Skill Sharing---Preparation of Antibodies in Mouse Ascites

At present, a large number of methods for preparing monoclonal antibodies mainly include two major systems, one is an in vivo mouse antibody production method and the other is an in vitro culture method. So far, the method of producing monoclonal antibodies in animals has been generally used. In view of the fact that most animal hybridomas are obtained by fusion of myeloma cells of BALB/c mice with spleen cells of the same strain, the animals used are of course BALB/c mice are preferred. In the method, the hybridoma cells are inoculated into the peritoneal cavity of the mouse, the hybridoma is grown in the peritoneal cavity of the mouse, and ascites is produced, so that a large amount of ascites monoclonal antibody can be obtained and the antibody concentration is high. It can be seen that the method is simple and economical to operate. However, the various mixed proteins (including Ig) of mice are often mixed in the ascites, so in many cases, it can be used after purification, and there is a danger of contaminating animal viruses, so it is best to use SPF grade mice.

Mice have different sensitivities to different hybridoma cells, some die one week after injection, some may have bloody ascites, and some may receive ascites 2-3 times. Generally under the abdomen:

Prepare 2 mice per group, 8-10 weeks old, preferably in the same line as the mice that prepare the monoclonal antibody; pre-use the sensitizer before inoculation of the hybridoma cells, you can use paraffin oil (one week in advance) or incomplete Freund's Adjuvant (three days in advance);

The number of the first inoculated cells can be controlled at 106. If there is less ascites and death, the number of cells inoculated in the second mouse antibody should not exceed 5*105; the number of injected cells is small, the formation of ascites is slow but the titer is higher. ;

On the 7th day, the ascites and survival of the mice were closely observed. If the vitality is good and there is obvious mobile ascites, the ascites can be placed about 2 times; once the mice are found to be restricted in activity, the ascites should be sacrificed immediately;

Alcoholic cotton balls and syringes (2-5ml) and sterilized eppendorf tubes are used for abortive ascites (because the ascites are still separated, it is best to use this for centrifugation). The method is to wipe the position of the needle with the alcohol cotton ball (probably at the right side of the midline of the abdomen), then let the air in the syringe and then push the needle slowly, then push the syringe slowly back, the pressure in the abdominal cavity is slowly The ascites will flow out, and you should gently lift it up when you put the needle. It feels like no obstacles.

Execution of ascites is to use two hemostatic forceps, scissors 2 small, 2 dice. First use a pair of scissors to gently cut a small opening in the abdominal cavity of the mouse, then use two hemostatic forceps to gently open the two sides to expose the abdomen, then use another pair of scissors and tweezers to cut the endothelium, and use a gun or glass straight tube. Ascites can be taken. It is also recommended to place the eppendorf tube.