sepharose and Plasmacytoma

A rapid, efficient method is described for cloning of lymphocyte hybridoma cells. The method utilizes ultra-low gelation temperature agarose.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Clone Cells; Culture Techniques; Female; Hybridomas; Immunoassay; Lymphocytes; Mice; Mice, Inbred Strains; Plasmacytoma; Sepharose; Temperature

Optimal conditions for the formation of hybridoma clones in soft agarose are described. The hybridization frequency is shown to be highly dependent on the pH of the polyethylene glycol (PEG) solution used for fusion and on the cloning medium. Maximal numbers of clones are obtained when the PEG solution used for fusion is at pH 8.0-8.2.

Topics: Animals; Antibody-Producing Cells; Cell Fusion; Clone Cells; Culture Media; Hybrid Cells; Hydrogen-Ion Concentration; Mice; Myeloma Proteins; Neoplasms, Experimental; Plasmacytoma; Polyethylene Glycols; Sepharose

Topics: Binding Sites; Cell Line; Chromatography, Affinity; Kinetics; Microchemistry; Peptide Hydrolases; Plasmacytoma; Pronase; Protein Binding; Ribonucleases; Sepharose

The preparation and specificity of antibodies specific for the ligand-binding site of HOPC 8, a phosphorylcholine (PC)-binding mouse myeloma protein, are described. Antiserum to HOPC 8, prepared in rabbits, was adsorbed with an HOPC 8-Sepharose immunoadsorbent and anti-binding site antibodies were eluted with PC. These antibodies reacted with HOPC 8 but not other myeloma proteins, including those with PC-binding specificity different from HOPC 8; the specificity of this anti-HOPC 8 antibody for the combining site region of HOPC 8 was shown by the fact that 1) the interaction of the anti-HOPC 8 antibody preparation with HOPC 8 was completely blocked by PC and 2) the antibody preparation failed to bind TEPC 15 in which the combining sites had been blocked by covalently bound PC groups. Moreover, these anti-binding site antibodies did not react with isolated heavy or light chains, indicating the requirement for a heavy-light chain interaction. By contrast an idiotypic antiserum to HOPC 8 prepared in A/J mice did bind affinity-labeled TEPC 15 and the reaction with HOPC 8 was only marginally hapten inhibitable. Both of the idiotypic determinants detected by these two antisera were present on anti-PC antibody raised in BALB/c mice;

Topics: Adsorption; Animals; Antibodies; Antibody Specificity; Antigens, Bacterial; Binding Sites, Antibody; Dinitrophenols; Epitopes; Female; Hemagglutination Inhibition Tests; Immune Sera; Iodine Radioisotopes; Lysine; Mice; Mice, Inbred A; Mice, Inbred BALB C; Myeloma Proteins; Organophosphorus Compounds; Phosphorylcholine; Plasmacytoma; Rabbits; Radioimmunoassay; Sepharose; Serum Albumin, Bovine; Streptococcus pneumoniae