Rabbit monoclonal antibodies are commercially available from Knights group [11]; however, a number of experts are interested in generating their own rodent monoclonal antibodies. 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. Keywords: mouse monoclonal antibody, iliac lymph node, cell fusion, tail base immunization I.?Introduction Since monoclonal antibodies were Rabbit polyclonal to CD14 first produced in mice 30 years ago [7], mouse monoclonal antibodies have been produced using sensitized lymphocytes from your mouse spleen. The use of mouse lymph node lymphocytes for the production of mouse monoclonal antibodies has not been extensively examined [2, 3, 8], however they can be utilized for cell fusion. The only limiting factor to date has been the small quantity of lymph node lymphocytes able to be obtained from a mouse, which is usually insufficient for cell fusion [2, 9]. In ITE 1995, we explained a novel method, known as the rat lymph node method, in which medial iliac lymph node lymphocytes from rats immunized with an antigen emul-sion via the hind footpads are used as fusion partners for cell fusion [5]. This produces approximately 10-fold the number of specific antibody-producing hybridomas than the more conventional method using mouse or rat splenic lymphocytes [5]. However, this method cannot be used in mice, since injection of an antigen emulsion into ITE the hind foot-pads of mice does not result in sufficient enlargement of draining lymph nodes, such as the popliteal, inguinal, and iliac lymph nodes, to enable cell fusion. Through trial and error, we discovered that intramuscular injection of the tail base with an antigen emulsion enlarges mouse iliac lymph nodes very effectively, and that lymph node lymphocytes from two immunized mice are sufficient for a single cell fusion attempt. The mouse method using lymphocytes from enlarged lymph nodes exhibited similar characteristics to the rat lymph node method [5], with the following advantages: (1) one injection of antigen emulsion was sufficient, (2) the lymph nodes were ready for use two weeks after injection, and (3) a high yield of positive ITE hybridomas was obtained. II.?Materials and Methods Animal immunization Chicken ovalbumin (Grade VII, Sigma, St. Louis, ITE MO, U.S.A.) and Freunds total adjuvant (Difco Labora-tories, Detroit, IL, U.S.A.) were used for making antigen emulsion. Freunds total adjuvant (0.6 ml) and 1.5 mg/ml chicken ovalbumin solution (0.3 ml) were emulsified in two 1-ml Luer-lock glass syringes with a double-ended locking needle connector. After emulsification was completed, one of the glass syringes made up of the emulsion was disconnected from your locking connector and a 27G3/4 needle attached. Eight-week-old female mice were used. BALB/cAnNCrlCrlj mice (BALB/c) were purchased from Charles River Laboratories Japan, Inc., Yokohama, ITE while C3H/HeNJcl mice (C3H/He), and closed colony Jcl: ICR mice (ICR), were purchased from Clea Japan, Inc., Tokyo. Mice were anesthetized and injected intramuscularly at the right and left tail base with an emulsion (0.1 ml in total) containing 50 g chicken ovalbumin and Freunds total adjuvant. Nine BALB/c mice were injected subcutaneously at the tail base with the same amount of antigen emulsion. The mice were kept in polycarbonate plastic cages containing solid wood shavings for bed linens. All animal experiments were conducted in accordance with the Guidelines for Laboratory Animal Experiments of the Shigei Medical Research Institute. Cell fusion A mouse myeloma cell collection, SP2/0-Ag14 [10], was utilized for cell fusion. Mice were sacrificed 11, 14, 21, and 28 days after injection. Iliac lymph nodes were collected and lymph node lymphocytes from 2 mice were pooled and used for each cell fusion attempt. Cell number was decided using a blood corpuscle counting chamber after lymph nodes were exceeded through a 200-mesh stainless steel wire mesh. Cell fusion was performed according to the method of -Kishiro [5], after which the cells were cultured in four 96-well.