ISSN / EISSN: 0272457X / 0272457X
Published by: Mary Ann Liebert Inc
Total articles ≅ 1,677
Latest articles in this journal
Hybridoma, Volume 20, pp 285-285; https://doi.org/10.1089/027245701753179901
Hybridoma, Volume 20, pp 284-284; https://doi.org/10.1089/027245701753179893
Hybridoma, Volume 20, pp 283-283; https://doi.org/10.1089/027245701753179884
Hybridoma, Volume 20, pp 281-282; https://doi.org/10.1089/027245701753179875
Hybridoma, Volume 20, pp 273-279; https://doi.org/10.1089/027245701753179866
Amidase from Pseudomonas aeruginosa was purified by anionic exchange chromatography and used to immunise female Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. A selected IgM subclass MAb was purified from in vitro hybridoma cell line supernatant by a two-step anionic exchange chromatography. The MAb was specific for amidase from P. aeruginosa as determined by Western blotting and recognized the native and denatured forms of the enzyme.
Hybridoma, Volume 20, pp 257-263; https://doi.org/10.1089/027245701753179848
Generation of three monoclonal antibodies (MAbs) to the major oncoproteins of human papillomavirus (HPV) was accomplished by an intense prime/boost regimen. Mice were primed with expression vectors expressing either the E6 or E7 oncoproteins of HPV-16 followed by boosting with a vaccinia virus construct and a replication-defective E1-deleted adenoviral recombinant of the human strain 5, and last, with baculovirus-derived HPV-16 E6 and E7 proteins in incomplete Freunds' adjuvant. Splenocytes were then fused with a myeloma cell line. The vaccination protocol generated one anti-E7 MAb of the IgM isotype and two anti-E6 MAbs of the IgG1 subisotype. The MAbs were tested for functionality in standard laboratory assays and found to detect the E6 and E7 proteins, respectively. The E7 MAb cross-reacted with the HPV-1a E7 oncoprotein. The binding sites of the MAbs were mapped to defined regions of each viral protein.
Hybridoma, Volume 20, pp 237-242; https://doi.org/10.1089/027245701753179811
Polydeoxyribonucleotide kinase (PNK) is a mammalian DNA repair enzyme that has the capacity to phosphorylate 5′ DNA termini and dephosphorylate 3′ DNA termini. A series of murine monoclonal antibodies (MAbs) was raised against the full-length recombinant human PNK. Seven of these antibodies were selected and characterized by enzyme immunoassay, Western blot analysis, and their capacity to immunoprecipitate PNK. The epitope location was defined by cyanogen bromide digestion and by using a truncated PNK for Western blot analysis. All of the MAbs recognize a single 60-kDa protein in human cell extracts. PNKs from calf, monkey, and Chinese hamster cell and tissue extracts were also detected by some or all of the MAbs. These antibodies can be successfully used for the cellular, biochemical, and functional analysis of PNK in different mammalian cell lines.
Hybridoma, Volume 20, pp 231-236; https://doi.org/10.1089/027245701753179802
The monoclonal antibody (MAb) A6H, originally developed to fetal renal tissues, was found to be highly reactive to renal cell carcinoma and was subsequently demonstrated to co-stimulate a subpopulation of T cells. The A6H antigen had not been identified heretofore. Antigen from detergent extracts of renal cell carcinoma cells (7860) was immunoabsorbed with A6H-agarose, and the resin-bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen had a molecular weight of approximately 120 kDa as determined by Western blots. The 120-kDa protein band was excised and subjected to in-gel tryptic digestion, and the resulting peptides were separated and analyzed by liquid chromatography tandem mass spectrometry (LC MSMS). The tandem mass spectra of the eluting peptides were used in combination with the SEQUEST computer program to search a human National Cancer Institute (NCI) protein database for the identity of the protein. The target antigen was shown to be dipeptidyl peptidase IV (DPP IV), which is also known as the cluster differentiation antigen CD26. Flow analysis of the expression of the A6H antigen and of CD26 on 7860 cells and on peripheral blood lymphocytes supported the identification of the A6H antigen as DPP IV. Recognition that the A6H antigen is DPP IV/CD26 afforded the opportunity to compare previous studies on A6H with those on other anti-CD26 antibodies in terms of expression in cancer cell lines and various tissues and as co-stimulators of T-cell activation.
Hybridoma, Volume 20, pp 223-229; https://doi.org/10.1089/027245701753179794
Monoclonal antibodies (MAbs) directed to Lewisx (Lex) and related carbohydrate sequences have been invaluable in anticipating biological roles for these oligosaccharides by detecting the remarkable changes that occur in their expression from the earliest stages of embryogenesis, through development and sequential stages of cell differentiation and maturation. A notable impact has been in the molecular dissection of ligand-receptor interactions in key cell adhesion events at the initial stages of leukocyte recruitment in inflammation, and almost certainly in the metastasis of epithelial tumours. Antibodies that recognise Lex and the 3′-sialyl forms were observed to identify leukocyte subsets; these were subsequently found to match those recognized by the leukocyte-endothelium adhesion molecules, the E- and P-selectins. We now describe a MAb (rat hybridoma MIN/3/60) raised to 3′-sulpho-Lex, a carbohydrate sequence which, in vitro, is bound not only by the E-, L-, and P-selectins, but also by the cysteine-rich domain of the macrophage endocytosis receptor. We observe that MIN/3/60 is bispecific, however; it binds 3′-sulpho-Lea as well as 3′-sulpho-Lex. Nevertheless, our exploratory studies reveal that it may be a useful histochemical reagent when used in conjunction with a monospecific antibody to 3′-sulpho-Lea. The MIN/3/60 antibody reveals a sub-population of epithelial glycans in the crypts of Lieberkühn in normal human colon.
Hybridoma, Volume 20, pp 265-272; https://doi.org/10.1089/027245701753179857
Eight monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) were characterized. Five clones are IgG1, two clones are IgM and one clone is IgG2b; all have kappa light chain. The affinities are in the range of 1.1 × 10-7 ~ 2.4 × 10-9 M; the affinities of two IgM clones could not be estimated because of their low enzyme-linked immunoadsorbent assay (ELISA) signal. Each clone was constructed as single-chain Fv (scFv) and expression was performed in E. coli. Four clones out of 8 could express scFv soluble to culture media and the expression was confirmed further by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The nucleotide and amino acid sequences of VH and VL of four scFvs were deduced and their family and subgroup were analyzed. We found that the clones that do not express the scFv have aberrant kappa chain (incorrect V/J recombination or stop codon); in contrast, their heavy chain sequences proved correct. The E. coli-expressed scFvs showed 1.5 × 3.4-fold lower affinities (2.8 × 10-8 ~ 3.6 × 10-9 M) than those of hybridoma-derived parental antibodies except the one clone (C5), which exhibited ~ 10-6 M of affinity.