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Biomedical subjects

J Zeuthen

Publications and source records attributed to J Zeuthen.

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

Monoclonal antibodies reactive with components in serum-free conditioned medium from a human breast cancer cell line (MCF-7).

Approximately 10,000 primary hybridomas were generated after immunization with either serum-free conditioned medium (SFCM) or extracts from the human breast cancer cell line MCF-7. A total of 11 different monoclonal antibodies (MAbs; 8 generated against SFCM and 3 generated against cell extracts) were selected on the basis of high specificity in cell-binding ELISA for human breast cancer cell lines. The 8 different MAbs obtained by immunization with SFCM all reacted with secreted components in SFCM from MCF-7 cells and 4 of these MAbs reacted with glycolipids extracted from MCF-7 cells. 1 of these MAbs (S2) also recognized a secreted glycoprotein of approximately 77 kilodaltons (kDa). The remaining 4 MAbs did not show specificity solely for carbohydrate determinants. 1 of these MAbs (S7) recognized a secreted protein of approximately 41 kDa. The 3 MAbs raised against cell extracts from breast cancer cells reacted with cytoplasmic antigens in immunofluorescence but also reacted with a secreted component in SFCM from MCF-7 cells. Immunoblotting experiments with proteins from cell extracts and with proteins in SFCM showed that these antibodies all reacted with a protein of a molecular weight of approximately 40 kDa. Our results suggest that this component is cytokeratin 19 or proteolytically processed cytokeratin 19.

Antibodies, Monoclonal↗
PubMed1988

Human-human hybridomas and human monoclonal antibodies obtained by fusion of lymph node lymphocytes from breast cancer patients.

Lymphocytes from lymph nodes obtained from breast cancer patients undergoing mastectomy were fused with the 0467.3, UC729HF2, or KR-12 human cell lines, totaling 42 fusions with lymphocytes from 23 patients. A total of 1696 human-human hybridomas were generated, 675 (39.8%) of which produced human IgG and/or IgM. Seventy-three human hybridomas produced antibodies binding to autologous malignant breast tissue and/or MCF-7 cells, as assayed by immunohistology or by cell-binding enzyme-linked immunosorbent assay. Twelve of these hybridomas, all reacting with malignant breast tissue, were subcloned to stabilize the production of human immunoglobulin. The reaction patterns of these 12 human monoclonal antibodies were investigated further by immunohistology on formalin-fixed, paraffin-embedded tissues. The reaction patterns of the various antibodies showed substantial variation and the antibodies reacted with a varying frequency with antigens expressed by different malignant breast tumors. One of these antibodies, MAC 40/43 (IgM), reacted with malignant breast and colon carcinomas and other epithelial derived neoplasms but did not react with normal breast tissue or with other normal and malignant tissues tested, except for a weak reaction with certain normal epithelial tissues. The antigen defined by MAC 40/43 was identified as a Mr approximately equal to 47,000 glycoprotein.

Antibodies, Monoclonal↗
PubMed1988

Applications of immunoperoxidase techniques in specificity testing of monoclonal antibodies (Mabs) against von Willebrand factor (vWf).

The present communication describes the production of a new series of murine Mabs against von Willebrand factor (vWf) in which specificity was tested using immunoperoxidase techniques. Seven Mabs showed specific reactivity with native and disaggregated vWf, whereas no binding was found to material from patients with severe homozygous (or doubly heterozygous) von Willebrand's disease (vWd) or factor VIII coagulant antigen (VIII:Ag). These Mabs are thought to carry separate specificities as only slight or no competitive activity was detected. Four Mabs partially inhibited the ristocetin-induced platelet agglutination and three interacted with vWf-binding to type I collagen. All antibodies bound to the complete range of vWf multimers of normal plasma. Excellent binding and detection properties of Mabs were found in asymmetrical two-site enzyme linked immunosorbent assays (ELISA) for quantitation of vWf antigen (vWf:Ag). One particular antibody (Mab vWf-33) discriminated vWf material from a number of subtype II vWd plasmas tested.

Animals↗
PubMed1988

Immunological characterization and detection of the major core protein p24 of the human immunodeficiency virus (HIV) using monoclonal antibodies.

Eight different monoclonal antibodies (MAbs) were raised against a lysate of the HTLV-IIIb isolate of human immunodeficiency virus (HIV). All eight MAbs recognized the major core protein p24 as well as the gag precursors p39 and p55. Three different epitopes were defined by the eight MAbs when an antigen-catching ELISA was used as the test system. An antigen-catching ELISA for p24 was developed by use of two of the MAbs defining two different epitopes. This ELISA system was applied to the detection of p24 in culture supernatants from lymphocyte cultures of 13 different HIV isolates. The present p24 detecting ELISA proved useful for characterization of different isolates of HIV. Further, two MAbs from the present panel of antibodies were demonstrated to be sensitive and specific probes for the immunohistological detection of p24 protein in tissue sections of lymphoid tissue.

Animals↗
PubMed1987

Plasminogen activator inhibitor-1 is the primary inhibitor of tissue-type plasminogen activator in pregnancy plasma.

The aim of the present work was to clarify to what extent plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) contribute to the increase in plasma inhibition of tissue-type plasminogen activator (t-PA) observed during pregnancy. It was demonstrated that a monoclonal antibody against PAI-1 almost completely quenched inhibition of single-chain t-PA and most of the inhibition of two-chain t-PA in plasma during the third trimester of pregnancy. The remaining inhibition of two-chain t-PA was to a great extent abolished by a PAI-2 antibody. The second order rate constant (k1) for inhibition of single-chain t-PA by the inhibitor neutralized by the PAI-1 antibody was about 4.8.10(6) M-1.s-1. The conversion of single-chain t-PA to the two-chain form increased the reaction rate with the inhibitor about 3-fold. These kinetic data are comparable with those obtained with PAI-1 in non-pregnancy plasma or with purified PAI-1. From the above results it is concluded that PAI-1 is the primary inhibitor of both single-chain and two-chain t-PA and that PAI-2 is the secondary inhibitor of two-chain t-PA in pregnancy plasma. The concentration of reactive PAI-1 versus gestation age was assayed in plasma from 6 women by binding of PAI-1 to 125I-labelled single-chain t-PA followed by quantitation of the labelled t-PA-PAI-1 complex after separation by SDS-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Female↗
PubMed1987

Immunochemical determination of acetylcholinesterase in amniotic fluid--an evaluation of eleven monoclonal antibodies.

Eleven monoclonal antibodies and a polyclonal rabbit antiserum were evaluated with respect to reactivity with acetylcholinesterase (AChE, EC 3.1.1.7) from erythrocytes and brain. Employing our enzyme antigen immunoassay for AChE five selected antibodies were evaluated with regard to their clinical usefulness in the prenatal diagnosis of neural tube defects (NTD). Of these, one antibody preferentially bound the enzyme from human brain, and discerned better than the others pathological samples (anencephaly, spina bifida and encephalocele) from normal ones. With this antibody no false positive values were obtained even if amniotic fluid samples were blood contaminated.

Acetylcholinesterase↗
PubMed1987

Distribution of the Burkitt's lymphoma-associated antigen (BLA) in normal human tissue and malignant lymphoma as defined by immunohistological staining with monoclonal antibody 38.13.

The distribution of the Burkitt's lymphoma-associated antigen (BLA) or globotriaosylceramide (Pk antigen) in normal human tissues and in 194 haematopoietic neoplasms was demonstrated by immunoperoxidase labelling of frozen tissue sections with monoclonal antibody 38.13. Staining was seen in most tissues of the body and was most pronounced in the epithelial compartments. In normal lymphoid tissue only dendritic reticulum cells, sinus-lining cells, macrophages and endothelial cells stained whereas lymphocytes were unlabelled. Among neoplasms, BLA was expressed strongly in 4/5 Burkitt's lymphomas. Rather weak expression was seen in 8/120 of the other B-cell lymphomas which included cases of pre-B-stage, mid-stage and secretory-stage B-cell maturation. Expression of BLA was also found in 3/54 T-cell lymphomas (all 3 of activated T-cell phenotype), 1 case of Hodgkin's disease and in 1 case of monocytic sarcoma. We found no correlation between the expression of BLA and any particular surface Ig-type in the B-cell lymphomas. We conclude that BLA is neither a tumour-specific antigen, nor a typical B-cell differentiation antigen. The results concerned with the antigen distribution in human tissues and haematopoietic neoplasms preclude the use of 38.13 as a reagent for diagnosis or specific in vivo immunotherapy of Burkitt's lymphoma.

Antibodies, Monoclonal↗
PubMed1986

Synthesis, processing, and secretion of rat immunoglobulin E made in Xenopus oocytes.

Rat immunoglobulin E (IgE) synthesized in Xenopus laevis oocytes, injected with rat plasmacytoma mRNA, was analysed by specific immunoprecipitation and SDS-polyacrylamide gel electrophoresis under reducing as well as non-reducing conditions. The results indicate that the oocytes will translate and correctly process the rat IgE heavy and light chains, resulting in secretion of a correctly assembled, normal immunoglobulin molecule. The normal, extensive glycosylation of the IgE heavy chain (e-chain) is faithfully carried out by the oocytes; therefore, this posttranslational modification is apparently of an unspecific nature, and does not depend upon a mechanism specific for plasma cells.

Animals↗
PubMed1986

Immunological relationship between the fast-acting plasminogen activator inhibitors from plasma, blood platelets and endothelial cells demonstrated with a monoclonal antibody against an inhibitor from placenta.

Two plasminogen activator inhibitors (I and II) were demonstrated in human placenta. The complex between inhibitor I and tissue-type plasminogen activator was purified by immunoadsorption to solid-phase anti-activator antibodies. The purified complex (Mr 95.000) was used for immunization of mice and subsequent production of monoclonal antibodies. One antibody (F37), which reacted with both free and complex-bound inhibitor I, was used for further study by a method involving binding of the antibody to protein A-Sepharose, immunoadsorption of antigen and analysis of the resulting supernatant by SDS-polyacrylamide gel electrophoresis and enzymography. The analysis showed that F37 reacted with the fast-acting plasminogen activator inhibitors recently demonstrated in plasma, blood platelets and endothelial cells, indicating that these inhibitors and inhibitor I share a common epitope. Inhibitor II did not react with F37. Inhibitor II is identical to the placenta inhibitor previously described by others. It reacted selectively with polyclonal antibodies against that inhibitor.

Antibodies, Monoclonal↗
PubMed1986

Identification of an Epstein-Barr virus-coded thymidine kinase.

We have demonstrated the presence of an Epstein-Barr virus (EBV)-coded thymidine kinase (TK) by producing biochemically transformed, TK-positive mammalian cell lines using either microinjection of whole EBV virions or calcium phosphate-mediated transfection of the SalI-B restriction endonuclease fragment of EBV DNA. Analysis of these cell lines showed that: (i) EBV DNA was present in the cell lines, (ii) sequences from the SalI-B restriction endonuclease fragment of EBV were expressed, (iii) a TK activity was present and (iv) a protein with antigenic cross-reactivity with the herpes simplex virus (HSV) TK was produced. The identity of the EBV TK gene was determined by demonstrating that a recombinant plasmid, which expressed the protein product of the BXLF1 open reading frame as a fusion protein, could complement TK- strains of E. coli. A comparison of the predicted amino acid sequences of the TK proteins of EBV and HSV-1 revealed significant regions of homology.

Amino Acid Sequence↗
PubMed1986

Simultaneous presence of translocations t(14;18) and t(2;8) in a case of chronic lymphocytic leukemia.

We report a patient with classical chronic lymphocytic leukemia of IgM kappa phenotype and a stable clinical course, in which repeated chromosome analyses of blood lymphocytes revealed the coexistence of t(14;18), a marker often associated with follicular low grade lymphocytic lymphomas, and t(2;8), a variant of the t(8;14) typically seen in Burkitt's lymphoma. Both these translocations involve immunoglobulin gene regions, the t(2;8) being almost always found in patients with kappa light chain restriction. However, in an EBV-immortalized cell line of this patient, most karyotypes contained t(14;18) alone, without the t(2;8). This suggests that t(14;18) was the primary cytogenetic event, and that t(2;8) evolved subsequently. As a secondary cytogenetic event, the t(2;8) may not share the grave clinical consequences of a primary t(2;8) as seen in Burkitt's lymphoma and related disorders.

Aged↗
PubMed1986

Monoclonal antibodies to K88ab, K88ac and K88ad fimbriae from enterotoxigenic Escherichia coli.

K88ab, K88ac and K88ad fimbriae derived from enterotoxigenic Escherichia coli strains involved in porcine colibacillosis were used to immunize BALB/c mice. Several hybridomas secreting monoclonal antibodies (MAbs) against the three intact K88 fimbriae subtypes were produced by fusion of spleen cells from these mice with P3-X63-Ag8.653 myeloma cells. Hybridomas producing MAbs with affinity for all 3 E. coli K88 subtypes proved to be the most frequent (248/303), but subtype-specific monoclonals (39/303) as well as MAbs reacting with two but not with the third subtype (16/303) were also produced. The antibody-containing culture supernatants from 71 selected hybridomas were characterized by enzyme-linked immunosorbent assay (ELISA) titrations, ELISA inhibition experiments and further examined by immunoblotting. Derivation of several MAbs specific for one of the E. coli fimbrial antigens, K88ab, K88ac or K88ad, was of interest in view of the extensive sequence homology in their primary structures. Specific binding of the MAbs to fimbriae on the surface of K88-positive E. coli strains was indicated by agglutination tests and visualized by immuno gold labeling and electron microscopy. The present MAbs against K88 fimbriae have potential veterinary applications for diagnosis and treatment of porcine colibacillosis. Preliminary results indicate the therapeutic value of oral administration of murine ascitic fluid containing anti-K88 MAbs to piglets experimentally infected with E. coli K88.

Animals↗
PubMed1986

Enzyme-linked immunosorbent assay for human urokinase-type plasminogen activator and its proenzyme using a combination of monoclonal and polyclonal antibodies.

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for human urokinase-type plasminogen activator (u-PA) and its inactive proenzyme (pro-u-PA) was developed. A monoclonal antibody was used as solid-phase antibody, while rabbit antibodies against human u-PA followed by peroxidase-conjugated third antibody were used for detection of bound u-PA. No reaction was observed with tissue-type plasminogen activator or with a variety of other human proteins. The assay was used for quantitation of u-PA in human urine and in culture fluid from human tumor cells. The recovery of added pro-u-PA was greater than 95%. A good agreement with the results obtained by enzymatic assays was found. The detection limit was less than 0.1 ng per ml, both for u-PA and pro-u-PA. The advantages of the use of ELISA compared with enzymatic assays and radioimmunoassays for quantitation of u-PA and pro-u-PA in biological samples are discussed.

Antibodies↗
PubMed1986

Two-site time-resolved immunofluorometric assay of human insulin.

We describe a two-site "sandwich"-type time-resolved immunofluorometric assay for human insulin, based on use of two monoclonal antibodies with different specificities. The first antibody is immobilized on the surface of microtiter plate strip wells, the other is labeled with Eu3+. Serum samples can be assayed with one incubation step; two incubation steps are required when plasma samples are assayed. After the immunoreactions are complete, the bound fraction of Eu3+-label is quantified by dissociating it in a fluorescence-enhancement solution and measuring its fluorescence with a fluorometer with time-resolution. The sensitivity of the assay is 0.24 micro-int. units/mL. The standard curve is linear from 0.24 to 2400 micro-int. units/mL.

Animals↗
PubMed1986

A T-helper cell x Molt4 human hybridoma constitutively producing B-cell stimulatory and inhibitory factors.

A human T-T hybridoma clone with helper cell phenotype was established from fusions between a HPRT- variant of the human T-cell lymphoma Molt4, and PPD-activated normal human T-lymphocytes. The hybrid clone (MP-6) was characterized with regard to expression of markers and lymphokine secretion. The T-T hybridoma was positive for Leu 3a, and thus of T-helper cell lineage. The transferrin receptor (T-9) and the interleukin 2 (IL-2) receptor (Tac) were also expressed as judged by immunofluorescence analysis using monoclonal antibodies. The hybridoma produces B-cell stimulatory factor (BSF) with proliferation and maturation activities, growth inhibitory factor (GIF), leukocyte migration inhibitory factor (LIF) constitutively under serum free conditions, but no detectable interferons (IFN-alpha, IFN-beta, IFN-delta), nor interleukin 2 (IL-2). Weak interleukin 1 (IL-1)-like activity was found. The B-cell stimulatory factor (BSF) induced solid phase-anti-mu triggered resting B-cells obtained from human spleen, tonsil or peripheral blood to proliferate and to secrete IgM and IgG. Without anti-mu triggering the BSF had no proliferation inducing effect. The BSF was characterized and partially purified using ammonium sulphate precipitation, Blue-Sepharose, HPLC hydrophobic interaction and HPLC gel filtration chromatography. The BSF was heat labile at 56 degrees C and was present in two forms, one with high and one with intermediate hydrophobicity. The more hydrophobic form of BSF has a molecular weight of 12K-14K. Kinetic studies of the lymphokine secretion revealed that BSF was produced in detectable amounts in low density (0, 2 X 10(6) cells/ml) 18-24 h cultures. In 48 h to 72 h cultures there was a significant influence of growth inhibitory activities (GIF) produced. GIF, with an apparent MW of 90K could be absorbed out on certain tumor cell lines or on Blue-Sepharose. Further absorption analysis of BSF activities show that anti-mu triggered B-cells but neither resting B-cells nor T-cells could absorb BSF activity.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1985

Plasminogen and tissue-type plasminogen activator bind to immobilized fibronectin.

Fibronectin immobilized onto polystyrene surface was found to bind plasminogen and tissue-type plasminogen activator (t-PA) but only slightly the urokinase type as determined using mono- and polyclonal antibodies against the activators. Of the defined fibronectin fragments tested, the Mr 120,000-140,000 fragment was found to bind both plasminogen and t-PA. Proteolytically modified plasminogen (Lys-plasminogen) bound considerably better than the native form (Glu-plasminogen). Experiments with 125I-plasminogen yielded Kd = 9.1 X 10(-8) M for the binding to immobilized fibronectin. The partially or completely inactive single-chain form of t-PA (pro-t-PA) bound considerably better than the activated two-chain form. Lysine at greater than 3 mM inhibited the binding of plasminogen. The interaction was independent of calcium ions. CaCl2 (greater than 0.5 mM) and NaCl (greater than 0.2 M) inhibited the binding of pro-t-PA and of t-PA. Fibronectin-bound t-PA retained its ability to activate plasminogen. The observed interactions may operate in directional proteolysis localizing plasminogen and plasminogen activator to degrade fibronectin-containing extracellular matrix including fibrin clots.

Epitopes↗
PubMed1984

Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody.

The human 66 000 mol. wt. plasminogen activator (HPA66; tissue-type plasminogen activator) has been purified from melanoma cells by a one-step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a one-polypeptide chain form with small amounts (15%) of a form containing two polypeptide chains held together by one or more disulphide bridges. The one-chain form was converted to the two-chain form by catalytic amounts of plasmin. During the conversion, the enzyme activity of HPA66, as measured by an [125I]plasminogen conversion assay and with a chromogenic substrate, increased linearly with the percentage of the two-chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the two-chain form, while the one-chain form had no measurable enzyme activity (detection limit approximately 5% of the activity of the two-chain form). Together with previous findings of inactive proenzymes to murine and human approximately 50 000 mol. wt. (urokinase-type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive one-chain proenzymes which are converted to active two-chain enzymes by limited proteolysis, thus demonstrating a third step in a cascade reaction leading to extracellular proteolysis.

Antibodies, Monoclonal↗