Although the above outlined modifications have increased the fusion frequencies, the other major parameters determining the successful outcome of a fusion is the expansion of clones of B lymphocytes secreting specific antibody prior to removal from the immune mouse. The route of administration and distribution of antigen will determine the microenvironment in which the antigen interacts with antigen presenting cells, B and T cells (Basten et al, 1977). Since subsets of B and T cells inoculate through preferred microenvironments, the major part of an immune response to antigen may well occur at one particular site in preference to another, eg spleen or lymph node, leading to an enrichment of B cells of interest. The use of cells from such a site may enhance the quality of a fusion. A study comparing different fusion protocols based on three different routes of administration of antigen (footpad, intradermal and subcutaneous) and two different sources of lymphocytes (lymph node and splenic) as fusion cells, with insulin as the antigen and following a single injection, shows that the major portion of B cells producing the immune response to insulin was localised in the lymph node after footpad immunisation and that the quality of a fusion could be enhanced by using lymph node cells (Mirza et al, 1987).
The site of administration of an antigen affects its route of lymphatic drainage and this in turn will determine the site at which antigen-presenting cells
process it (Basten et al, 1976). Additionally, the microenvironment in which the antigen presenting cells actually present antigen to lymphocytes may determine which subsets of lymphocytes are activated (Balfour et al, 1981).
Antigen presenting cells in the spleen and lymph nodes differ in that unlike spleen B cells, lymph node cells play an essential role in the activation of T helper cells (Janeway et al, 1987). Thus, it is likely that a greater degree of specific Ty-B cell collaboration results from the intimate contact that occurs between these cells in the lymph node, as compared to the spleen. The lymph node microenvironment may thus represent an enriched source of cells secreting antigen-specific antibody. T cells isolated from lymph nodes at the peak of the primary immune response induce greater help than splenic T cells for in vitro antibody formation by splenic B cells from mice that have been primed and boosted with antigen intraperitoneally (Jensen and Kapp, 1984), thus suggesting that lymph node T cells, highly enriched for helper activity, may be responsible for the differences reported. Enlarged popliteal and inguinal lymph nodes, as seen with hind footpad immunisation, produced better fusion efficiencies than hind leg intradermal immunisations. Since there was no gross swelling or ulcerations of the footpads reported after plantar immunisations and the mice were not incapacitated in any way, this technique may be used to enhance the production of antigen specific monoclonal antibodies (Mirza et al, 1987).
Other routes of immunisation, especially when limited amounts of antigen are available, include in vitro immunisation of splenic cells (Reading, 1986; Erkman et al, 1987; Gratecos et al, 1987), single-shot intrasplenic immunisation (Thorpe et al, 1986) and an abdominal lymph node immunisation technique (Raymond and Suh, 1986).
Intrasplenic immunisation was introduced by Nilsson et al (Nilsson et al, 1987). They showed that a total amount of about 80ng of bovine serum albumin dispersed in four depots, with the antigen immobilised on activated
sepharose beads, resulted in serum antibodies (Nilsson et al, 1987). It is easier to obtain an antibody response using minute amounts of immunogen by the intrasplenic route than by the intraperitoneal or intravenous routes (Larsson and Nilsson, 1988; Ambrosius and Schenderlein, 1986). An antibody response was also obtained when the immunogen was immobilised on nitrocellulose paper as a carrier for immunisation (Nilsson et al, 1987; Larsson and Nilsson, 1988). The success of intrasplenic immunisation seems to be due to the deposition of the immunogen directly into the spleen, thus avoiding the losses associated with systemic injections, and to immobilising the immunogen within the spleen, thus prolonging its exposition to the antigen-presenting cells. A requirement of the technique is that no toxic or irritating additive is used within the spleen, since this would affect spleen cell function. The choice of matrix for intrasplenic immunisation depends on the type of antigen and on the way in which the antigen can be recovered (Nilsson and Larsson, 1990).
In 1978, Hengartner et al reported the first use of spleen cells sensitised in vitro and fused with myeloma cells to lead to specific monoclonal antibody production (Hengartner et al, 1978). Since then, this system of immunisation in culture has been used to produce monoclonal antibodies to several highly conserved proteins (Pardue et al, 1983). The main attraction of this route of immunisation is its ability to produce antibodies to ’self antigens which are not produced in vivo due to suppression or tolerance. Two major culture systems were described for initiation of primary immune responses in vitro (Mishell and Dutton, 1966, 1967; Marbrook, 1967). Modifications of these original systems have also been reported (Schreier and Lefkovits, 1979). The nutritional aspects of the culture systems have also been improved and the serum source identified as the single most important aspect of the medium (Shiigi and Mishell, 1975). All of the different aspects of this technique and its modifications have been reviewed by Reading (Reading, 1982).