Fine mapping of HIV-1 Nef-epitopes by monoclonal antibodies.

A panel of newly isolated murine monoclonal antibodies is described which are specific for the Nef protein of the human immunodeficiency virus type 1 (HIV-1). Epitope mapping using recombinant Nef-related proteins, synthetic peptides and lipopeptides showed 3 independent antigenic determinants located within the regions of amino acids 83-93, 175-190 and 86-166 of the Nef protein. None of the monoclonal antibodies reacted with recombinant Nef proteins of HIV-2.

Human monoclonal IgM antibodies from foetal B-cell hybridomas directed against a surface antigen on human tumour cells.

In order to assess the existence of B lymphocytes capable of producing anti-tumour antibodies in non-tumour-bearing individuals, human lymphocytes derived from foetuses and adults were fused with the heteromyeloma cell line CB-F7. By indirect immunofluorescence, 29 out of 4,472 IgM-producing hybridomas (from 8 foetuses and 8 adults) were shown to produce antibodies which bind to colon carcinoma lines Colo205 and SW620, Raji lymphoma cells and small cell carcinoma of the lung. In vitro growth of tumour cells recognized by these antibodies was inhibited.

Oligonucleotide fingerprinting as a means to identify and survey long-term cultured B cell hybridomas and T cell lines.

Common problems encountered during cell culture are cross-contamination, instability, and inadvertent exchange of cells. Here we report on the application of oligonucleotide fingerprinting as a simple and efficient method to screen hybridomas and T cell lines. Among the fingerprint probes tested, the simple repetitive oligonucleotide (CAC)5/(GTG)5 proved to be most useful for obtaining many fragments specific for each cell line.

Lymphocyte surface marker expression on hybridomas secreting human monoclonal antibodies.

The expression of human leucocyte markers on the surface of hybridoma cell lines producing human monoclonal antibodies was studied using immunofluorescence analysis (FACS). We tested 36 different hybridoma cell lines from fusions of lymphocytes of different organs of fetal and adult organisms with the mouse myeloma line P3 X63 Ag8.653 or the mouse-human heteromyeloma line CB-F7 (IgM-, IgG-, and nonproducer) with a panel of 21 murine monoclonal antibodies against human differentiation and activation antigens.

Expression of the HIV-1 Nef protein in the baculovirus system: investigation of anti-Nef antibodies response in human sera and subcellular localization of Nef.

The nef gene of HIV-1 was expressed in insect cells using the eucaryotic baculovirus system. The recombinant Nef protein frequently reacted with seropositive sera of HIV-1 and HIV-2 infected patients. Anti-Nef antibodies in HIV-1 seronegative high risk groups individuals were only occasionally seen.

IgG human monoclonal anti-DNA autoantibodies from patients with systemic lupus erythematosus.

We describe the production of six mouse-human heterohybridomas secreting human IgG anti-dsDNA antibodies derived from patients with systemic lupus erythematosus (SLE). Peripheral blood cells used for fusion experiments were from patients who were shown to have high numbers of anti-DNA secreting B cells in the peripheral blood. All monoclonal antibodies bind to dsDNA in ELISA systems, five are reactive with Crithidia lucilae kinetoplasts and three precipitate dsDNA in the Farr assay.

Characterization of human lymphocytes separated from fetal liver and spleen at different stages of ontogeny.

Membrane markers on human lymphocytes separated from fetal liver and spleen were studied. Depending on the period of intrauterine development, a growing percentage of T- and B-lymphocytes (up to 16% and 45%, respectively) among spleen cells was seen, but in liver the number was low independent of the gestational age (T cells less than 10% and B cells less than 15%). The majority of early CD3+ spleen cells (21st-28th week) expressed TCR alpha beta but not TCR gamma delta, although a significant proportion of these cells was still lacking CD4, CD8, and CD5 differentiation antigens, suggesting their immaturity.

Human hybridomas derived from CD5+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM (kappa) antibodies.

Great numbers of CD5+ B lymphocytes were detected in the peripheral blood of patients with B-CLL. To study the antibody repertoire of this immune cell subpopulation on a monoclonal level, we fused the lymphocytes derived from five different donors to a highly efficient HAT-sensitive heteromyeloma line (CB-F7). A fusion frequency of up to 10(-5) allowed us to analyse hundreds of initial hybridoma lines per fusion.

A solid-phase enzyme immunoassay for the detection of tetanus toxin using human and murine monoclonal antibodies.

Three human and three murine monoclonal antibodies were tested for their reactivity to tetanus toxin and toxoid and used to establish an enzyme immunoassay specific for tetanus toxin. The dissociation constants of the monoclonal antibodies were between 3.91 x 10(-9) and 8.

Ultrastructural localization of gold-labeled anti-IgM-antibodies and type A retrovirus particles in endoplasmic cisternae and vesicles of IgM-producing human B cell hybridomas.

Simultaneous appearance of IgM and type A retro-virus particles in endoplasmic cisternae and vesicles of human hybridomas has been demonstrated for the first time by immunogold-labeling at ultrastructural level. A suspected link between type A particle and IgM gene-expression in hybridoma cells could not be substantiated in this study.

Polyclonal stimulation of human B lymphocytes derived from fetal liver and spleen cells at different stages of ontogeny.

The functional capacity of human lymphocytes derived from fetal liver and spleen at different stages of ontogeny (16-34 weeks of gestation) was studied using in vitro models (increase in cell volume, [3H]thymidine incorporation, Ig secretion) reflecting various stages of activation induced by mitogens (LPS, PWM) in vitro. Lymphocytes differed in their reactivity to LPS depending on the period of intrauterine development: cells from the early liver could respond with enhanced IgM production whereas lymphocytes derived from this organ after more than 25 weeks failed. The opposite was found to apply to spleen cells: only lymphocytes derived from the organ after more than 25 weeks showed significant LPS-induced in vitro differentiation.

Immunochemical quantification of Cu/Zn superoxide dismutase in prenatal diagnosis of Down's syndrome.

Cu/Zn superoxide dismutase (SOD) was quantified by enzyme immunoassay for prenatal diagnosis of Down's syndrome. Overall, 154 samples of amniotic fluid, 72 samples of amniotic cells and 31 samples of chorionic tissue were investigated. Due to the large biological variance of the SOD concentrations in normal pregnancies (range for amniotic fluid 10.

Production and characterization of monoclonal antibodies against human Cu/Zn superoxide dismutase and the establishment of a super-rapid enzyme-linked immunosorbent assay (SURALISA).

Murine monoclonal IgG1 antibodies directed against four different epitopes of human Cu/Zn superoxide dismutase (SOD) were produced by immunization with recombinant Cu/Zn SOD. The antibodies reacted well with the recombinant protein and Cu/Zn SOD purified from human erythrocytes, with binding constants ranging from 8.8 X 10(9) to 2.

Strategies in the development of human monoclonal antibodies.

A high-efficiency, HAT-sensitive heteromyeloma fusion line CB-F7 has been developed from an 8-Azaguanin-treated Ig-non-secreting human X mouse heterohybridoma. The use of this line allowed us to produce human hybridomas more successfully by fusion of cell material from blood, lymph node or spleen. A polyspecific repertoire of IgM isotype was detected among the hybridomas obtained from the spleen.

Multireactive human monoclonal antibodies.

Human monoclonal antibodies were tested using different immunochemical procedures for their reactivity with various antigens. The great majority of human monoclonal IgM antibodies (15 out of 24) turned out to bind to a whole series of recognized antigens (DNA, keratin, tetanus toxin, ricin etc.).

[Structure and function of the immunoglobulin Vh gene in man].

The present study provides a survey on the structure and functions of human Vh-genes. The more than 100 individual gene segments on chromosome 14 are classified, according to sequence homologies, into 6 families. They differ very much in size and contain pseudogenes, form an interspersed cluster, exhibit homologies with mouse genes and have phylogenetically developed from a single primordial gene.

The hybridization of EBV-immortalized human B-lymphocytes with a human-mouse heteromyeloma cell line.

The combination of Epstein-Barr-Virus (EBV)-permitted immortalization and somatic hybridization (fusion with a myeloma partner) may be the method of choice to produce human monoclonal antibodies. We show here that the fusion of EBV-infected human B-lymphocytes to the HAT-sensitive, ouabain-resistent heteromyeloma (human x mouse) fusion line CB-F7, resulted in stable growing hybridomas producing much more immunoglobulin than the parental lymphoblastoid lines. A more efficient clonability was shown for hybridoma cultures too.

Immortalization of magnetically separated human lymphocytes by electrofusion.

Human lymphocytes from peripheral blood (MNC) were separated on magnetic beads for the presence of different surface markers. Cells from positive and negative fractions were successfully immortalized by electrofusion with the heteromyeloma line CB-Fu2. B cells, which were separated on anti-CD 19 coated beads, could be immortalized at a rate between 10(-5) and 10(-4) even if the fusion was conducted with just a few hundred thousand cells.

Development of specific IgG-producing human hybridomas by fusions of lymphocytes from actively immunized persons.

More than 13,500 initial hybridoma lines derived from fusions of lymphocytes from non-boosted persons were tested for IgG production against Tetanus Toxoid. However, only 2 were found to produce IgG monoclonal antibodies of the desired specificity. Peripheral blood lymphocytes were then taken from actively immunized donors 3, 7, 14 or 60 days after boosting and fused to the HAT-sensitive heteromyeloma cell line CB-F7.

Cryopreservation of newly formed human and mouse hybridoma cells.

The aim of this study was to develop an optimal technique for cryopreservation of newly formed human and mouse hybridoma cells immediately after fusion. It was shown that human hybridoma cells could be frozen most successfully at a cooling rate of 5 K/min whereas mouse hybridomas at 1 K/min. The percentage of FCS in cryopreservation media (30 or 90%) had no influence on the recovery of both types of hybridomas.

Development of specific human mab's by a small scale electrofusion technique: the influence of some physical and chemical factors on hybridoma yield of human peripheral blood lymphocytes XCB-F7 fusions.

A fusion chamber and an appropriate procedure are described which allow to fuse a sample of 15 to 25 microliters of cell suspension every two minutes. The cells can be observed throughout the process. They are not exposed to mechanical stress after the fusion pulse.

[Chemical characterization of Escherichia coli K 1 capsular polysaccharide].

The exact chemical characterization of the prepared Escherichia coli K 1 capsule polysaccharide is necessary and a prerequisite for using this antigen as a screening antigen in the production of monoclonal anti-K 1-antibodies. The K 1-antigen, prepared by phenol-water-extraction, was analyzed by protein, RNA, and neuraminic acid determination. An addition, the antigen was subjected to elementary analysis, infrared- and 13C-NMR-spectroscopy, and gelchromatography.

Occurrence of hybridomas producing multispecific IgM in human x (human-mouse) fusions with lymphocytes from different human immune compartments.

Human lymphocytes derived from peripheral blood, the spleen and lymph nodes were fused to the HAT-sensitive heteromyeloma cell line CB-F7. The Ig-producing initial cell lines were selected, and the supernatants were further analyzed for specific antigen binding (ELISA). IgM-antibodies were found which reacted with self- and non-self antigens of different molecular origin (nucleotides, proteins, carbohydrate structures).