Liu Yang, You Guangju, Shi Jialei, Gao Li, Li Xiaoqi, Cao Hong, Wang Yongqiang, Zheng Shijun J
College of Veterinary Medicine, National Key Laboratory of Veterinary Public Health Security, China Agricultural University, Beijing, China.
Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Appl Microbiol Biotechnol. 2025 May 6;109(1):110. doi: 10.1007/s00253-025-13503-4.
Mycoplasma synoviae infection is a chronic disease of poultry with significant economic impacts. An efficient diagnostic tool for M. synoviae infection is in great demand. This study aimed to develop a novel indirect enzyme-linked immunosorbent assay (iELISA) method based on antigens identified by pull-down assay combined with mass spectrometry. Using these methods and anti-M. synoviae serum, we identified an uncharacterized protein with a molecular weight of 53 kDa (named P50 protein) and then established a recombinant P50 protein-based ELISA (rP50-ELISA) to detect antibodies against P50 protein. A receiver operating characteristic (ROC) analysis was performed to estimate the optical density (OD) cut-off value that maximized the sensitivity (Se) and specificity (Sp) of the rP50-ELISA, which had a mean Se of 93% (95% confidence interval (CI) = 86.25-96.57%) and a mean Sp of 100% (95% CI = 91.80-100%), with an area under the curve (AUC) of 0.9979 (95% CI = 99.41-100%). The rP50-ELISA showed no cross-reactivity with antibodies against other avian pathogens. Serum samples from 164 clinical chickens were tested with the rP50-ELISA, and the results revealed a high concordance rate of 93.29% with commercial diagnostic kits. KEY POINTS: • Screening for the major antigen of M. synoviae for ELISA development. • The P50 protein was selected as a coating antigen for ELISA. • rP50-ELISA was successfully developed for detecting anti-M. synoviae antibodies with high sensitivity and specificity.
鸡滑液囊支原体感染是一种对家禽具有重大经济影响的慢性疾病。目前迫切需要一种高效的鸡滑液囊支原体感染诊断工具。本研究旨在基于通过下拉试验结合质谱鉴定的抗原开发一种新型间接酶联免疫吸附测定（iELISA）方法。利用这些方法和抗鸡滑液囊支原体血清，我们鉴定出一种分子量为53 kDa的未表征蛋白（命名为P50蛋白），然后建立了基于重组P50蛋白的ELISA（rP50-ELISA）来检测针对P50蛋白的抗体。进行了受试者工作特征（ROC）分析以估计使rP50-ELISA的灵敏度（Se）和特异性（Sp）最大化的光密度（OD）临界值，其平均Se为93%（95%置信区间（CI）=86.25-96.57%），平均Sp为100%（95%CI=91.80-100%），曲线下面积（AUC）为0.9979（95%CI=99.41-100%）。rP50-ELISA与针对其他禽病原体的抗体无交叉反应。用rP50-ELISA检测了164只临床鸡的血清样本，结果显示与商业诊断试剂盒的一致性率高达93.29%。要点：•筛选鸡滑液囊支原体的主要抗原用于ELISA开发。•选择P50蛋白作为ELISA的包被抗原。•成功开发了rP50-ELISA用于检测抗鸡滑液囊支原体抗体，具有高灵敏度和特异性。
