Development of an internalin-based double-antibody sandwich quantitative ELISA for the detection of Listeria monocytogenes in slaughterhouse environments

Qing Cao Wenjing Shi Yanquan Wei Jiayu Wang Zhonglong Wang Qian Chong Qianqian Guo Kunzhong Zhang Huitian Gou ^* Huiwen Xue ^*

• College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, Gansu Province, China

Listeria monocytogenes causes zoonotic listeriosis with a high mortality rate, which is frequently detected in slaughterhouse processing environments and animal-based food. To enable the specific, rapid, and cost-effective detection of L. monocytogenes in environments and animal-based food, we developed a double-antibody sandwich quantitative ELISA (DAS-qELISA) method. This method is based on monoclonal antibodies targeting internalin G (InlG), a surface macromolecular protein of L. monocytogenes with demonstrated immunogenicity in previous studies. The antibody pair 1D2-2H10 was selected for following studies using sandwich ELISA. The DAS-qELISA method was then established and optimized using this antibody pair. The detection limits of the method were determined to be 32 ng/mg for the InlG protein and 7875.83 CFU/mL for L. monocytogenes. The accuracy of the method was evaluated across various bacterial concentrations, with results falling within 91.56%-107.07% and a coefficient of variation (CV) of less than 10%. Compared to traditional methods, this approach requires only 12 h of bacterial enrichment and incubation to achieve 100% accuracy. These findings suggest that the DAS-qELISA developed in this study offers a rapid, accurate, and cost-effective tool for detecting L. monocytogenes in environmental and product samples from animal.