ABSTRACT
Antibodies are molecules secreted by plasmocytes; these are differentiated from B lymphocytes following previous contact with a specific antigen. The number of different antibodies produced by humans is extremely large, incorporating between 107 and 108 distinct molecules, but any one B lymphocyte only produces antibodies against a single specific antigen. After immunization or contact with the antigen, the B cells which recognize each antigenic determinant in an antigen molecule will produce clones, thus increasing the number of lymphocytes specific for that antigen. Although the antibodies produced by all of these lymphocyte clones will be specific for the same antigen, these antibodies will not necessarily be structurally identical, that is, they are polyclonal antibodies. The result of this process is an increase in the level of specific antibodies in the serum or body secretions of the immunized individual. Even immunization with highly purified antigens results in the production of polyclonal antibodies, so that the composition and properties of immune sera vary with each preparation (Abbas et al., 2000). Attempts to produce homogeneous antibodies in vitro arose almost as
soon as lymphocytes and their biological properties were discovered. However, B lymphocytes normally do not survive in cell cultures. For survival, they must undergo a malign transformation, such as that induced by the Epstein-Barr virus (Steinitz et al., 1977). Some tumors of B lymphocytes, the multiple myelomas, can arise spontaneously and others can be induced by the administration of mineral oil. These tumors, whether spontaneous or induced, can be adapted to in vitro cultivation, and they will secrete highly homogeneous antibodies (monoclonal antibodies, mAbs) since they originate from a single tumor cell (Horibata and Harris, 1977). However, even if all antibodies are homogeneous in a myeloma culture, it is impossible to predict what this specificity will be. The production in vitro of mAbs with a predetermined specificity has
only been possible since the advent of the technology of hybridomas, which was introduced by Kohler and Milstein in 1975. These hybridomas are the products of in vitro fusion of myelomas with normal B lymphocytes. The fusion products preserve the capacity for self-propagation in a culture, as well as the secretion of the antibodies of interest, characteristics inherited from the parent myeloma and the normal B lymphocyte, respectively. The myelomas used in such fusions generally involve cell lines from B-lymphocyte tumors developed in mice or rats (Cotton and Milstein, 1973; Ko¨hler and Milstein, 1975b; Shulman et al., 1978), while
the B lymphocytes come from mice or rats previously immunized with the antigen of interest. After the fusion of the myeloma with the normal B lymphocyte, the hybridomas of interest can be selected and cloned to obtain an unlimited quantity of homogeneous antibodies that are highly specific (Ko¨hler and Milstein, 1975a). The production of such mAbs using hybridoma technology has made it
possible to detect and quantify a wide variety of molecules produced by live organisms in highly specific and sensitive assays, thus making possible enormous advances in various areas of biological research. The specificity of these mAbs has also facilitated the improvement of clinical diagnoses, as well as creating expectations about their use as a therapeutic agent for human diseases. One problem with the use of these antibodies in humans, however, is
the immunogenicity (the ability to induce an immune response) of murine mAbs, and various associated adverse reactions. Some mouse mAbs have, however, been licensed for use in human patients (Lin et al., 2005). The adverse reactions can be controlled to a certain extent by ‘‘humanization’’ of the murine reagents (produced by cells transfected with antibody genes), as well as by the use of recombinant DNA techniques to produce antibody fragments preserving the antigen-binding capacity of the original antibody molecules (Huhalov and Chester, 2004). Only recently has the establishment of a line of human myeloma cells (called Karpas 707H) been reported, and this is proving useful in the production of hybridomas via fusion with B lymphocytes obtained from immunized or infected humans (Karpas et al., 2001; Vaisbourd et al., 2001). Obtaining all these highly specific mAbs, whether murine, humanized, or antibody fragments, is dependent upon the production of a hybridoma. Both hybridomas and humanized antibody producer cells can be culti-
vated indefinitely in conventional cultures, usually containing fetal bovine serum in the medium. In the supernatants of these cultures, the quantity of mAbs varies from 20 to 100 g/ml of protein, depending on the cell and the system of cultivation. There are, however, in vivo methods of obtaining mAbs, and these
produce much larger quantities. One of these is intraperitoneal administration of the hybridomas into histocompatible animals (of the same cell line as the parents of the hybridoma) or immunodeficient animals (individuals with no functional immune system). These receptor animals will develop ascitic tumors containing from 1 to 40 mg/ml of the mAb secreted by the hybridoma (Kretzmer, 2002). However, despite the fact that this method is well documented and has been used widely in the past, the procedure is presently being rejected because of ethical concerns over the use of laboratory animals. This has led to an increased interest in methods for the in vitro
production of large quantities of mAbs. Submersed cultivation in bioreactors is one possibility. In contrast with traditional methods, this procedure uses large quantities of culture medium, increasing the scale of the process and making possible the production of virtually unlimited quantities of mAbs by a given hybridoma. In this chapter, the fundamentals of hybridoma technology are dis-
cussed and the most widely used protocols and procedures are presented.
