Production of murine monoclonal antibodies against human thyroglobulin using an in vitro immunization procedure in serum-free medium.

A serum-free in vitro immunization method for the generation of hybridomas producing specific antibodies to an antigen is described. The method was tested with human thyroglobulin as antigen. The serum-free medium used (Yssel et al., 1984) consisted of Iscove's modification of Dulbecco's modified Eagle's medium, supplemented with albumin, transferrin, insulin, ethanolamine and linoleic, oleic and palmitic acids. An optimal response was obtained when splenocytes from BALB/c mice were cultured for 3 days in the presence of 1.5 nM thyroglobulin and thymocyte-conditioned medium prior to fusion with SP2/0 myeloma cells and seeding of the fused cells in microtitre plates. The frequency of positive wells, defined as the number of wells secreting anti-(thyroglobulin) antibodies/number of viable cells used for the fusion, was 1.6 X 10(-6) +/- 0.25 X 10(-6) (mean +/- SD; n = 4). Eight stable clones producing anti-(thyroglobulin) antibodies were isolated. One clone (3D12) produced antibodies reacting only with human thyroglobulin. The antibodies produced by the other clones reacted with human, murine and porcine thyroglobulins. Seven of the clones produced antibodies of the IgM class and one clone produced IgG. The specificity of 3D12 (IgM) for human thyroglobulin and the absence of any reactivity with murine thyroglobulin provides evidence for a primary response of splenocytes in culture to the presence of an antigen.