recovery of biological activity. The biological activity (specific reactivity) was checked by antigen enzyme-linked immunosorbent assay with recombinant antigen. The human monoclonal antibodies could not be selectively eluted from the hydroxyapatite but elution could be effected from the protein A Superose at pH 4.0 and from protein G at pH 3.0. The eluted immunoglobulin G was distributed over a broad pH range when protein G Superose was used. Biologically active material could be obtained from protein A Superose and protein G Sepharose fast flow.

Antibodies, Monoclonal↗
PubMed1989

Isolation of human monoclonal antibody isoproteins by preparative isoelectric focusing in immobilized pH gradients.

A method for preparative isolation of human monoclonal antibody isoproteins is described in the present paper. A human monoclonal antibody directed against the transmembrane protein gp 41 from the human immunodeficiency virus (HIV-1) was used in this study. The antibody belongs to the IgG1 subtype and exhibits antibody dependent cellular cytotoxicity. The resolving power of conventional preparative protein separation techniques such as ion-exchange chromatography, chromatofocusing and lectin affinity chromatography is too poor for a complete separation of isoproteins. The more sophisticated technique of chromatofocusing on FPLC-based material (Mono P, Pharmacia) did not satisfy our expectation. With semipreparative IEF in immobilized pH gradients we were able to prepare the different isoproteins of a human monoclonal antibody in milligram amounts. No significant difference between the single isoproteins with respect to specificity and avidity to the recombinant antigen (rec gp 160) was detected. Therefore, we assume that the separation conditions did not influence the immunochemical nature of the antibody and significant denaturation and/or precipitation of the IgG did not occur. Furthermore the method affords preparative separation with resolution equivalent to analytical runs. Experiments for scale up and further characterization of isoproteins (carbohydrate composition, amino acid analysis, half life times etc.) are in progress.

Animals↗
PubMed1988

The high efficiency, human B cell immortalizing heteromyeloma CB-F7. Production of human monoclonal antibodies to human immunodeficiency virus.

This paper describes the construction of a new heteromyeloma cell line designated CB-F7. The cell line was derived from xenogeneic somatic cell hybridization between normal human B lymphocytes and the murine HAT-sensitive P3X63Ag8/653 cell line. CB-F7 cells were characterized by rapid cell growth (doubling time about 16 h) and high cloning efficiencies in culture medium supplemented with 10% or 5% fetal calf serum, respectively. The karyotype of the cells consists of about 75-78 chromosomes as well as two chromosomal fragments. Fusions of the cells with human peripheral blood cells resulted in approximately 2-6 clones per 10(5) seeded lymphocytes. Furthermore, the cells are ouabain resistant and therefore suitable for fusions with EBV-transformed lymphoblastoid cell lines. Using CB-F7 as the parental cell line a number of specific human mAb producing hybrids were established. For the first time, we describe here the generation of hybrids secreting human monoclonal antibodies to human immunodeficiency virus (HIV). Two monoclonal antibodies of IgG type and one of IgM type reacted with the major core protein p25 and one IgG antibody reacted with the transmembrane protein gp41.

Antibodies, Monoclonal↗
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

Production kinetics of monoclonal antibodies.

Two typical patterns of kinetics of monoclonal antibody (mAb) production are observed. Hybridomas which produce high mAb-titers do not show any feed back inhibition upon secretion of mAb into the culture supernatant over long periods of cultivation. "Low producing hybridomas" generally display high initial rates of mAb production, however, after a few hours the mAb secretion is stopped at a certain low concentration level in the order of some 100 mg mAb per ml. To illustrate the case that the high initial production rates of mAb of low producing hybridomas could be maintained for a period of at least 24 hours, mAb titers comparable to those produced by high-mAb-titer producing hybridomas were achieved.

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↗
PubMed1985

Stabilizing effect of reduced cultivation temperature on human x mouse hybridomas.

Interspecies hybridomas, such as human X mouse hybrid cells, usually show a high degree of unstability for monoclonal antibody (mAb) production. We investigated growth and specific rate of mAb-production during a period of 52 days after recloning human-mouse hybridomas at cultivation temperatures of 37 degrees C and 32 degrees C respectively. The lower cultivation temperature stabilized the overall IgM-mAb-production rate (IgM/ml) over the total cultivation period. At 37 degrees C incubation temperature the overall IgM-mAb-production was reduced to 1/3 due to loss of heavy chain production. This is visible from an increase of the kappa/mu-chain ratio. At 32 degrees C incubation temperature the specific IgM-mAb-production rate (IgM/cell X time) was stabilized, whereas at 37 degrees C incubation temperature a decrease of 80% was observed.

Animals↗
PubMed1985

Monoclonal ELISA for the determination of BNYV-virus.

Monoclonal antibodies have been developed against the BNYV-virus (beet-necrotic-yellow-vein) which causes the rhizomania disease in sugar beet (Beta vulgaris). The B103 hybridoma produced predominantly IgG2a at greater than 100 micrograms/ml. IgM-production could not, however, be ruled out. In a polyclonal-monoclonal indirect sandwich ELISA, the B103-antibody bound to BNYVV of Austrian, German, French and Bulgarian origin at a sensitivity of 1 ng/ml or less. Comparison of sugar beet samples in ELISA and immuno-electronmicroscopy showed good correlation, demonstrating the good performance of B103 antibody for broad application in BNYVV diagnosis.

Antibodies, Monoclonal↗
PubMed1983

Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

Antibodies, Monoclonal↗