Preparation of Monoclonal Antibodies

It is known that there are two ways for cells to synthesize DNA: D and S, in which D can be blocked by aminopurine. There are two DNA synthesis pathways in human lymphocytes, but they generally do not divide and proliferate, and can be eliminated after culture.

There is no S pathway in mouse myeloma cells, which can be eliminated by blocking D pathway with aminopurine. Only when hybridoma cells block D pathway with aminopurine can they have S pathway, so they can continue to divide.

The peritoneal cells of mice contain macrophages, which can not only eat food, but also remove dead and broken cells and microorganisms. 8 ~ 10 weeks old mice of the same strain were decapitated and killed, and 10 ~ 15 ml complete culture medium was injected intraperitoneally under aseptic operation. After rubbing the abdomen with sterile thumb several times, the peritoneal fluid was sucked back and the cell concentration was adjusted to 1× 105/ml. Generally, a mouse peritoneal fluid can obtain 3 ~ 5× 106 cells, which can be used for one fusion. Two kinds of mouse peritoneal macrophages can also be mixed. In addition, spleen cells and thymus cells of homologous mice can also be used as feeder cells.

After PEG treatment, the selectively cultured two kinds of parental cells can form a mixture of various cell components, including unfused free parental cells, fusion between myeloma cells, fusion between immune B cells and fusion between myeloma cells and immune B cells. Only the latter can form hybridomas and should be screened out for cloning and culture. HAT medium is usually used, which contains hypoxanthine H, aminopterin A and thymidine T.

Most laboratories add HAT culture solution on the day of fusion or the next day. Generally, HAT medium is used within 7 ~ 14 days after fusion, and then HT medium is used. 2 ~ 3 weeks after fusion, the liquid was changed, and the old liquid of12 in the culture hole was sucked out and replaced with new liquid. After 3 ~ 5 days, change the liquid 1 time for 2 weeks. After 3 ~ 5 days, the unfused and self-fused cells gradually died, and clones appeared around 1 week, and continued to proliferate. When the colony at the bottom of the hole is >: 3mm or 1/2, the supernatant can be taken for antibody activity detection, and fresh HT culture solution can be added at the same time. In a good fusion test, about 70 ~ 80% of stomata have clonal growth. All wells growing clones need to take culture supernatant for antibody activity detection. After 3 weeks of culture, the common complete culture solution can be used.