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Biomedical subjects

B Micheel

Publications and source records attributed to B Micheel.

At least 19 recordsLinked to original sources
PubMed1997

Fast isolation of RNA to detect expression of tumor markers.

The expression status of several tumor-related proteins is of great interest in clinical examination and research. As a completion to conventional antibody staining, RT-PCR is often used today. Reliable isolation of RNA from a low number of cells is very often a critical stage of such an examination. We demonstrate here a simple and fast method to isolate RNA from only 10,000 cells and applied it to the detection of CEA, c-ERB-B2, and mdr-1 as often studied models for tumor markers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Proteasome alpha-type subunit C9 is a primary target of autoantibodies in sera of patients with myositis and systemic lupus erythematosus.

Autoantibodies occur in low frequencies among patients with myositis characterizing only distinct subsets of this disease. Most of these known antibodies are directed to enzymatically active complexes. The 20S proteasome represents an essential cytoplasmatic protein complex for intracellular nonlysosomal protein degradation, and is involved in major histocompatibility complex class I restricted antigen processing. In this study we investigated whether the 20S proteasome complex is an antibody target in myositis and in other autoimmune diseases. 34 sera of poly/dermatomyositis patients were assayed for antiproteasomal antibodies using enzyme-linked immunosorbent assay, immunoblot, and two-dimensional non-equilibrium pH gradient electrophoresis (NEPHGE). Sera was from patients with systemic lupus erythematosus (SLE), mixed connective tissue disease, and rheumatoid arthritis; healthy volunteers served as controls. In 62% (21/34) of the cases sera from patients with myositis and in 58% (30/52) of the cases sera from patients with SLE reacted with the 20S proteasome. These frequencies exceeded those of sera from patients with mixed connective tissue disease, rheumatoid arthritis, and healthy controls. The alpha-type subunit C9 of the 20S proteasome was determined to be the predominant target of the autoimmune sera in myositis and SLE. Lacking other frequent autoantibodies in myositis, the antiproteasome antibodies are the most common humoral immune response so far detected in this disease entity.

Antibodies, Monoclonal↗
PubMed1996

Development of an enzyme immunoassay for the measurement of human tumour necrosis factor-alpha (hTNF-alpha) using bispecific antibodies to hTNF-alpha and horseradish peroxidase.

The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved in several pathological processes, and human recombinant TNF-alpha (hrTNF-alpha) is available for testing in preclinical and clinical research. The purpose of this work was the creation of tetradomas producing bispecific anti-hTNF-alpha/anti-HRP (horseradish peroxidase) antibodies and the development of a rapid and sensitive solid-phase enzyme immunoassay. Monoclonal antibodies obtained against hrTNF-alpha could recognize both natural and recombinant hTNF-alpha. The four chosen hybridomas produced IgG1 with an affinity constant of the order of 10(-9) M. Three of them recognized different epitopes. The clone selected for fusion with the hybridoma producing anti-HRP antibodies secreted antibodies against portion 30-50 of the hTNF-alpha N-terminal amino acid residues as found by Western-blot analysis with mutant and chimaeric proteins. The tetradoma producing bispecific anti-hTNF-alpha/anti-HRP antibodies was identified using a fluorescence-activated cell sorter. Bispecific antibodies were isolated by hydroxyapatite chromatography. A sandwich ELISA was developed: one of the monoclonal anti-TNF-alpha antibodies was absorbed to the solid phase as the catcher and was detected by a bispecific anti-hTNF-alpha/anti-HRP antibody. The detection limit of the assay was 1 ng/ml. With such ELISA, the level of hTNF-alpha could be conveniently estimated in different samples containing either natural or recombinant hTNF-alpha in an experimental environment or in clinical trials.

Antibodies, Bispecific↗
PubMed1995

Interaction of B and T lymphocyte subsets with high endothelial venules in the rat: binding in vitro does not reflect homing in vivo.

Lymphocytes continuously migrate through the body, and their efficient extravasation from the blood via high endothelial venules (HEV) is essential for initiating an appropriate immune response. Most investigations have focused on the lymphocyte/HEV interaction in vitro. However, to what extent such systems reflect the situation in vivo is not known. It is also unclear whether lymphocyte subsets immigrate into the HEV in proportion to their presence in the blood, and whether import capacity is limited by the HEV. When rat mesenteric lymph node lymphocytes were incubated in vitro on cryostat sections, the well-known preferential binding of B lymphocytes to HEV of Peyer's patches (PP) and T cells to HEV of axillary lymph nodes (axLN) was observed (axLN vs. PP: B lymphocytes 21.2 +/- 5.0% vs. 40.6 +/- 11.0%, T lymphocytes 84.6 +/- 6.3% vs. 56.5 +/- 12.9%). However, when labeled mesenteric lymph node lymphocytes were injected and their location within the HEV was analyzed 15 min later, no preferential interaction was seen. After injection of labeled thoracic duct lymphocytes, the percentage of labeled cells among B and T lymphocytes in the blood was significantly different (4.4 +/- 0.9% vs. 8.9 +/- 3.6%), whereas that in HEV of axLN (19.0 +/- 6.4% vs. 16.6 +/- 6.0%) and PP (30.6 +/- 6.1% vs. 33.9 +/- 4.4%) was comparable. Although the number of injected lymphocytes was similar in magnitude to the total blood lymphocyte pool, after injection there was no increase in lymphocyte numbers in the HEV. Thus, the adhesion assay in vitro does not completely reflect immigration into HEV in vivo. In addition, our data suggest that both the availability of lymphocyte subsets in small venules and the immigration rate into HEV are actively regulated in vivo.

Animals↗
PubMed1995

Lymphocyte traffic through lymph nodes and Peyer's patches of the rat: B- and T-cell-specific migration patterns within the tissue, and their dependence on splenic tissue.

The migration routes of lymphocyte subsets through organ compartments are of importance when trying to understand the local events taking place during immune responses. We have therefore studied the traffic of B, T, CD4(+), and CD8(+ )lymphocytes through lymph nodes and Peyer s patches. At various time points after injection into the rat, labeled lymphocytes were localized, and their phenotype characterized in cryostat sections using immunohistochemistry. Morphometry was also performed, and the recovery of 51Cr-labeled lymphocytes in these organs was determined. B and T lymphocytes entered the lymph nodes via the high endothelial venules in similar numbers. Most B lymphocytes migrated via the paracortex (T cell area) into the cortex (B cell area), and then back in substantial numbers into the paracortex. In contrast, T lymphocytes predominantly migrated into the paracortex and were rarely seen in the cortex. No obvious differences were seen between various lymph nodes and Peyer s patches and the routes of CD4(+) and CD8(+)lymphocytes. After injection of lymphocytes into animals with autotransplanted splenic tissue, the number of B lymphocytes that had migrated into the B cell area of lymph nodes and of Peyer s patches was significantly decreased, whereas CD4(+) lymphocytes migrated in larger numbers into the T cell area of both organs.

Animals↗
PubMed1995

The accessibility of thiophosphorylated groups in DNA fragments to the enzymatic activity of ligases and restriction endonuclease Bbs I.

The aim of this paper was to test the possibility to ligate and hydrolyse DNA sequences containing thiomodified ends and bonds. T4 DNA ligase was shown to ligate DNA fragments regardless of whether it contains phosphorylated or thiophosphorylated 5'-end. But the cleavage of an internally thiomodified phosphodiester bond was found to be totally inhibited when using the non-palindromic restrictase Bbs I. The special properties of this restriction endonuclease should allow the development of an oriented cloning strategy when combined with T4 ligase and a thiophosphorylation of DNA fragments.

Base Sequence↗
PubMed1994

A flow cytometric long-term cytotoxicity assay.

A method to evaluate cytotoxic effects applicable over a wide range of incubation times has been developed. It is based on quantification by flow cytometry of dead and viable target cells stained by covalently binding the fluorescent dye fluorescein isothiocyanate (FITC). The staining with FITC did not affect cell viability and growth parameters and was stable enough to identify target cells for at least 2 days. The flow cytometric analysis of the cell mixture was performed in a test system with activated CD8+ lymphocytes as effector cells and melanoma M21 cells as targets in the presence of appropriate bispecific antibodies and revealed a rather complex pattern composed of several distinct cell subsets which were identified by use of antibodies to lymphocyte antigens. The assay compared favourably with results from a conventional 51Cr release assay obtained after 4 h and 8 h of incubation and from a target cell adherence assay obtained after 24 h of incubation. The application of the method described herein is especially advantageous for the evaluation of long-term cytotoxic effects. Furthermore, it provides valuable multi-parameter information which is useful for elucidating mechanisms of cytotoxicity.

Animals↗
PubMed1994

A simple and sensitive method to study effects mediated by soluble lymphokines as demonstrated by the interaction of CD4+ and CD8+ cell subsets during T cell activation.

A method is described for the study of lymphokine-mediated cellular interactions using triple wells, which permits co-culture of cell subpopulations without direct physical contact. The triple wells are constructed by slitting the walls to half height between three adjacent wells of a 96-well microtiter plate. The cells under study are positioned in the outer two wells, whereas the middle well serves to separate the cells. The half slits permit the wells to be treated independently before filling the triple well with the culture medium and prevents cell leakage thereafter. The feasibility of the method was established by studying the interaction of isolated CD4+ and CD8+ T cell subsets during T cell proliferation induced by immobilized anti-CD3 and anti-CD28 monoclonal antibodies.

CD4-Positive T-Lymphocytes↗
PubMed1994

Production of monoclonal antibodies against epitopes of the main coat protein of filamentous fd phages.

Three monoclonal antibodies (MAbs) were produced which react with epitopes of the main structural coat protein (pVIII) of filamentous fd phages as demonstrated by solid-phase fluorometric enzyme immunoassays and by immunoelectron microscopy. The antibodies are of the IgG1, IgG2a and IgG2b immunoglobulin subclasses. Since they also react with recombinant phages expressing antigen fragments in their pIII region they may be suitable reagents for the demonstration and isolation of filamentous phages used in recombinant protein technology.

Animals↗
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↗