Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver.

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.

Human catechol-O-methyltransferase: cloning and expression of the membrane-associated form.

A cDNA clone for human catechol-O-methyltransferase (hCOMT; S-adenosyl-L-methionine:catechol O-methyltransferase; EC 2.1.1.

Monoclonal antibodies recognizing both soluble and membrane bound catechol-O-methyltransferase.

Both cytosolic, soluble and membrane-bound catechol-O-methyl-transferase (COMT) from pig and rat liver or kidney were recognized by mouse monoclonal antibodies (MAbs) raised against soluble COMT isolated from pig liver. In ELISA, the MAbs Co 16 and Co 54 reacted better with the pig than with the rat enzyme. The MAb Co 60 showed good reactivity with both pig and rat COMT.

Monoclonal antibodies against antigens on breast cancer cells.

Of 360 mAb obtained in a cell fusion experiment with the spleen cells of a mouse immunized with a mixture of different human breast carcinoma cell lines, 30 mAb were selected which reacted more strongly with tumor cells than with (noncancerous) fibroblasts. These mAb were tested for reactivity with additional types of cancerous and noncancerous tissues. Two mAb showed high tumor selectivity, but the corresponding epitopes on individual tumor cells were heterogeneously expressed.

Immunohistochemical characterization of an anti-epithelial monoclonal antibody (mAB lu-5).

A mouse monoclonal antibody (mAB lu-5) was prepared using a lung cancer cell line as an antigen. The selected clone produces an IgG with a gamma-1 heavy chain and a kappa-light-chain. Immunohistochemical testing of mAB lu-5 on 117 normal tissue biopsies and 474 tumours revealed reactivity with an intracytoplasmic, formaldehyderesistant antigen present in most epithelial and mesothelial cells, but absent in mesenchymal cells.

A rapid and highly sensitive solid-phase enzyme immunoassay specific for human fibronectin using a characterized monoclonal antibody.

A solid-phase enzyme immunoassay (EIA) was developed for the quantitation of human fibronectin in body fluids and cell culture media. In the assay a human fibronectin-specific murine monoclonal IgG1 (f-33) was used as capture antibody and polyclonal rabbit anti-fibronectin as detector antibody. The antibody showed no reactivity to purified monkey, dog, rabbit, horse, sheep, mouse, bovine or chicken fibronectins.

Monoclonal antibody against the N-terminal end of human plasma fibronectin.

Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment.

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them.

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not.

Human fibroblasts X mouse cell hybrids, containing a human 11/X translocation, do not express human fibronectin.

Using a monoclonal antibody specific for human fibronectin (FN), we screened hybrid clones derived from the fusion of FN+ human fibroblasts, carrying a 11/X translocation, and FN-, HPRT- mouse cells for the production of this glycoprotein. Since no hybrid clone retaining the human der 11 chromosome was found to produce any human fibronectin, the segment of chromosome 11 included in the rearranged chromosome (11qter leads to 11p13) probably does not carry the structural locus for fibronectin.

Production of hybridomas secreting monoclonal antibodies to the human leukocyte interferons.

Thirteen monoclonal antibodies to human leukocyte interferon have been obtained. They exhibit different patterns of binding to purified leukocyte interferon species that are consistent with the structural multiplicity of the human leukocyte interferons. These antibodies will be useful as probes into the structure of the human leukocyte interferons, for their purification, and for rapid assay of leukocyte interferon.

High frequencies of antigen-specific hybridomas: dependence on immunization parameters and prediction by spleen cell analysis.

Hybridomas producing antibodies against soluble antigens have in most cases been difficult to establish. After fusion of myeloma cells with spleen cells obtained from mice immunized with a soluble protein, hybridomas secreting specific antibodies have been observed to occur very rarely among non-specific hybridomas. We found that the frequency of specific hybridomas correlates directly with the increase over background of the frequency of blast and/or plasma cells in the spleen (measured by cell size analysis) after antigenic stimulation.

Complementation between genetic variants affecting the response of chicken leukocytes to concanavalin A.

Birds of the partially inbred G-B1 chicken line can be classified as either high or low responders to Con A, on the basis of the amount of 3H-thymidine incorporated by Con A-containing cultures of their peripheral blood leukocytes. The pattern of inheritance of the high and low responder traits suggests that the variation in response is due to genetic polymorphism at a single autosomal locus. However, the allele responsible for the low responder trait of the G-B1 line is not identical to the allele of the previously described Mr1 locus carried by the inbred low responder CC line, since (CC x G-B1 low responder)F1 birds are uniformly high responders to Con A.

Analysis of the alloimmune properties of a recombinant genotype in the major histocompatibility complex of the chicken.

In a population of (CB X CC)F1 X WB hybrids, a chicken was found with a presumably recombinant haplotype, BR1, whose antigenic products detectable by hemagglutination contained determinants derived from both parental haplotypes, i.e. B1 (from CB) and B2 (from CC).

I-region-associated determinants: expression on mitogen-stimulated lymphocytes and detection by cytotoxic T cells.

We have studied the expression of Ia-antigens, controlled by genes in the I-region of the H-2 complex, on phytohemagglutinin (PHA)-stimulated lymph node cells and on lipopolysaccharide (LPS)-stimulated spleen cells, and have compared these two types of cell populations as targets for killer cells in the cell mediated lympholysis (CML) assay. PHA targets are almost completely insensitive to complement-mediated lysis by anti-Ia sera while the majority of LPS targets are killed. T cell-mediated lysis against I-region determinants was also detected, and these determinants, in contrast to H-2K and H-2D CML determinants, seem to be much more strongly expressed on LPS-stimulated cells.

Further miniaturization and automation of in vitro lymphocyte cultures.

A microtechnique (10 mul culture volume) for in vitro lymphocyte cultures is described, which, compared with current miniaturized techniques, permits a four to 20-fold reduction of both medium volume and cell numbers. Furthermore, two alternative procedures for harvesting and washing radioisotope labelled cells are described. The first, making use of a conventional harvesting device, collects cells on glass fibre filters in a two to three-fold shorter time than that needed for the current semi-automatic collectors.