基于IgY的ELISA定量检测甲鱼系统性败血症球状病毒

摘要

Based on chicken immunoglobulin of yolk (IgY), we have developed an indirect ELISA for detection of soft-shelled turtle systemic sepsis spherical virus (STSSSV). Chicken IgY against STSSSV is used as the primary antibody, while an HRP conjugated goat anti-chicken IgG is used as the secondary antibody. By comparing ELISA with hemagglutination inhibition test (HI), the linear relationship between OD492nm values and the dilution of STSSSV, as well as log 2 of the dilutions is established and analyzed, base on which a quantitative ELISA is developed. The presented method has been applied to test various infected samples including sera, liver, feces and culturing water. The results show that the minimum detection limit of the developed ELISA method is 3.98 ng·mL-1, and there has been no cross-reaction to other aquatic virus.The detection range of quantitative ELISA is limited to 0.031~4μg·mL-1. Sera contains the highest viral load. Even in latent period, STSSSV in sera can be detected, which is helpful for early diagnosis.%为了对甲鱼系统性败血症球状病毒(STSSSV)进行快速检测,以抗 STSSSV 卵黄抗体(egg yolk immunoglobulin, IgY)为一抗,以羊抗鸡IgG(HRP-IgG)为二抗,建立STSSSV检测的间接酶联免疫吸附(ELISA)与血凝抑制(HI)实验,对两者进行了比较.运用 ELISA 对感染病毒甲鱼的血清、肝脏、粪便、及饲养环境水样进行了STSSSV检测.用ELISA分析了病毒稀释倍数及其2的对数值与OD492nm值间的线性关系,建立STSSSV的ELISA定量检测方法.结果显示： ELISA较HI更灵敏,其对STSSSV的检测灵敏度为3.98 ng·mL-1,能够在携带该病毒尚未显症的隐性感染的甲鱼血清和甲鱼粪便与养殖水中检测出STSSSV. ELISA对其他水产病毒无交叉反应,特异性强. ELISA定量检测限度范围为0.031~4μg·mL-1.本研究建立的间接ELISA方法为STSSSV的定性、定量提供了准确有效的检测手段.