【摘要】 为建立鸡传染性喉气管炎病毒（ILTV）抗原的检测方法，利用ILTV gJ蛋白的2株单克隆抗体，1株用作捕获抗体，另1株经辣根过氧化物酶（HRP）标记后用作检测抗体，采用棋盘法优化反应条件，建立了一种检测ILTV抗原的双抗夹心ELISA方法，并对其进行了性能评估。结果显示：捕获抗体的最佳包被质量浓度为2μg/m L，酶标单克隆抗体的最适稀释度为1:4 000；临界值为0.295 6，当待测样品OD_{450 nm}值大于0.295 6且P/N值大于2时，判为阳性；建立的双抗夹心ELISA方法可特异性检测ILTV抗原，而对其他禽类常见抗原的检测结果均为阴性；最低检测限为49.4 ELD₅₀/0.1 mL，批内和批间变异系数均小于10%。48份临床样品的检测结果显示，该方法与行业标准中PCR方法的符合率为93.75%。结果表明，本研究建立的ILTV双抗夹心ELISA方法特异性强、灵敏度高、重复性好，可用于ILTV抗原检测。该方法的建立为该病的检测、监测以及防控提供了技术支撑。更多还原

【Abstract】 In order to detect avian infectious laryngotracheitis virus（ILTV）pathogen,a double-antibody sandwich ELISA was established using two monoclonal antibodies（mAbs）against ILTV gJ protein,specifically,one mAb served as the capture antibody,while the other was labeled with horseradish peroxidase（HRP）as the detection antibody,and reaction conditions were optimized via checkerboard titration,followed by evaluation on its performance. The results showed that the optimal coating concentration of the capture antibody was 2 μg/mL,and the optimal dilution of the enzyme-labeled mAb was 1:4 000;the critical value was determined to be 0.295 6,the samples pending for test were considered to be positive when the OD_{450 nm} exceeded 0.295 6 and the P/N ratio was larger than 2;the established method could be used to specifically detect ILTV,other than other common avian pathogens;its detection limit was 49.4 ELD₅₀/0.1 mL,and the coefficients of variation of the intra-and-inter assay were less than 10%. For 48 clinical samples,the result of the established method was 93.75% coincident with that of PCR specified by industry standard.In conclusion,the double-antibody sandwich ELISA established in the study could be used to detect ILTV antigen with its advantages of strong specificity,high sensitivity and good repeatability,supporting future detection,monitoring,prevention and control of the disease.更多还原