Monoclonal Antibodies: A Tool for Molluscan Pathology

-Hybridoma technology is reviewed, and the characteristics of monoclonal antibodies are compared with those of polyclonal antibodies. The contribution of monoclonal antibodies to molluscan pathology is developed with special emphasis on their use as diagnostic tools. The results of studies with monoclonal antibodies prepared against the protozoan oyster pathogen Bonamia ostreae are briefly described. The development of hybridoma technology as elaborated by Kohler and Milstein (1975) has made an impact in many fields ofbiological research such as immunology, biochemistry, and pathology (Yelton and Scharff 1981; Krakauer 1985; Seiler et al. 1985). In pathology, mouse monoclonal anti­ bodies have been used in diagnosis (Van Der Auwera 1987) and therapy (Blythman et al. 1981; Frankel 1985). In this article, we briefly review hybridoma technology. We compare the properties of monoclonal and polyclonal antibodies, and we consider the prospects for use of monoclonal anti­ bodies in molluscan pathology, especially for the diagnosis of infectious diseases. Hybridoma Technology and Production of Monoclonal Antibodies The principle of hybridoma technology is the continuation of the nonproliferative line of anti­ body-producing lymphocytes by fusing the lym­ phocytes with a tumor cell !ine (myeloma cells; Figure 1). Hybrid cells, called hybridomas, are obtained which retain both the ability of individ­ uallymphocytes to secrete antibody and the abil­ ity of myeloma cells to grow without limit. Thus, the homogenous antibody derived from a single clone of hybridomas is called a monoclonal anti­ body (MAB). The properties of the lymphocyte can also be retained by infecting the lymphocyte with a transforming virus or by transfecting it with tumorigenic DNA (Schonherr and Houwink 1984). The production of MAB has been reviewed by Kennett et al. (1980), Goding (1983), Pau et al. (1983), Schonherr and Houwink (1984), and Pa­ olucci et al. (1986). The different steps in the production are shown in Figure 2. !mmunization, Preparation of Cel/s, and Fusion A mouse is immunized by successively inject­ ing it with antigenic preparations (Figure 2). Pu­ rified antigen is not necessary, but the hybridiza­ tion yield is partly conditioned by the level of sensitization of the animal. After the last injec­ tion, lymphocytes isolated from the spleen are fused with myeloma ceUs either by chemical treat­ ment with polyethylene glycol (Paolucci et al. 1986) or by electrical treatment (Vienken and Zimmermann 1985). Selection ofHybridomas Because the yield of stable hybridomas from parent cells is low, about 10_10 , the nonfused myeloma cells deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT-) must be kept from overgrowth. The HGPRT-deticient cells cannot grow in a medium containing hypoxanthine aminopterin thymidine, so this medium is used to select the HGPRT+ hybridomas after fusion. Selection of Hybridomas Producing Specifie Antibody The hybridomas must be screened as early as possible to distinguish and eliminate those pro­ ducing nonspecitic antibody from those producing specitic antibody. The screening technique must be rapid, simple, and suitable for the large number of hybridomas grown in the wells of microculture plates. The techniques used most frequently are radioimmunoassay (RIA), enzyme-linked im­ munosorbent assay (ELISA), and immunofluores­ cence (IF). Screening is a key step in lymphocyte hybridization, and its success depends entirely on the availability of purified antigen.