medium (see below). Fused cells are incubated in HAT medium (hypoxanthine-aminopterin-thymidine medium) for roughly 10 to 14 days. Aminopterin blocks the pathway that allows for nucleotide synthesis. Hence, unfused myeloma cells die, as they cannot produce nucleotides by the de novo or salvage pathways because they lack HGPRT. Removal of the unfused myeloma cells is necessary because they have the potential to outgrow other cells, especially weakly established hybridomas. Unfused B cells die as they have a short life span. In this way, only the B cell-myeloma hybrids survive, since the HGPRT gene coming from the B cells is functional. These cells produce antibodies (a property of B cells) and are immortal (a property of myeloma cells). The incubated medium is then diluted into multi-well plates to such an extent that each well contains only one cell. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are thus monoclonal antibodies. The next stage is a rapid primary screening process, which identifies and selects only those hybridomas that produce antibodies of appropriate specificity. The first screening technique used is called ELISA. The hybridoma culture supernatant, secondary enzyme labeled conjugate, and chromogenic substrate, are then incubated, and the formation of a colored product indicates a positive hybridoma. Alternatively, immunocytochemical, western blot, and immunoprecipitation-mass spectrometry. Unlike western blot assays, immunoprecipitation-mass spectrometry facilitates screening and ranking of clones which bind to the native (non-denaturated) forms of antigen proteins. Flow cytometry screening has been used for primary screening of a large number (~1000) of hybridoma clones recognizing the native form of the antigen on the cell surface. In the flow cytometry-based screening, a mixture of antigen-negative cells and antigen-positive cells is used as the antigen to be tested for each hybridoma supernatant sample. The B cell that produces the desired antibodies can be cloned to produce many identical daughter clones. Supplemental media containing interleukin-6 (such as briclone) are essential for this step. Once a hybridoma colony is established, it will continually grow in culture medium like RPMI-1640 (with antibiotics and fetal bovine serum) and produce antibodies. Multiwell plates are used initially to grow the hybridomas, and after selection, are changed to larger tissue culture flasks. This maintains the well-being of the hybridomas and provides enough cells for cryopreservation and supernatant for subsequent investigations. The culture supernatant can yield 1 to 60 μg/ml