The aim of our work was to produce bispecific antibodies using biological and chemical methods. Bispecific antibodies recognise two antigens simultaneuosly. The bispecific antibodies produced were used in the development of novel ELISA and immunocytochemical techniques for the detection of antigens present on the peripheral blood cells of patients with Chronic Lymphocytic Leukemia, (CLL). Cytokine production in these patients was also determined.
Biological production involved the production of triomas which were formed by the fusion of hybridoma cells with immunized splenocytes. This method required backselecting the hybridoma cells for HAT-sensitivity, a procedure which was performed firstly by growing the cells in increasing concentrations of 8-azaguanine and, secondly, by treatment of the hybridoma cells with the mutagenic reagent, ethyl methanesulfonate. The cells were then fused with horseradish peroxidase-immunized mouse and rabbit splenocytes. The chemical method chosen involved the activation of thiol groups on Fab" fragments of one antibody. These Fab" fragments were reacted with the second reduced Fab" fragment to form the heterogeneous F(ab'y)2 bispecific antibody.
Studies were also performed on the cytokine, interleukin-6 (IL-6). These included measurement of the concentration of IL-6 in the plasma and in conditioned medium (CM), prepared from the peripheral blood lymphocytes of patients with CLL. The IL-6 levels were measured by an ELISA and by a bioassay using an IL-6-dependent cell line. The levels of circulating IgG present in the plasma of these patients were also determined by ELISA, but did not correlate. The effects of feeder cell layers and various medium supplements, including both IL-6-CM and CLL plasma, on the growth of hybridoma cells were also studied. The growth of hybridoma cells which are of B-cell origin under such conditions may give indications of how the various growth factors present in CLL plasma affect B-cell growth in these patients.