【正文】 drug immediately.) Seed the cells in 96 well microtiter dishes, 250 ul per well, 8 plates per fusion. First clones may be seen in 710 days. First screen will usually start after 2 weeks, with a second and third, if necessary, a few days later. Screening ELISA Materials: Plates 96 well Dynatech Immulon, type 2. (Fisher 170221199).PBS, TBSPBS + % Tween 20 or TBS + % Tween solution = 2% BSA (type V ) in PBS. (Add % azide for longer storage.)Elisa buffer = 2% BSA + % Tween 20 in PBS ( azide optional ).Enzyme linked antibody = Horseradish peroxidase1Substrate = ABTS 100X (from Zymed 002022)1HRP buffer: 100mM Na Citrate pH ( 490 mg citric acid + 720 mg Na citrate dehydrate + 50 ml H2O, pH ).Hydrogen peroxide 30% ( 1000X )Protocol: ABSORBTION OF ANTIGEN Dilute Ag to 10 ug/ml in PBS. Add 100 ul of Ag solution to each well. Leave covered with saran wrap, at 4176。 C IMDM to 10 ml, without disturbing pellet. After adding , swirl the tube gently to mix and dilute the PEG. Do not disturb the cells. Spin at 1000 rpm for 5 min. Aspirate medium, resuspend cells by tapping. Slowly add 5 ml of 37176。 this required large numbers of immunized animals and did not immortalize the antibodyproducing cells, so it required repeated animal use to obtain more antibodies. Development of the hybridoma technology has reduced the number of animals (mice, rabbits, and so on) required to produce a given antibody but with a decrease in animal welfare when the ascites method is used. ? Step 1: Immunization of Mice and Selection of Mouse Donors for Generation of Hybridoma Cells ? Mice are immunized with an antigen that is prepared for injection either by Cell Fusion and Selection Solutions: IMDM plete + 15% MCM, PBS, PEG 50% ( w/v ), Aminopterin Prepare a T150 flask with IMDM plete and keep it in the incubator for fused cells. Isolation of spleen cells Sacrifice mouse by cervical dislocation. Immerse mouse in 70% Ethanol. Remove spleen ( on the left side ) and transfer into a small petri dish which contains IMDM at room temp. Clean fat from the spleen and transfer the spleen into an empty dish. Using sharp tipped forceps, one end is punctured. A curved forceps is used to hold down the intact end, and the spleen is gently rubbed towards the opened end with another set of forceps. The cells from inside the spleen will ooze out with very little damage. Stop the process when you are left with a nearly empty, transparent skin. Collect the cells by rinsing with IMDM. Transfer cell suspension to a 15ml conical tube and let the cell debris settle out (approx. 5 min.). Remove the cell suspension (without disturbing the settled cell debris) and transfer to a 50ml conical tube. Add an additional 30ml of IMDM and pellet the cells at 1200 rpm for 5 10 min. Aspirate the medium, resuspend pellet and wash again with 30ml of IMDM. One immunized spleen has approx. 10^8 cells. After this wash the cells are ready for the fusion. Myeloma cell preparation: It is essential that the myelomas be free of debris, rounded and refractive under phase contrast, and that they are harvested in log or late log phase growth (between and 9 x10^5 cells/ml). Thaw cells 7 days before scheduled fusion. Myeloma do not grow well after being in culture for more than a few weeks. Make sure you refreeze cells for future use. We have been using a ratio of 2 spleen cells : 1 myeloma cell. However, workers have been using a ratio from 1 to 10 spleen cells per myeloma successfully. For one spleen we harvest 5x10^7 cells. It is advisable to do this spin at the same time that the second wash of the spleen cells is done. The fusion The washed myeloma and spleen cells are pooled in 30ml of PBS (room temp.) and spun gently at 1000 rpm for 10 min. Aspirate the PBS and resuspend pellet gently by tapping the tube. Volume should be approx. ml. Set a timer. Add an equal volume of PEG solution, slowly, dropwise, with gentle tapping, over min