Development of an ELISA for quantification of the antimicrobial peptide piscidin 4 and its application to assess stress in fish

Antimicrobial peptides (AMP) are an integral component of innate immunity. One of the most widespread AMP in fish are the piscidins, which have potent, broad-spectrum activity against viruses, bacteria, fungi, and parasites. The widespread phylogenetic distribution of piscidins suggests that they might play an important host defense role in many fish. Quantifying their expression is important in understanding how and where they function. Using a novel piscidin (piscidin 4) that we recently isolated from commercially cultured hybrid striped bass (white bass, Morone chrysops Rafinesque, female x striped bass, Morone saxatilis Walbaum male), we optimized the conditions for measuring this piscidin via sandwich ELISA. We used an antibody to the highly conserved amino terminus of all piscidins as the capture antibody and a peroxidase-labeled antibody specific for the carboxy terminus of piscidin 4 as the detecting antibody. Specificity of the detecting antibody was confirmed by lack of cross-reactivity with other piscidins in ELISA, as well as specificity for piscidin 4 in tissue extracts via Western blotting. The accuracy of the test, defined as piscidin 4 recovery, was 96-103%. Precision, measured by the coefficient of variation, was 13-19%, and parallelism, determined by linearity of the response, had an r(2) > 0.99. The ELISA paralleled the results obtained via Western blotting. Piscidin 4 levels expressed in gill tissue of healthy hybrid striped bass were well within concentrations that are lethal to important fish pathogens. Mean gill piscidin 4 in healthy hybrid striped bass was significantly greater than in either nutritionally stressed fish or in diseased (ectoparasite-infested) fish, suggesting that piscidin 4 can be significantly downregulated with stress or disease. These data suggest that the piscidin 4 ELISA might be a useful indicator of disease susceptibility, providing a new, sensitive tool for rapid screening of population health. (C) 2009 Elsevier Ltd. All rights reserved.