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Biomedical subjects

E Wenisch

Publications and source records attributed to E Wenisch.

26 records · Page 2Linked 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↗
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↗
PubMedDate not supplied

Purification to single isoforms of a secreted epidermal growth factor receptor in a multicompartment electrolyzer with isoelectric membranes.

A purified, size-homogeneous (100 kDa), desialylated form of a truncated, soluble form of the epidermal growth factor receptor secreted by A431 human tumor cells has been found, by isoelectric focusing in immobilized pH gradients, to consist of two major isoforms (with pIs of 6.96 and 6.71), one intermediate form (pI 6.45) and a number (> 10) of minor components. The two major components have been purified to charge homogeneity by isoelectric focusing in a multicompartment electrolyzer with buffering isoelectric membranes having the following pI values: 5.90, 6.63, 6.76, 6.92, 7.05 and 7.35. Such single pI species are presently used for attempts at crystal growing.

ErbB Receptors↗
PubMedDate not supplied

Pilot scale production of a human monoclonal antibody against human immunodeficiency virus HIV-1.

Human monoclonal antibodies against the transmembrane protein gp41 of HIV-1 were isolated and purified on a pilot scale. A purification scheme was established for the production of human monoclonal antibodies on the gram scale. 50 1 of culture supernatant can be treated in one purification cycle. The hybridomas were mass cultured in an airlift fermenter. The culture broth was clarified by microfiltration and chromatographed on CM-Sepharose fast flow and protein A Superose. Scale up of the high performance affinity chromatography from 1 ml protein A Superose up to 40 ml is described. All desalting steps were performed by gel filtration on Sephadex G-25 coarse. The yield of the whole purification procedure is in the range of 50-60%. The purity is higher than 99.9%. DNA and reverse transcriptase could not be detected. The whole method is designed as a basis for scale up to industrial scale. Results from quality control assays have proven the validity of this approach.

Antibodies, Monoclonal↗