Optimal conditions for the generation of monoclonal antibodies using primary immunisation of mouse splenocytes in vitro under serum-free conditions.
We have further optimised the serum-free in vitro immunisation system described by Ossendorp et al. (J. Immunol. Methods 91, 257, 1986) for the generation of hybridomas secreting specific antibodies. Thyroglobulin and the hapten 2-phenyl-5-oxazolone coupled to chicken serum albumin were used as antigens. For an optimal outgrowth of antigen-specific B cells the presence of T cells, thymocyte-conditioned medium and antigen are required. The addition of supernatant from EL-4 cells (stimulated by phorbolmyristate acetate) inhibits the outgrowth of antigen-specific B cells. Using six-well plates with a surface area of 10 cm2 per well, an optimal IgM response was obtained when 10(7) splenocytes in a total volume of 2 ml/well were cultured for 3 days in the presence of antigen and thymocyte-conditioned medium. Increasing the concentration of cells whilst maintaining a constant surface area resulted in a decreased response.
Department of Biochemistry, Faculty of Medicine, University of Amsterdam, The Netherlands.
M
M
De Boer
F A
FA
Ossendorp
G
G
Van Duijn
G H
GH
Ten Voorde
J M
JM
Tager
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D003470:Culture Media; D006801:Humans; D007114:Immunization; D007378:Interleukins / Q000032:analysis; D008214:Lymphocytes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D013154:Spleen / Q000276:immunology*
D000911:Antibodies, Monoclonal; D003470:Culture Media; D007378:Interleukins