INTERVALOS DE CONFIANZA, ERROR MÁXIMO Y TAMAÑO DE

The Milstein and Kohler technique produces ’immortal’ clones of cells making single antibody specificities by fusing normal antibody forming cells with an appropriate B-cell tumour line. Therefore, mice immunised with an antigen develop spleen cells making antibodies to specific epitopes on the antigen, which appear in the serum. The spleen is removed and the individual cells fused in polyethylene glycol with constantly dividing, immortal, B-tumour cells selected for a purine enzyme, hypoxanthine guanine phosphoribosyl transferase (HGPRT) deficiency, and also often for their inability to secrete immunoglobulin. The basis of this selection involves a ’salvage’ pathway for DNA synthesis (Fig 3.1). Most cells can make DNA by de novo synthesis or via an alternative ’salvage’ pathway, using endogenous or exogenous source of preformed bases. Cells are grown in the presence of a lethal purine analogue 8-aza or 6-thioguanine dye, until HGPRT deficient mutants arise. These mutants are ’immortal’ but can only synthesize ribonucleotides by de novo synthesis and not by the salvage pathway. The tumour ’parent’ used in the hybridoma techniques are HGPRT deficient and so are unable to grow in HAT (hypoxanthine, aminopterin, thymidine) medium. HAT contains a folic acid analogue aminopterin which blocks de novo synthesis and these cells are unable to use hypoxanthine and so die. The hybrid cells can grow in HAT medium because DNA from the normal partner provides the information to synthesise HGPRT and the tumour cell DNA provides the ’message’ for

unrestricted proliferation (Fig 3.2).

In this technique many cells show cytoplasmic fusion and a lower proportion complete the nuclear fusion required to produce tetraploid hybrids. Although this procedure results in a heterogenous mixture of fused and unfused cells, plasmacytoma cells tend to ’rescue’ large, recently activated B lymphocytes. After dispensing the hybridoma mixture into culture wells, the selective medium positively selects for the fusion hybrids. The culture supernatants are then tested for antibody activity after 1 - 3 weeks and positive cultures cloned by conventional cell cloning techniques.

3.1.1.1 The plasmacytoma line for fusion

P3/X63-Ag8 is an 8-azaquanine-resistant subline of the plasmacytoma MOPC 21, induced in a BALB/c mouse by the injection of mineral oil. This cell line is special in that it tends to fuse spontaneously and can grow at very low cell densities, thus facilitating the recovery of fusion hybrids. However, this line has the disadvantage that it synthesizes and secretes the MOPC 21 myeloma protein and so hybrid cells will secrete myeloma and antibody molecules, as well as inactive hybrid molecules. Spontaneous variants of P3/X63-Ag8 have been selected that neither secrete nor synthesize immunoglobulin molecules, but still retain the ability to rescue normal antibody-producing cells. P3/X63- Ag8.653 is a variant that does not synthesize or secrete immunoglobulin chains.

3.1.2 Heterohybridomas

Much of the progress and use of hybridoma technology has been limited to rodents due to the lack of suitable myeloma cell lines in other species. There have been attempts to generate monoclonal antibodies in other species by fusing lymphocytes from the species of interest to a mouse myeloma line (Yarmush et al, 1980; Tucker et al, 1981; Murphy et al, 1986). However the efficiency of generating hybrids is low and of those that do grow only a few secrete specific antibody and they appear to do so for only a short period of

time. Several studies have been reported on techniques to improve the fusion efficiency and stability of these heterohybridoma cell lines (Flynn et al, 1989; Verbanac et al, 1993; Capparelli et al, 1990). Reports have shown that these problems can be reduced by back-fusing lymphocytes from the species of interest to an already generated, aminopterin-sensitive, heterohybridoma (Anderson et al, 1987; Groves et al, 1987). The secondary hybridomas thus generated appear to be more stable in tissue culture, they live longer and secrete antibody for longer periods of time and at higher concentration. Another study discusses the development of a suitable aminopterin-sensitive sheep/mouse heterohybridoma fusion partner for the routine production of sheep monoclonal antibodies (Flynn et al, 1989). The report also evaluated the source of lymphocytes used as fusion partners by taking cells from efferent lymph, lymph nodes or peripheral blood. They showed that lymphocytes prepared from either efferent lymph or lymph node on the fourth day following antigenic stimulation have high fusion efficiencies, and both were vastly superior to peripheral blood lymphocytes. It is suggested that blast cells present in lymphoid tissues due to antigenic stimulation were the main cell types involved in the generation of viable antibody-secreting sheep x sheep X mouse heterohybridomas.

In a study describing the development of rabbit-mouse hybridoma cell lines (Yarmush et al, 1980) hybrids were obtained by fusion of rabbit lymphoid cells with the following mouse myeloma lines: P3/X63-Ag8 (Kohler and Milstein, 1975), 45.6TG1.7 (Weitzman et al, 1976), P3-NS-I-Ag4-l (Kohler and Milstein, 1976), P3/X6-Ag8UI (Yelton et al, 1981) and SP2/0-Agl4 (Kohler et al, 1978). Yields of hybrids and of hybrids secreting antibody were highest when P3/X63-Ag8UI was used, P3/X63-Ag8, 45.6TG1.7 and P3-NS-l-Ag4-l gave intermediate results and SP2/0-Agl4 showed the lowest frequency. Also a comparison of peripheral blood lymphocytes, lymph node cells and spleen cells showed that the relative frequencies of secreting hybrids were significantly lower with peripheral blood lymphocytes or lymph node cells than with spleen cells.

One of the major drawbacks to the production of heterohybridomas has been the instability of the other species’ chromosome in the fused cell (Kuo et al, 1985). The inclusion of normal rabbit serum in the heterohybridoma culture medium has been reported to increase the number of cultures secreting specific antibody as well as the stability of antibody secretion upon subcloning (Raybould and Takahashi, 1988). They report that hybridoma stability, ie the retention of rabbit immunoglobulin chromosomes and/or the ability to produce secretory rabbit immunoglobulin, appears to be dependent on some undefined factors present in normal rabbit serum.