Lymphocytes from Enlarged Iliac Lymph Nodes as Fusion Partners for the Production of Monoclonal Antibodies after a Single Tail Base Immunization Attempt

A novel method of preparing hybridomas producing mouse monoclonal antibodies was ­established, called “the mouse iliac lymph node method”. Lymphocytes from enlarged iliac lymph nodes from mice injected intramuscularly at the tail base with an emulsion of antigen and Freund’s adjuvant were used for cell fusion. For the most part, lymph node lymphocytes from two mice were used for a single cell fusion attempt. Ovalbumin was used as the antigen and the results of fusion were compared with the results of a previous report (Cell Struct. Funct. 20; 151–156, 1995). Approximately 100 positive wells producing antibody of interest were identified using this method. By comparison, approximately 10 positive wells were identified using the more conventional mouse spleen method after three immunization injections. The relative proportions of hybridomas producing IgM, IgG1, IgG2a, IgG2b, and IgG3, following fusion using iliac lymph node lymphocytes obtained 14 days after injection were 14.0%, 78.7%, 3.2%, 3.5% and 0.5%, respectively. This method demonstrated the following advantages: (1) a single injection of the antigen emulsion was sufficient, (2) the lymph nodes were ready for use 14 days after injection, and (3) a high yield of positive hybridomas was obtained.