8-AG (also known as 8-nitroguanine) is a basic cytotoxic factor. The synthesis of cellular DNA mainly involves two pathways: the primary pathway and the remedial pathway. The HGPRT enzyme (i.e., hypoxanthine-guanine ribosyltransferase) is the primary synthase for purines in the remedial pathway. Under normal circumstances, cells can synthesize DNA through both of these pathways simultaneously. 8-AG is also a purine and can naturally be regarded as a raw material for DNA synthesis. However, since it is toxic, its presence can kill cells. Myeloma cells with HGPRT enzyme deficiency cannot synthesize DNA through the salvage pathway, and thus can survive. Therefore, all sp2/0 cells screened by 8-AG were HGPRT enzyme-deficient strains.

The titer before fusion was approximately 160,000. The threshold standard was 0.2 and the maximum value of twice the negative serum value. Three fusions were performed. The positive serum (the serum of the mice before fusion) had positive results, but all the results on the board were negative. Each time the customer fuses five plates, Sp2/0 cells are cultured separately with HAT, and all of them die approximately seven days later It can be suggested that the client increase the number of plates made after fusion to between 10 and 20, and at the same time appropriately increase the concentration of HAT medium and switch to other batches of Sp2/0 cells.

8-AG (also known as 8-nitroguanine) is a basic cytotoxic factor. The synthesis of cellular DNA mainly involves two pathways: the primary pathway and the remedial pathway. The HGPRT enzyme (i.e., hypoxanthine-guanine ribosyltransferase) is the primary synthase for purines in the remedial pathway. Under normal circumstances, cells can synthesize DNA through both of these pathways simultaneously. 8-AG is also a purine and can naturally be regarded as a raw material for DNA synthesis. However, since it is toxic, its presence can kill cells. Myeloma cells with HGPRT enzyme deficiency cannot synthesize DNA through the salvage pathway, and thus can survive. Therefore, all sp2/0 cells screened by 8-AG were HGPRT enzyme-deficient strains.

The number of plates made after fusion can be increased to between 10 and 20. At the same time, the concentration of HAT medium can be appropriately increased, and other batches of Sp2/0 cells can be used.

Possible reasons: (1) The presence of glycerol, urea, organic solvents, etc. in the antigen buffer will affect the emulsification effect (2) The concentration of the antigen is too low. The following are the experimental results of the equal volume mixture of 1.5mg/ml antigen and Frexner's complete adjuvant and emulsification for 5 minutes: The following are the experimental results of the equal volume mixture of water and Freund's complete adjuvant and emulsification for 15 minutes: