Hypoxanthine, aminopterin

The cell fusion mixture is transferred to a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Unflised myeloma cells are unable to grow as they lack HGPRT. Unflised normal spleen cells can grow but their proliferahon is limited and they eventually die out. The hybridoma cell can proliferate in the HAT medium as the normal spleen cell supplies the enzyme which enables the hybridoma to utilize extracellular hypoxanthine. 

Select HPGRT+ cells by culturmg the mixture in a medium containing HAT (hypoxanthine, aminopterin, and thymidine), which is growth inhibitory to HPGRT+ cells. Therefore, the myeloma cells will die in this medium while the hybridoma cells proliferate. The unfused lymphocytes will die due to their limited lifespan. 

Fig. 3.2 Hybridoma technique for the production of monoclonal antibodies. Spleen (milt) cells, which have been taken from mice (being immunized with an antigen X) contain anti-X-antibody-producing B cells. These cells are fused with myeloma cells in the presence of polyethylene glycol (PEG) and then taken to the HAT (hypoxanthine-aminopterin-thymi-dine) medium. HAT will induce death to myeloma cells because of the absence of the enzyme hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT). Hybridoma cells, how-...

Conversely, HPRT4 cells can be selected for in the presence of hypoxanthine-aminopterin-thymidine medium. It should be possible to select for HPRT cells in normal HPRT+ persons with one of the analogues, and this was one of the first systems used to detect spontaneously occurring mutants in freshly cultured cells from humans.88 388... 

Fig. 13.3. Metabolic cooperation between BHK21/C3 cells. Growth of BHK-HPRT-and BHK-TK- cells separately and in mixed (1 1) culture, in medium containing hypoxanthine, aminopterin and thymidine (HAT medium). HPRT = hypoxanthine phosphoribosyl transferase TK = thymidine kinase. (Reproduced from Pitts, 1971, with kind permission of the author and publisher.)...

Hypoxanthine, Aminopterin, and Thymidine supplement (100X HAT, Sigma). 

Fig. 4. Multistep methods for eliciting specific antibody-producing cells (hybridomas). HAT, hypoxanthine, aminopterin, and thymidine PEG, polyethylene glycol.

Hybridomas are generated 3 d following an iv booster injection of 10 pg of DT in phosphate-buffered saline (PBS) and selected using Hy medium with 10% fetal calf serum plus hypoxanthine, aminopterin, and thymidine (HAT) (6,16). 

HAT Medium - A cell culture medium augmented by hypoxanthine, aminopterin, and thymidine, which selects for cells that have a functional purine salvage pathway. 

HAT Selection - The compounds hypoxanthine, aminopterin (see here), and thymidine (H,A, and T, respectively) can be used to select for cells having functional salvage pathways. Aminopterin inhibits dihydrofolate reductase, which blocks de novo purine and thymidine synthesis. Only cells which can utilize thymidine (pyrimidine salvage) and hypoxanthine (purine salvage) can grow in this medium. 

FIGURE 52-2 Generation of monoclonal antibodies. Mice are immunized with the selected antigen, and spleen or lymph node is harvested and B cells separated. These B cells are fused to a suitable B-cell myeloma that has been selected for its inability to grow in medium supplemented with hypoxanthine, aminopterin, and thymidine (HAT). Only myelomas that fuse with B cells can survive in HAT-supplemented medium. The hybridomas expand in culture. Those of interest based upon a specific screening technique are then selected and cloned by limiting dilution. Monoclonal antibodies can be used directly as supernatants or ascites fluid experimentally but are purified for clinical use. HPRT, hypoxanthine-guanine phosphoribosyl transferase. 

Once the fusion has taken place, it is necessary to eliminate any unfused myeloma cells and to select only hybrid cells secreting antibody. This is primarily achieved by the use of hypoxanthine aminopterin thymidine (HAT) media and cells that are deficient in the enzyme responsible for incorporation of hypoxanthine into DNA. Figure 3 illustrates this process. The unfused splenocytes are not immortal and naturally die off in culture. The elimination of the unfused myeloma cells is carried out by the initial use of mutant myeloma cells selected for a deficiency in the enzymes hypoxanthine guanine phosphoribosyl transferase (HGPRT) and thymidine kinase (TK), rendering them unable to use the salvage pathway for nucleic acid synthesis. The myelomas will die off... 

In order to determine whether restriction is due to a lack of host factor as opposed to inhibition by a host factor of viral functions, it was decided to hybridize MDBK with L-cells. We have introduced an HGPRT mutation into the MDBK cells, and utilized a TK strain of L-cells. Hybrids were selected in HAT medium (hypoxanthine, aminopterin, thymidine), clones were selected, grown up, and tested for virus yield. Table 2 summarizes the data obtained. 

SV40, simian virus 40 HAT, hypoxanthine aminopterin thymidine PM A, phorbol myristate acetate LPS, lipopolysaccharide. 

The selective medium, HAT contains hypoxanthine, aminopterin, and thymidine. This medium allows the selection and growth of hybridomas which are HGPRT+ and have a normal functioning salvage pathway. However, HAT is unable to support the growth of the HGPRT" myelomas because the de novo pathway is inhibited and the salvage pathway cannot function because of the defective enzyme. 

Among the technical problems is the selection of the hybrid cells from the myeloma cells, both of which are equally rapid in growth. The biochemical trick, upon which the technique relies, is the use of mutant non-secreting myeloma cells which are deficient in hypoxanthine phosphoribosyltransferase (HPRTase ) and a culture medium containing a mixture of hypoxanthine, aminopterin and thymidine (HAT medium). Aminopterin blocks the synthesis of both purines and pyrimidines. In the presence of aminopterin, HPRTase" cells die because they cannot utilize the pertinent salvage pathway. Since B-lymphocytes contain HPRTase, the fused cells survive by utilizing the hypoxanthine and thymidine in the culture medium. The unfused B-lymphocytes are also unaffected but are rapidly outgrown by the hybrid cells. 

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