The development of highly sensitive ELISA techniques has identified many more antigen positive fish than had previously been identified by FAT or culture techniques (Meyers et al., 1993; Gudmundsdottir et al., 1993). The antigen-positive samples that cannot be confirmed using another technique should be interpreted with caution. It should not be assumed that these fish will at some point develop clinical BKD, as the fish might harbour cross-reactive substances which interfere with the ELISA and/or antigen may be present but the bacteria may be non-viable.
The sensitivity and specificity of the ELISA relies heavily on the quality of antisera used. Numerous reports of cross-reactive organisms have been documented in the literature, for both polyclonal antibodies and MAbs. Cross-reactive Gram-positive, Gram-negative and Gram-undetermined organisms have been identified using FAT (Bullock et al., 1980; Evelyn et al., 1981; Austin et al., 1985; Yoshimizu et al., 1987; Brown et al, 1995; Teska et al., 1995). In general, the cross-reactive component(s) have not been identified but may include common carbohydrate molecules, such as galactose (Fiedler and Draxl, 1986) or heat-shock proteins (Wood et al., 1995). Dixon (1987) found that preadsorption of polyclonal antisera with Rothia dentocariosa and Bacillus sphaericus increased specificity of the ELISA. In addition to cross-reactivity with microorganisms, cross-reactivity of polyclonal antisera has been observed in the ELISA with feather meal components present in certain commercial diets (Pascho et al., 1991a) and with fish serum components (Turaga et al., 1987a). Thus, affinity purification of polyclonal antisera is probably necessary to ensure adequate specificity. The use of MAbs may increase the specificity of the immunoassay, as these antibodies recognize precise, selected epitopes. Judicious choice of MAbs is necessary, as Arakawa et al. (1987) identified a MAb that recognized a cross-reactive determinant on R. salmoninarum and three other Gram-positive organisms.
Since cross-reactive antigens exist, it is essential that independent assays are utilized to confirm ELISA results. In our opinion, Western blotting is an ideal technique for the confirmation of R. salmoninarum p57 in ELISA-positive samples, since p57 can be identified on the basis of both molecular mass and
antigenic identity (Turaga et al., 1987b; Sakai et al., 1990; Wiens et al., 1990). A limitation of the Western blot has been the sensitivity (Rockey et al., 1991a; Griffiths et al., 1991; Olivier et al., 1992), however, we have increased the sensitivity 50-100-fold using a chemiluminescent development system (Wiens, 1992). The presence of p57 in samples was detectable at levels as low as 13 ng ml-1 of tissue homogenate and 10 ng ml-1 in ovarian fluid. Disadvantages
36 24 12 p57 Standard (ng ml-1)
36 24 12 p57 Standard (ng ml-1)
Fig. 7.2. (and opposite) (a) Schematic diagram of the theory and practice of the monoclonal antibody-based ELISA described in Rockey et al. (1991a). An individual well of an ELISA plate is depicted after each step in the assay. (1) The wells are coated with a primary monoclonal antibody (MAb 4D3) and the unbound sites on the plate are then blocked with a non-specific protein, bovine serum albumin (not shown). (2) Serial dilutions of a standard p57 preparation or dilutions of clinical samples are added. The primary antibody 4D3 binds to an amino-proximal epitope on p57, specifically retaining p57 but not other proteins in the sample. (3) A secondary biotinylated MAb (3H1) is used to recognize a specific epitope in the middle of p57. B, biotin. (4) MAb 3H1 is biotinylated, which is bound subsequently by an avidin-enzyme conjugate. (5) A chromogenic substrate is added and the colorimetric reaction is measured on an ELISA reader. (b) Photo of an ELISA plate after substrate addition. The dark colour of the substrate indicates the presence of R. salmoninarum antigen. On the bottom of the plate (rows F, G, H) is a dilution of a p57 standard (six wells per dilution), while kidney homogenates of indivdual fish samples are on the remainder of the plate rows A-E (samples are in duplicate). Comparison of the colour intensity of the clinical samples with the standard allows for precise quantification of antigen levels in fish tissues. The image was supplied courtesy of Dr John Reddington, Diaxotics, Wilton, Connecticut.
of this technique are that it is still three- to fourfold less sensitive than the monoclonal-based ELISA and, secondly, even if antigen is confirmed, the presence of viable organisms is still in question. Therefore, it is recommended that, if putative R. salmoninarum-free stocks are identified as ELISA-positive, Western blot and culture techniques be used to confirm the presence of p57 and viable R. salmoninarum. Using this type of combined approach for detecting R. salmoninarum, Griffiths et al. (1996) have found that incubation of ovarian fluid cellular debris in selective kidney disease medium (SKDM) broth, followed by Western blotting, increased the total numbers of positive samples by 32% over SKDM agar culture or indirect FAT (IFAT).
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