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Background <p>Listeriosis is a severe zoonotic disease caused by <i>Listeria monocytogenes</i> (<i>L. monocytogenes</i>), which poses a significant threat to both livestock and public health. Efficient detection of <i>L. monocytogenes</i> infections in humans and various animal species requires the development of a specific and sensitive method for clinical diagnosis.</p> Results <p>In this study, a competitive enzyme-linked immunosorbent assay (cELISA) was developed for the detection of anti-ActA antibodies in serum, based on a horseradish peroxidase-labeled monoclonal antibody 3G11 (HRP-MAb 3G11). The assay parameters, including the coating antigen concentration, HRP-MAb 3G11 titer, serum dilution factor, blocking buffer, and blocking incubation time, were optimized for the detection of ActA-specific antibodies. Receiver operating characteristic (ROC) analysis identified 27.50% as the optimal cutoff value for the percentage of inhibition (PI), yielding a diagnostic sensitivity of 100% and a diagnostic specificity of 93.33%. This assay exhibited no cross-reactivity with positive sera specific for other common pathogens, demonstrated stable reproducibility, and achieved a diagnostic accuracy of 95.7% compared with a commercial indirect LLO-based ELISA kit. The cELISA kit was used to detect 930 ovine serum samples, revealing an overall seropositivity rate of 24.84%.</p> Conclusions <p>The cELISA kit developed in this study provides a specific, sensitive, and repeatable serological method for the rapid detection of <i>L. monocytogenes</i> infection.</p>

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Development and application of ActA-based competitive ELISA for the specific diagnosis of ovine listeriosis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月20日

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  • Chao Chen,
  • Beiru Wang,
  • Mengdi Zhao,
  • Fanzeng Meng,
  • Suojun Jiang,
  • Xin’an Jiao,
  • Yuelan Yin

摘要

Background

Listeriosis is a severe zoonotic disease caused by Listeria monocytogenes (L. monocytogenes), which poses a significant threat to both livestock and public health. Efficient detection of L. monocytogenes infections in humans and various animal species requires the development of a specific and sensitive method for clinical diagnosis.

Results

In this study, a competitive enzyme-linked immunosorbent assay (cELISA) was developed for the detection of anti-ActA antibodies in serum, based on a horseradish peroxidase-labeled monoclonal antibody 3G11 (HRP-MAb 3G11). The assay parameters, including the coating antigen concentration, HRP-MAb 3G11 titer, serum dilution factor, blocking buffer, and blocking incubation time, were optimized for the detection of ActA-specific antibodies. Receiver operating characteristic (ROC) analysis identified 27.50% as the optimal cutoff value for the percentage of inhibition (PI), yielding a diagnostic sensitivity of 100% and a diagnostic specificity of 93.33%. This assay exhibited no cross-reactivity with positive sera specific for other common pathogens, demonstrated stable reproducibility, and achieved a diagnostic accuracy of 95.7% compared with a commercial indirect LLO-based ELISA kit. The cELISA kit was used to detect 930 ovine serum samples, revealing an overall seropositivity rate of 24.84%.

Conclusions

The cELISA kit developed in this study provides a specific, sensitive, and repeatable serological method for the rapid detection of L. monocytogenes infection.