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:

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.

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.

融合之前的效价大约16万，阈值标准是0.2和两倍阴性血清值取最大值，做了三次融合，阳性血清（融合前小鼠的血清）是有阳性结果，但板子上全部是阴性结果。客户每次融合的板子做5块，Sp2/0细胞单独用HAT培养，大约7天后全部死亡 可以建议客户增加融合后做板子的数量，在10-20块之间，同时适当增加HAT培养基的浓度，换用其他批次Sp2/0细胞。

8－AG（即8－氮鸟嘌呤）是一种碱性细胞杀伤因子。细胞DNA的合成主要是两个途径，一个是主要途径，一个是补救途径，而HGPRT酶（即次黄嘌呤－鸟嘌呤核糖转移酶）是补救途径嘌呤的主要合成酶，正常情况下，细胞可以同时通过这两种途径来合成DNA。而8－AG也是一种嘌呤，自然也可以被认为是一种合成DNA的原料，但由于它是有毒的，它的存在会杀死细胞。而HGPRT酶缺陷的骨髓瘤细胞不能通过补救途径合成DNA，所以就可以存活。因而经8－AG筛选后的sp2/0细胞都是HGPRT酶缺陷株。