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Biomedical subjects

B Micheel

Publications and source records attributed to B Micheel.

At least 37 records · Page 2Linked to original sources
PubMed1994

Surface display of antibodies.

To screen antibody libraries that contain many millions of different clones, a selection system is required with an efficiency comparable to that of the immune system. This can be achieved by displaying antibodies on the surface of microorganisms containing the antibody's gene, analogous to the expression of the IgM antigen receptor on the surface of unactivated B-lymphocytes. Specific clones can then be selected using immobilized antigens. The minor coat protein of filamentous phages, pIII, which initiates the infection of E.coli by binding to their F-pili, and the major coat protein, pVIII, have been used as carriers for displaying antibodies on the phage surface. Recombinant antibodies have also been targeted to the cell surface of bacteria by fusing them with outer membrane components derived from lipoproteins, OmpA and an IgA protease. However, only the pIII system has been routinely used for screening antibody libraries. Here we describe the various antibody surface display systems and the screening of antibody libraries generated from the gene repertoire of lymphocytes and by gene synthesis. Finally, we have made a short comparison of the bacterial production of Fabs versus single chain antibodies (scFv).

Journal Article↗
PubMed1993

Hybrid hybridomas producing bispecific antibodies to CEA and peroxidase isolated by a combination of HAT medium selection and fluorescence activated cell sorting.

A combination of fluorescence-activated cell sorting and HAT medium selection has been used to establish bispecific antibody (biAbs)-producing hybrid hybridomas. For this purpose hypoxanthine-guanine phosphoribosyl transferase (HGPRT)-deficient mutants were isolated from a hybridoma line (D11-DG2) producing anti-CEA antibodies by 8-azaguanine treatment. The resulting HAT-sensitive hybrid cells were stained with the fluorescence marker tetramethyl rhodamine isothiocyanate (TRITC) and fused by polyethylene glycol (PEG) with HAT-non-sensitive unstained hybrid cells producing antibodies to horseradish peroxidase (POD). Fluorescent fused hybrid hybridomas as well as non-fused stained anti-CEA cells were separated from the unstained anti-POD cells using a fluorescent activated cell sorter (FACS). Finally, non-fused enzyme-deficient anti-CEA cells were eliminated by cultivation in HAT selection medium which permits only an outgrowth of HAT-resistant hybrid hybridoma cells containing the genes for producing both antibodies.

Animals↗
PubMed1993

Monoclonal antibodies to human chorionic gonadotropin (HCG) and their use in two-site binding enzyme immunoassays.

A panel of mouse monoclonal antibodies (MABs) was produced against human chorionic gonadotropin (HCG) and its isolated beta-subunit (beta-HCG). According to their binding specificities the antibodies could be divided into HCG-specific and cross-reactive MABs. The HCG-specific antibodies reacted with antigenic sites on holo-HCG or holo-HCG and beta-HCG, or exclusively with the non-associated beta-HCG chain. The cross-reactive antibodies reacted with either HCG and luteinizing hormone (LH) or with HCG, LH, follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH). According to the binding specificities of the MABs and their reciprocal inhibition detected in two-site binding enzyme immunoassays (EIA), altogether 13 epitopes (including the 3 hidden epitopes detectable only on free non-associated beta-HCG) were distinguished by the antibodies described here. Antibody combinations resulting in most effective and specific HCG- or beta-HCG-determination were used as clinical assays and proved their reliability and correctness for monitoring patients with HCG- and/or beta-HCG-producing tumors before and after therapy.

Animals↗
PubMed1992

Bispecific IgA/IgM antibodies and their use in enzyme immunoassay.

Two hybrid hybridomas secreting polymeric bispecific antibodies to human chorionic gonadotropin and calf intestinal alkaline phosphatase were produced by fusion of IgA- and IgM-secreting mouse hybridomas. Both hybrid antibodies were purified from ascitic fluid by size exclusion chromatography. An IgM-like fraction was shown to exhibit bispecific activity. Bispecificity was completely lost following mild reduction and alkylation. Both bispecific antibodies were used to develop a sensitive enzyme immunoassay for hCG.

Alkaline Phosphatase↗
PubMed1991

Immunoaffinity purification of human alpha-fetoprotein (AFP) using monoclonal antibodies.

A two-step immunoaffinity isolation procedure for human alpha-fetoprotein (AFP) was developed resulting in highly purified AFP at high yield. A monoclonal antibody immunoadsorbent was used in the first step. Elution of AFP was carried out at alkaline pH by a solution of 0.5 mol/l ammonia containing 0.5 mol/l sodium chloride. To remove impurities caused by the first step, an anti-mouse immunoglobulin antibody immunoadsorbent was applied in the second step.

Antibodies, Monoclonal↗
PubMed1991

Production and characterization of monoclonal antibodies against carcinoembryonic antigen (CEA).

In the experiments presented here 22 monoclonal antibodies (MoAbs) were produced which reacted with the tumor marker carcinoembryonic antigen (CEA). Eleven of the MoAbs reacted neither with peripheral blood granulocytes nor with purified spleen NCA-60 kDa and were therefore regarded as "CEA-specific". Only three antibodies of this group reacted exclusively with CEA-180 kDa. Eight MoAbs reacted with CEA-180 kDa and with CEA-like substances of lower molecular mass (of 160 kDa and/or 120 kDa) present in colon carcinoma cells as determined by immunoblotting. These molecules seem to be different from the classical non-specific cross-reacting antigens (NCAs) present in peripheral blood granulocytes. In contrast to that, the other 11 anti-CEA MoAbs recognized in addition to CEA-180 kDa also NCAs on granulocytes. Six of them were reactive with a purified spleen NCA-60 kDa preparation. These MoAbs bound also to reduced and alkylated CEA-180 kDa (CEA r/a), i.e. they recognize sequential epitopes. All 22 MoAbs reacted with CEA expressed in different human tumor cell lines as determined by immunocytological analysis. But six of them did not bind to the surface of these cells when tested in a radioimmuno-binding assay. It was concluded that the epitope(s) recognized by these antibodies are involved in cell membrane anchoring of the CEA-molecules.

Antibodies, Monoclonal↗
PubMed1990

An experimental immunoscintigraphic study with anti-CEA monoclonal antibody (DG2).

Monoclonal antibody D11-DG2 (DG2) against carcinoembryonic antigen (CEA) was examined for suitability for radioimmunodetection of human tumors grown in nude mice. Antibodies DG2 and a control antibody of the same IgG1 subclass were labeled with 131I and injected into mice bearing one of three types of CEA-containing tumors (cell lines LS 174T, HT-29 and Rec S) and/or a CEA-negative tumor (Rec R). Gamma-camera imaging and distribution studies revealed that CEA-containing tumors selectively accumulate DG2 but Rec R does not. As the tumors differ in CEA-content, the highest accumulation of 131I-DG2 (corresponding to the best scintigraphic imaging) was found in LS 174T tumors, intermediate in Rec S and lowest in HT-29 tumors. The mean tumor-to-blood ratios on the sixth day after antibody administration were 4.6, 3.2, and 2.1, respectively, in the control experiments the value of this parameter was always lower than 1. The results showed the applicability of DG2 for immunoscintigraphic studies in patients. Furthermore, a positive correlation was found between the uptake of anti-CEA antibody and CEA-content in the tumors.

Animals↗
PubMed1989

[Immunoscintigraphy in experiments and clinical practice: its possibilities, advantages and limitations].

The authors present their experience gained in preparing, isolating and labeling antibodies with radionuclides for the purpose of using them in immunoscintigraphy. The experimental part includes results obtained with different labeled antibodies and their F/ab/2 fragments in distribution studies, involving also immunoscintigraphic imaging of tumors. The clinical part presents results of immunoscintigraphy obtained with the commercial antibody kits Iodomab and Imacis in patients with tumors of the digestive tract.

Animals↗
PubMed1988

Production and ELISA application of bispecific monoclonal antibodies against fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP).

Hybrid hybridomas producing bispecific monoclonal antibodies reacting with both horseradish peroxidase (HRP) and fluorescein isothiocyanate (FITC) were obtained by fusing two hybridoma lines and selecting the fused cells using a fluorescence activated cell sorter (FACS). FITC was used to label different monoclonal antibodies and the bispecific antibodies acted as a linking agent between FITC-labelled antibody and the marker enzyme HRP. This system was used in enzyme immunoassays for the detection of different antigens. The results suggest a wide application of bispecific anti-FITC/anti-HRP antibodies as a detection system in EIA.

Animals↗
PubMed1987

Bispecific antibody-producing hybrid hybridomas selected by a fluorescence activated cell sorter.

Hybrid hybridomas (tetradomas) producing bispecific monoclonal antibodies reacting with both horseradish peroxidase (HRP) and human alpha-fetoprotein (AFP) were obtained by fusing two hybridoma lines and selecting the fused cells by a fluorescence activated cell sorter (FACS III). The hybridoma cells were labelled before fusion with fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC) respectively and heterofluorescent cells were sorted out after fusion. Several clones were found to produce bispecific antibodies, and one clone, designated T1, was subjected to growth in ascitic fluid in mice in order to obtain large quantities of hybrid antibodies. Bispecific antibodies could be separated from the monospecific antibody populations by one-step hydroxylapatite chromatography. SDS-polyacrylamide gel electrophoresis demonstrated that the hybrid antibody molecules contained the heavy chains of both anti-HRP and anti-AFP origin. The bispecific antibodies were used to build up a sensitive two-site binding enzyme immunoassay.

Animals↗
PubMed1986

Differences of K562 leukemia cell clones in the pattern of monoclonal antibody binding and NK cell susceptibility.

Fifty-three K562-clones were tested against three monoclonal anti-K562-antibodies (MoAbs). The individual NK-cell sensitivity of thirty-one K562-clones was evaluated simultaneously. It was found that considerable differences exist in the antibody binding between single clones and the parental cell line indicating antigenic heterogeneity in the K562 line. Some K562-clones showed increased NK-cell sensitivity in comparison with the uncloned cell line. However, no correlation was observed between both investigated parameters.

Antibodies, Monoclonal↗
PubMed1986

Search for CEA-like molecules in polymorphonuclear leukocytes of non-human primates using monoclonal antibodies.

The monoclonal anti-CEA antibody ZIK-A42-A/C1 which reacts with NCA of human polymorphonuclear leukocytes was found to bind also to polymorphonuclear blood leukocytes of the following non-human primates tested: hamadryas baboon (Papio hamadryas), stump-tailed monkey (Macaca arctoides), pig-tailed monkey (Macaca nemestrina), and rhesus monkey (Macaca mulata). No binding was observed to mononuclear blood leukocytes. It was concluded that non-human primates contain CEA-like substances in their polymorphonuclear leukocytes as humans do and that these substances carry some identical epitopes.

Animals↗
PubMed1985

Monoclonal antibodies against the human leukemia cell line K 562.

Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

Antibodies, Monoclonal↗
PubMed1985

Reactivity of monoclonal anti-K 562 antibodies with cells of leukemia- and lymphoma-patients.

For further characterization, monoclonal anti-K 562 antibodies (1) were tested against blood or bone marrow cell samples of patients with various leukemias and lymphomas. One antibody, ZIK-C1-A/D9 (also designated Y) reactive in previous tests exclusively with K 562 cells, but not with normal blood cells, exhibited a selective binding to cells of most AML-patients and CML-patients in myeloid blast crisis. Cells of patients with other hematopoietic malignancies were negative, except three single cases (one lymphosarcoma, one AUL and one hairy cell leukemia). Antibody ZIK-C1-B/H5 (short name H) detected an antigenic determinant, preferentially expressed on cells of AML and CML patients, but also on normal granulocytes and some mononuclear cells. Two additional monoclonal anti-K 562 antibodies, ZIK-C1-A/F5 (short name C) and 2B7, yielded specificities shared by a variety of normal and malignant hematopoietic cells.

Antibodies, Monoclonal↗