An enzyme-linked immunosorbent assay (ELISA) for the detection of Piscirickettsia salmonis in fish tissue samples was developed. The test uses a combination of different monoclonal antibodies specific against P. salmonis in the capture step of the assay. The antibodies 7G4, 6E2 and 2C1 chosen for the capture step are bound to the solid support with an adhesive protein purified from a bivalve mollusc, resulting in a high yield of adsorption and binding stability. The monoclonal antibody 7G4, used as a second antibody, is conjugated to horseradish peroxidase. The resulting ELISA test detected 7 different isolates of P. salmonis and does not cross-react with several other fish pathogens, revealing a high specificity and sensitivity. The test also detects P. salmonis in kidney tissue of infected coho salmon with 98% correlation with the immunofluorescence assay. KEY WORDS: ELISA · Salmon diseases · Piscirickettsia salmonis · Piscirickettsiosis · Monoclonal antibodies Resale or republication not permitted without written consent of the publisherDis Aquat Org 49: [33][34][35][36][37][38] 2002 reports describing detection of P. salmonis with immunofluorescence assays using polyclonal antibodies conjugated with fluorescein isothiocyanate , Barnes et al. 1998. Recently, we have developed a panel of monoclonal antibodies with high sensitivity and specificity like those tested in an immunofluorescence assay (Jamett et al. 2001). These monoclonal antibodies react with several isolates of P. salmonis, and the coccoid immunofluorescent shapes suggest that the antibodies recognize membraneassociated antigens (Jamett et al. 2001). This report describes the development of a highly specific sandwich enzyme-linked immunosorbent assay (ELISA), based on the use of monoclonal antibodies in the capture step of the assay and as a secondary antibody conjugated with horseradish peroxidase. MATERIALS AND METHODSIn addition to Piscirickettsia salmonis LF-89 (ATCC VR 1361), several other isolates of P. salmonis obtained from different places in the south of Chile were used. The names of these isolates and their origins are as follows: (1) an isolate from the kidney of steelhead trout from Calbuco, Puerto Montt, obtained by Fundación Chile and named P. salmonis-Fundación Chile; and (2) isolates from the kidney of coho salmon from Pargua and Ralún, Puerto Montt, obtained by Aquatic Health Laboratories and referred to as P. salmonis-Aquatic Health, Ralún 14 and Ralún 17, 052 and L047. A bank of support inocula was prepared from the P. salmonis isolates by resuspending infected CHSE-214 lysates in a mixture of 10% dimethylsulfoxide in fetal calf serum and storing them in liquid nitrogen.To determine the specificity of the ELISA assay, several bacteria maintained and grown as described previously (Jamett et al. 2001) were used as antigens (see Table 1).Growth and purification of Piscirickettsia salmonis. P. salmonis LF-89 and other isolates listed above were continuously propagated in the chinook salmon embryo cell line CHSE-214 (CR...