Corrales J, Gordon W L, Noga E J
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA.
Fish Shellfish Immunol. 2009 Aug;27(2):154-63. doi: 10.1016/j.fsi.2009.02.023. Epub 2009 Mar 4.
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 symbolxstriped bass, Morone saxatilis Walbaum male symbol), 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.
抗菌肽（AMP）是先天免疫的一个重要组成部分。鱼类中分布最广泛的一种抗菌肽是鱼杀菌素，它对病毒、细菌、真菌和寄生虫具有强大的广谱活性。鱼杀菌素广泛的系统发育分布表明它们可能在许多鱼类中发挥重要的宿主防御作用。量化它们的表达对于理解它们如何以及在何处发挥作用很重要。我们使用最近从商业养殖的杂交条纹鲈（白鲈，Morone chrysops Rafinesque，♀×条纹鲈，Morone saxatilis Walbaum♂）中分离出的一种新型鱼杀菌素（鱼杀菌素4），优化了通过夹心酶联免疫吸附测定（ELISA）测量这种鱼杀菌素的条件。我们使用针对所有鱼杀菌素高度保守的氨基末端的抗体作为捕获抗体，以及针对鱼杀菌素4羧基末端的过氧化物酶标记抗体作为检测抗体。通过ELISA中与其他鱼杀菌素无交叉反应以及通过蛋白质印迹法对组织提取物中鱼杀菌素4的特异性，证实了检测抗体的特异性。以鱼杀菌素4回收率定义的检测准确性为96 - 103%。以变异系数衡量的精密度为13 - 19%，通过响应线性确定的平行性，r²>0.99。ELISA与通过蛋白质印迹法获得的结果平行。健康杂交条纹鲈鳃组织中表达的鱼杀菌素4水平处于对重要鱼类病原体具有致死性的浓度范围内。健康杂交条纹鲈鳃中鱼杀菌素4的平均含量显著高于营养应激鱼或患病（有体外寄生虫感染）鱼，这表明鱼杀菌素4可能会因应激或疾病而显著下调。这些数据表明鱼杀菌素4 ELISA可能是疾病易感性的一个有用指标，为快速筛查群体健康提供了一种新的、灵敏的工具。
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