摘要:

ABSTRACTThe epitope for E44 monoclonal antibody (mAb) was mapped using mutated ETS1 proteins lacking different carboxy-terminal regions and by the employment of synthetic oligopeptides spanning the epitope region. This epitope lies around Arg211of the human ETS1 protein since substitution of Arg211by Gin211in the epitope region results in the loss of recognition of the mouse ETS1 protein by E44 mAb. Substitution of Leu214by valine214in the epitope region (as is found in the chicken ETS1 and viral Ets proteins) does not alter the capacity of the E44 mAb to recognize this antigen. Taken together, these results suggest that a specific ionic interaction is able to play a pivotal role in the recognition of the ETS1 protein by the E44 mAb.

ISSN:0272-457X    

DOI:10.1089/hyb.1994.13.1

年代:1994

数据来源: MAL

摘要:

ABSTRACTIn this study we describe the establishment of two hybridoma cell lines secreting human monoclonal antibodies to the 22-kD nucleocapsid protein (core, p22) of the hepatitis C virus (HCV). For this purpose we isolated B lymphocytes from an anti-HCV positive blood donor and infected them with Epstein–Barr (EBV). We obtained several lymphoblastoid cell clones secreting antibodies to the recombinant HCV core protein. The B-cell cultures were oligoclonally expanded and two of them were fused with the (mouse:human) heteromyeloma cell line K6H6/B5. The resulting stable hybridomas produce antibodies of the IgG1/kappa (UI/F10) and the IgM/kappa (UI/F11) isotype reacting specifically with the recombinant core protein p22. To identify the epitopes recognized by these antibodies we synthesized overlapping peptides (13-mer and 6-mer) from the amino terminus of the core amino acid sequence. Antibody reactivity to these peptides was analyzed in an immunoblot assay. Finally, we were able to define a linear epitope recognized by the UI/F10 antibody on the nucleocapsid protein. The antibody shows specificity to the sequence N-VYLLPR-C, which corresponds to the amino acids 34–39 of the core seque

ISSN:0272-457X    

DOI:10.1089/hyb.1994.13.9

年代:1994

数据来源: MAL

摘要:

ABSTRACTSeveral monoclonal antibodies (mAb) reactive against a high-molecular-weight growth factor from human glioblastoma cell lines have been produced by immunizing mice with partially purified preparations from conditioned media. Antibody-secreting colonies were selected by their capacity to bind35S-labeled glioma cell protein and by reactivity in indirect enzyme-linked immunoadsorbent assay (ELISA), using high-molecular-weight gel filtration fractions and preparative isoelectric focusing fractions containing growth factor activities. Two of the select mAbs (20F3 and 12A12) depleted mitogenic activity (>50% inhibition,p<0.05) from gel filtration fractions by immunoprecipitation, but could not neutralize mitogenic activity directly. Mitogenic activity recovered from affinity columns prepared with mAb 20F3 eluted at 48% and 52% acetonitrile from HPLC C4 reversed-phase columns. Immunoprecipitation of35S-labeled cell lysates with 20F3 followed by resolution with SDS-PAGE autoradiography revealed one unique protein of 170 kD. Established glioma cell line D-54 MG showed perinuclear and cytoplasmic staining with mAb 20F3. mAb 20F3 should prove useful in purification and characterization of these glioma-derived growth factor(s).

ISSN:0272-457X    

DOI:10.1089/hyb.1994.13.15

年代:1994

数据来源: MAL

摘要:

ABSTRACTMurine monoclonal antibodies were raised againstMycobacterium tuberculosis(H37Rv) employing conventional hybridoma procedure. The binding characteristics of the four selected monoclonal antibodies reactive toM. tuberculosiswere assessed by enzyme-linked immunosorbent assay (ELISA) using sonic extracts. The immunofluorescence test (IFT) was done using intactM. tuberculosis, 16 other mycobacterial species, and 10 bacteria of other genera. Monoclonal antibody A30 reacted strongly toM. tuberculosisboth in ELISA and IFT. Monoclonal antibody A25 showed restricted cross-reactivity with mycobacterial species whereas two other monoclonal antibodies B6 and H2 appeared broadly cross-reactive. In Western blot analysis, A30 reacted with the 30-kD protein antigen, A25 with the 18- and 28-kD protein bands, H2 with the 18-kD antigen alone, and B6 with three bands of 17–19, 22, and 28 kD ofM. tuberculosisH37Rv. A30 exhibited high reactivity with virulentM. tuberculosisH37Rv and a clinical South Indian Strain and minimal reactivity with avirulentM. tuberculosisH37Ra strai

ISSN:0272-457X    

DOI:10.1089/hyb.1994.13.21

年代:1994

数据来源: MAL

摘要:

ABSTRACTThe isotype of a monoclonal antibody is closely associated with its biologic activity. Certain immunoglobulin subclasses are more effective than others regarding their ability to execute complement-mediated lysis of cells, antibody-dependent cell-mediated cytotoxicity, and tumor localization. Many potential targets for cancer immunotherapy are tumor-associated antigens with high percentages of carbohydrate. Immunizations of mice with carbohydrate antigens usually produce IgM and IgG3antibodies. The use of different adjuvants in immunization protocols has been associated with the induction of isotype-specific antibody responses. In experiments reported here, we compare the use of four different adjuvants on the generation of an IgG immune response to the carbohydrate-rich, tumor-associated antigen, CA-195. We report the production of IgG1and IgG2amonoclonal antibodies (mAbs) to CA-195.

ISSN:0272-457X    

DOI:10.1089/hyb.1994.13.31

年代:1994

数据来源: MAL

摘要:

ABSTRACTD-type cyclins are necessary and rate-limiting for G1progression during the mammalian cell cycle. Cyclins D1, D2, and D3 are encoded by distinct genes and are expressed in proliferating cells in a lineage-specific manner. Monoclonal antibodies (mAbs) generated to bacterially produced recombinant D-type cyclins were able to react with the native proteins expressed in mammalian cells. One mouse and three rat mAbs immunoprecipitated cyclin D1 from mouse macrophages. Only rat mAbs reacted with human cyclin D1 and cross-reacted with cyclin D2 expressed in proliferating T lymphocytes and human tumor cell lines. A single rat mAb to cyclin D2 exhibited a pattern of reactivity reciprocal to that of rat mAbs to D1. Three rat mAbs reacted specifically with mouse or human cyclin D3, but did not cross-react with cyclins D1 or D2 from either species. Representative mAbs were useful for immunoblotting and detected D-type cyclins coprecipitating in complexes recovered with antiserum to cyclin-dependent kinase-4 (CDK4). Because these mAbs detect D-type cyclins in the nuclei of fixed permeabilized cells, they should prove useful in documenting cyclin overexpression in those human tumors in which the genes are amplified or are targets of specific chromosomal rearrangements.

ISSN:0272-457X    

DOI:10.1089/hyb.1994.13.37

年代:1994

数据来源: MAL

摘要:

ABSTRACTSeveral hybridoma cell lines producing murine monoclonal antibodies (mAbs) directed to theClostridium tyrobutyricumouter cell wall have been established and characterized. Whole bacteria, crude extract of cell wall, and polysaccharide fraction of crude extract have been used as immunogens. The immunizations were performed eitherin vivoorin vitroafter primingin vivo. Amongst the clones obtained, six hybridoma cell lines were selected. Four mAbs recognized only the immunizing strain (ATCC 25755), while two mAbs recognized all theC. tyrobutyricumtested strains. Three mAbs were IgM, one IgG3, and two IgG1isotypes. The antigens (proteins or polysaccharides) recognized by these mAbs have been characterized by Western Blot. These mAbs could be used for an early detection ofC. tyrobutyricumin milk.

ISSN:0272-457X    

DOI:10.1089/hyb.1994.13.45

年代:1994

数据来源: MAL

摘要:

ABSTRACTAn experiment was set up to assess the influence of some parameters on the production of ascites and monoclonal antibodies against circulating excretory-secretory antigens ofTaenia saginatacysticerci in mice. The following parameters were examined: time lapse between priming and the inoculation of hybridoma cells, age and body weight of the mice at the time of inoculation, number of cells injected IP, and the resulting antibody titers of the ascites. In this experiment the method used to prime the mice was the only factor having an influence on the amount of ascites produced. Injection of a higher number of hybridoma cells (2−4 × 106cells) coincided with higher antibody titers and resulted in an earlier ascites production. The antibody titer of the ascites was increasing with ti

ISSN:0272-457X    

DOI:10.1089/hyb.1994.13.53

年代:1994

数据来源: MAL

摘要:

ABSTRACTTwo hybridomas secreting monoclonal antibodies (mAbs) against human/rat corticotropin-releasing factor (CRF) have been produced by the cell fusion technique. Isotyping of the mAbs revealed that both belong to the IgG1subclass. Human serum containing CRF-binding protein inhibits the binding of CRF to both mAbs. Modification of lysine residue inhibits binding of the mAbs in a different manner. Affinity constants of binding with native and histidine-modified antigens have been determined by ELISA. The epitope specificity of the mAbs has been examined in competition experiments. No competition was detected, suggesting that the mAbs recognize different antigenic determinants. Two monoclonal antibodies can be employed in a two-site assay to measure CRF.

ISSN:0272-457X    

DOI:10.1089/hyb.1994.13.59

年代:1994

数据来源: MAL

摘要:

ABSTRACTSA novel immunization procedure for eliciting murine monoclonal antibodies (mAb) is described. A murine leukemia virus (MLV)-based retroviral vector, designed as a marker protein for the expression of human factor IX(FIX), was constructed. Direct injections of mice with 0.8–1.3 × 104retroviruses were carried out three times at 4-week intervals. Three out of 4 mice that had the viruses injected subcutaneously produced the antibodies against FIX, and 2 out of 3 mice injected intraperitoneally produced the antibodies. From 1 of the mice with the antibodies, an anti-human FIX murine monoclonal antibody, designated ACTIX (IgMktype), was produced. The immunization of mice by direct injection of viruses facilitated mAb production in many instances in which cDNAs are availab

ISSN:0272-457X    

DOI:10.1089/hyb.1994.13.65

年代:1994

数据来源: MAL