Yang Liu, Guangju You, Jialei Shi, Li Gao, Xiaoqi Li, Hong Cao, Yongqiang Wang, Shijun J. Zheng
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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. 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引用次数: 0
摘要
滑膜支原体感染是一种具有重大经济影响的家禽慢性疾病。滑膜分枝杆菌感染的有效诊断工具的需求很大。本研究旨在建立一种基于下拉法和质谱法鉴定抗原的间接酶联免疫吸附测定（iELISA）方法。利用这些方法和anti-M。我们鉴定了一种分子量为53 kDa的未知蛋白（命名为P50蛋白），并建立了基于重组P50蛋白的ELISA （rP50-ELISA）检测P50蛋白的抗体。采用受试者工作特征（ROC）分析，估计rP50-ELISA检测灵敏度（Se）和特异性（Sp）最大的光密度（OD）临界值，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与其它禽源病原体抗体无交叉反应性。对164只临床鸡的血清进行了rP50-ELISA检测，结果与市售诊断试剂盒的符合率高达93.29%。•筛选滑膜分枝杆菌的主要抗原用于ELISA开发。•选择P50蛋白作为ELISA的包被抗原。•成功开发了用于检测抗m抗体的rp50 elisa试剂盒。滑膜抗体具有高敏感性和特异性。
Indirect ELISA developed to detect antibodies against Mycoplasma synoviae P50 protein via immunoproteomic screening
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.
• 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.
期刊介绍：
Applied Microbiology and Biotechnology focusses on prokaryotic or eukaryotic cells, relevant enzymes and proteins; applied genetics and molecular biotechnology; genomics and proteomics; applied microbial and cell physiology; environmental biotechnology; process and products and more. The journal welcomes full-length papers and mini-reviews of new and emerging products, processes and technologies.
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