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The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against solamargine was produced by fusing splenocytes immunized with a solamargine-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. Extensive cross-reaction of anti-solamargine antibodies against solasonine appeared. Aglycone of solamargine, solasodine cross-reacted with anti-solamargine antibodies resulting in a 43.8% cross-reaction. Insignificant cross-reaction appeared with tomatine (2.06%). The full measuring range of the assay extends from 57.5 pmol ml(-1) to 11.5 nmol ml(-1) of solamargine.

A serum-free medium supplemented with a few growth factors was devised to grow lymphocyte hybridomas. The medium was developed with the hybridoma line MPCll-BL, a fusion product between a mouse plasmacytoma cell line (MPCllTG70na3) and mouse (BALB/c) spleen cells. In the process of developing the-medium, ethanolamine was found to be an essential growth factor for the hybridoma. Phosphoethanolamine at 10-fold higher concentration could substitute for ethanolamine. Long-term cultivation of the cells was achieved in the defined medium supplemented with insulin, transferrin, ethanolamine, and selenium. The defined medium supported the growth of various other mouse hybridoma cell lines, mostly at a rate comparable to that observed in a serum-containing medium. After one-step ammonium sulfate precipitation of the spent medium, more than 95% of the protein recovered was immunoglobulin as shown by NaDodSO/polyacrylamide gel electrophoresis.Traditionally, tissue culture media have required serum supplementation in order to support long-term culture of cells. Serum is a complex mixture of components which may vary according to the age, sex, and species of its source. Sato and his co-workers have systematically investigated the hormone and growth factor requirements ofa large number ofcell lines. They described completely defined serum-free media that promote growth comparable to serum-containing media and in which cells maintain their differentiated characteristics (1, 2). Among the lines investigated 'are the mouse myeloma MPC11 (3), rat neuroblastoma B104 (4), and human colon carcinomas (5).Fusion of a plasmacytoma and a splenic plasma cell can produce a hybridoma that continues to secrete splenic antibody (6), and the monoclonal antibodies produced have been useful for immunology, molecular biology, and cell biology research (6, 7). Cultivation of these cells in a defined medium would facilitate such studies by eliminating serum, the major source of protein contamination.We have developed a hormone-and growth factor-supplemented, serum-free medium for culturing mouse plasmacytomas and hybridoma cell lines of MPC1l-BL and SP2/0-BL' origins. In the process of defining this medium we identified two compounds, ethanolamine and phosphoethanolamine, as necessary growth-promoting materials. These compounds, which had originally been found to be essential for growth of a rat mammary carcinoma (8, 9) have been required for growth of all hybridoma lines tested. Analyses of proteins precipitated at 50% ammonium sulfate saturation of the spent medium of hybridoma cell cultures indicated that in most cases >95% of the proteins were IgG or IgM. MATERIALS AND METHODSCells and Cell Culture. Most ofthe hybridoma cell lines used in these studies were obtained from W. Raschke (Salk Institute, La Jolla, CA). For NaDodSOipolyacrylamide gel electrophoresis of antibodies, hybridomas between SP2/0 and BALB/c splenocytes immunized against S100 protein were used. These cell lines were ordinarily maintained in a 1:1 mixture of Ham's F-1...

The ratio of hapten to bovine serum albumin in an antigen conjugate was exactly determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against forskolin was produced by fusing splenocytes immunized with a forskolin-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.57%. A very small cross-reaction appeared with 1-deoxy, 9-deoxy and 1,9-dideoxy forskolin derivatives. The full measuring range of the assay extends from 6 ng to 200 ng of forskolin. The competitive ELISA assay used for this analysis was found to be more sensitive than TLC (10 micrograms), GLC (30 ng) and HPLC (1 microgram) methods.