A new and rapid method for the selection and cloning of antigen-specific hybridomas with magnetic microspheres.

A new and highly selective procedure is described for the rapid selection and cloning of antibody-secreting hybridomas with antigen-coated magnetic beads. Immune splenocytes were fused to Sp2 myeloma cells with a PEG/DMSO mixture. Cells were cultured in HAT and screened for the presence of specific antibody after 7-10 days. Hybridomas from the positive colonies were mixed with antigen-coated magnetic polymer beads and antigen-specific cells separated on a magnet. Cloning was carried out directly by limiting dilution with the magnetic beads still bound on the cells. No obvious toxic effects were observed. The antibodies established by this technique were of a high affinity (greater than 10(9) l/mol) and were generated in 50% of the usual process time. The procedure described here should greatly facilitate the process of obtaining hybridomas and expand the range of monoclonal antibodies available.

Human peripheral blood monocyte and bronchoalveolar macrophage cytotoxicity for cultured human lung tumor cells.

Human peripheral blood monocytes (PBM) and bronchoalveolar macrophages (BAM) were tested for cytotoxicity toward a cultured human lung tumor cell line, A549, using a 75Se-methionine post-labelling assay. Cytotoxicity of increasing numbers of PBM plateaud at an effector cell (E):target cell (T) ratio of 3:1. In contrast, BAM cytotoxicity was significantly lower than that of PBM at low E:T ratios but increased in a dose-dependent manner approaching 100% at an E:T ratio of 20:1, this increased cytotoxicity being due to cytolysis. PBM cytotoxicity appeared to be suppressed at least partly by a factor(s) liberated by PBM themselves. The different nature of the two effector cell populations' cytotoxic dose response curves and kinetic studies, and the inability of lipopolysaccharide to stimulate a level of PBM cytotoxicity attainable by BAM, suggested that the mechanism of cytotoxicity of the two cell populations differed or that BAM were more activated than PBM, or both.

Human bronchoalveolar macrophage cytotoxicity for cultured human lung-tumour cells.

Human bronchoalveolar macrophages were separated from other free lung cells by density sedimentation on Percoll gradients. They were then tested for cytotoxicity against the human lung adenocarcinoma cell line A549, using a Selenomethionine-75 post-labelling assay. The cytotoxicity of the macrophages increased as the effector:target cell ratio was increased, approaching 100% at 20:1. There was no significant difference in the cytotoxicity of macrophages isolated from the lungs of bronchial-carcinoma or non-carcinoma patients. The highly cytotoxic nature of the macrophages was not due to selection of a more potent cytotoxic subpopulation of macrophages on the Percoll gradient, nor to a generally elevated activation of the macrophages due to the pathological conditions in the patients' lungs. An attempt to determine whether low concentrations of macrophages could potentiate target-cell growth proved negative. Cytotoxicity of macrophages for cultured lung target cells was not restricted to A549 cells and is not in accordance with the view that defective bronchoalveolar macrophage cytotoxicity contributes to the emergence of bronchial neoplasia.

Further studies on the differences in cytotoxicity of human peripheral blood monocytes and bronchoalveolar macrophages for cultured human lung cells.

Previously reported differences between the cytostatic activity of human peripheral blood monocytes (PBM) and bronchoalveolar macrophages (BAM) for cultured human lung tumor cells have been further investigated. The differences are both quantitative and qualitative and are shown not to be due to the respective methods of purification. There was a varying contribution of cytolysis to the cytostasis detected by the 75selenomethionine post-labeling assay used. Bronchoalveolar macrophages were cytolytic when tested at both low and high E:T ratios but PBM were only cytolytic at the low E:T ratio. A variable dependence upon soluble cytostatic factor(s) was suggested, and there was evidence of heterogeneity in the factors released by the two populations. Cytostatic factor production by both populations appeared to be under similar regulatory constraints. In vitro maturation of PBM altered their cytostatic dose-response curve to one resembling that previously reported for BAM. It was also shown that sera from poor-prognosis lung tumor patients, which suppressed the in vitro maturation of PBM, also suppressed the in vitro cytostatic activity of PBM for cultured human lung tumor cells.

The effect of oral Mycobacterium vaccae on subsequent responses of mice to BCG sensitization.

It has been postulated that previous exposure to mycobacteria in the environment may be a contributing factor to the variable efficacy of BCG vaccination in protecting against human tuberculosis or leprosy, in different geographical regions. To test this hypothesis mice were given Mycobacterium vaccae in their drinking water for 3 weeks immediately, 27 days, or 54 days before they were injected subcutaneously with BCG. Spleen cells were examined, 50 days after injection of the mice, for ability to proliferate in vitro in response to killed mycobacteria added to the cultures. The results show that, depending on the timing of the exposure of the mice in vivo to M. vaccae before BCG injection and the dose of the mycobacterial challenge in vitro, oral administration of this species of environmental mycobacteria can either enhance, mask or interfere with the expression of sensitization by BCG. Thus, our data from mice support the hypothesis that the results of BCG vaccination for a particular human population are influenced by exposure to indigenous mycobacteria.

Ovine interleukin 12 has biological activity on ovine and human activated peripheral blood mononuclear cells.

Interleukin 12 (IL-12) is a heterodimeric cytokine composed of two subunits that form the biologically active p70 molecule, and is a potent inducer of the pro-inflammatory cytokine IFN-gamma. In this study the coding sequence for ovine interleukin 12 p35 and p40 subunits was derived by RT-PCR cloning. Ovine p35 and p40 cDNA sequences show a high level of similarity at the nucleic acid and protein levels when compared to corresponding bovine and human sequences. In particular, cysteine residues and N-linked glycosylation sites are conserved between species. Secretion of the IL-12 heterodimer from CHO cells co-transfected with ovine p35 and p40 cDNA was shown by immunoprecipitation of a 60 and 66 kDa protein from transfectant supernatant. In addition, the supernatant from co-transfected cells augmented the proliferation of Con A-activated ovine peripheral blood mononuclear cells (PBMNC). Cross-species activity was shown by the enhancement of proliferation of human phytohaemagglutinin (PHA)-activated PBMNC. Supernatants from co-transfectants of hu p35/ov p40 and ov p35/hu p40, to generate chimeric heterodimers, also demonstrated stimulatory activity. Human and chimeric IL-12-induced proliferation of activated PBMNC was inhibited using an anti-human IL-12 polyclonal antibody, however this antibody showed minimal inhibition of ovine IL-12. This study suggests that ovine IL-12 has biological properties similar to its human counterpart.