Establishment of an Indirect ELISA Antibody Detection Method for Equine Trypanosomosis Based on Nucleotide Triphosphate Diphosphohydrolase
ZENG Qing-Qin1,2,3, LI Ru-Song1, LI Hong-Ying4, WANG Xin-Le1, WANG Ying5, XIA Tian-Qi3, WANG Pu3, SHI Heng-Zhi3, ZHANG Lu-Ping3, ZHENG Ya-Dong3, ZHAO Xiu-Ling1,*, SONG Hou-Hui3,*
1 Ningbo Customs Technical Center/Ningbo Key Laboratory of Port Biological and Food Safety Testing, Ningbo 315000, China; 2 Animal Disease Prevention and Control Center of Jiangyou City, Sichuan Province, Jiangyou 621700, China; 3 College of Animal Science and Technology & College of Veterinary Medicine/Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province/Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics & Advanced Technology/Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management/China-Australia Joint Laboratory for Animal Health Big Data Analytics, Zhejiang A&F University, Hangzhou 311300, China; 4 Department of Cardiology, First Hospital of Hebei Medical University, Shijiazhuang 050031, China; 5 National Institute of Parasitic Diseases (Chinese Center for Tropical Diseases Research)/NHC Key Laboratory of Parasite and Vector Biology/WHO Collaborating Centre for Tropical Diseases/State Key Laboratory of Infectious Disease Prevention and Control, Chinese Center for Disease Control and Prevention, Shanghai 200025, China
Abstract：Trypanosomosis (Surra) by Trypanosoma evansi, which resides in the blood, is a protozoan disease in animals such as horses (Equus) and camels (Camelus). Accurate diagnosis is a crucial step in the effective prevention and control of Surra, but the existing diagnostic techniques have many drawbacks such as high false negative rates and high prices. Based on recombinant expression of nucleoside triphosphate diphosphate hydrolase (TeNTPDase) from T. evansi, in this study, an ELISA method was established for Surra by optimizing various reaction parameters, including antigen coating concentration and conditions, serum dilution and incubation conditions, conjugated second antibody dilution and incubation conditions, and evaluated its diagnostic efficacy. The results showed that the recombinant TeNTPDase was mainly expressed in the form of inclusion bodies, with a size of approximately 60 kD. The coefficient of variation of the established ELISA method in different batches of coating antigens were 3.50%~7.32% being less than 10%, and the coefficient of variation at different incubation time points at 37 ℃ were 3.89%~14.45% being less than 15%, indicating good repeatability and stability. Moreover, when using the established ELISA method to detect clinical serum samples of horses, it was found that the positive rate of Surra was 5.83% (7/120), slightly higher than that of the commercial test kit (3.33%), but the difference was not significant, with a coincidence rate of 92.5% (111/120). This study provides technical support for the effective prevention and control of Surra in horses.
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