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 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.

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. High yields of specific hybridomas were obtained simply by following a novel immunization technique consisting of several conventional preimmunization courses followed by 4 very high doses of antigen in saline on each of the last 4 days before fusion.

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. The determinant of the monoclonal antibody was mapped to the cell-binding region of the fibronectin molecule. This localization was based on the use of purified fragments of fibronectin, immunoblotting, EIA and inhibition of fibroblast adhesion and spreading by the antibody. The detection limit of the fibronectin assay was 2 ng/ml. The assay was used for the quantitation of fibronectin in human plasma, urine and cerebrospinal fluid specimens and culture media of human cells.

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. In addition, all three MAbs recognize the soluble (23 kDa) as well as the membrane-bound (26 kDa) forms of the COMT enzyme.

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. The responses to Con A of mixtures of CC and G-B1 low responder cells are not significantly higher than the responses of either component of the mixture alone. Thus, the gene products responsible for the complementation observed in F1 hybrids cannot complement extracellularly, and are not readily transferable from one cell to another.

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. The second takes advantage of the possibility of washing and fixing the cells in their culture wells and allows processing of more cultures with fewer manual operations. Various technical aspects of the micro-culture system are described and discussed with special reference to automation problems.

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. The mAb will be evaluated for in vivo applications.

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 2.1.1.6) was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (COMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The endogenous COMT activity, which was approximately 9.98 units per mg of protein in the untransfected cells, increased to 206 units per mg of protein upon transfection with a plasmid containing the COMT cDNA. The COMT activity of recombinant protein was inhibited competitively (IC50 = 700 nM) by the selective COMT inhibitor Ro 40-7592. An anti-COMT monoclonal antibody recognized, on immunoblots, a major polypeptide with apparent molecular mass of 29 kDa, in reasonable agreement with the predicted molecular mass. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A)+ RNA.