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Abstract <p><i>Mycoplasma synoviae</i> infection is a chronic disease of poultry with significant economic impacts. An efficient diagnostic tool for <i>M. synoviae</i> 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-<i>M. synoviae</i> serum, we identified an uncharacterized protein with a molecular weight of&#xa0;53&#xa0;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.</p> Key points <p>• <i>Screening for the major antigen of M. synoviae for ELISA development.</i></p> <p>• <i>The P50 protein was selected as a coating antigen for ELISA.</i></p> <p>• <i>rP50-ELISA was successfully developed for detecting anti-M. synoviae antibodies with high sensitivity and specificity.</i></p>

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Indirect ELISA developed to detect antibodies against Mycoplasma synoviae P50 protein via immunoproteomic screening

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月6日

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  • Yang Liu,
  • Guangju You,
  • Jialei Shi,
  • Li Gao,
  • Xiaoqi Li,
  • Hong Cao,
  • Yongqiang Wang,
  • Shijun J. Zheng

摘要

Abstract

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.