Preparation of Monoclonal Antibody Against EMA-1 and Development of Rapid Serological Detection Method for Theileria equi Infection, Xinjiang, China.
Abstract: The erythrocytic-stage surface protein equi merozoite antigen 1 (EMA-1) of Theileria equi is a major candidate for the development of a diagnostic antigen for equine piroplasmosis. In this study, BALB/c mice were immunized with purified recombinant EMA-1 to prepare monoclonal antibody (mAb) against T. equi EMA-1, and 1 mAb 5H2 was obtained that showed good reaction with infected red blood cells (RBC) in the indirect immunofluorescence assay (IFA). To develop a rapid serological detection method for T. equi infection in Xinjiang Uygur Autonomous Region, China, recombinant EMA-1 originating from the local T. equi strain and the mAb to EMA-1 were employed to develop an immunochromatographic test (ICT) to detect antibodies to T. equi in horse sera. The ICT showed high sensitivity and specificity and no cross-reaction with Babesia caballi. Ninety-two horse serum samples collected from Ili, Xinjiang, were tested by ICT and compared with the detection results of a commercial ELISA kit. The results showed that 56 of 92 (61%) serum samples were seropositive according to the ICT assay, and 50 (54%) samples were seropositive according to the ELISA kit. The ICT had a high coincidence (91.3%) but was more sensitive than the reference ELISA kit. To confirm whether the horses were infected by T. equi, 30 blood DNA samples from 92 horses were examined by PCR. The results showed that 14 of 30 (47%) horses were confirmed to be infected with T. equi by PCR, while 16 of 30 (53%) horses were seropositive by ICT. All PCR-positive horses were ICT-positive. The findings indicate that T. equi is endemic in Ili, Xinjiang, and that the ICT is reliable as a serological diagnosis method. The ICT developed in this study could be an efficient diagnostic tool to detect T. equi infection in horses in the Xinjiang area.
© American Society of Parasitologists 2020.
Publication Date: 2020-04-17 PubMed ID: 32296849DOI: 10.1645/19-98Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research article centers on the creation and evaluation of a quick and efficient method for detecting Theileria equi infection, a disease in horses, using a monoclonal antibody prepared against the equi merozoite antigen 1 protein. The findings reveal that the developed method, called an immunochromatographic test, offers high sensitivity and specificity, and is more effective than existing diagnostic tools.
Preparation of Monoclonal Antibody Against EMA-1
- The study aims to create a diagnostic tool using a monoclonal antibody prepared against a protein called equi merozoite antigen 1 (EMA-1) that’s found on the surface of the Theileria equi parasite during infection.
- BALB/c mice were immunized with a purified and recombinant form of EMA-1 to generate this antibody.
- Successful generation led to monoclonal antibody 5H2, which appeared reactive against the infected red blood cells of horses when analyzed using the indirect immunofluorescence assay (IFA).
Development of Rapid Serological Detection Method
- Employing the recombinant EMA-1 and 5H2, an immunochromatographic test (ICT) was designed for a fast detection method for Theileria equi infection in horses, specifically in the Xinjiang Uygur Autonomous Region, China.
- This ICT allows detection of antibodies to T. equi in horse serum and shows high sensitivity and specificity, with no instances of cross-reaction with Babesia caballi, a related parasite.
- A total of 92 horse serum samples were evaluated using ICT and compared with a commercially available Enzyme-Linked Immunosorbent Assay (ELISA) kit. The ICT identified more infected samples than the ELISA kit, demonstrating its increased sensitivity.
Efficacy of the ICT in Detecting T. equi Infection
- The ICT’s efficiency was further validated by examining horse blood DNA samples for signs of T. equi infection using Polymerase Chain Reaction (PCR) testing.
- Of 30 tested samples, 14 were confirmed to be infected with T. equi via PCR. Meanwhile, 16 samples returned seropositive using the developed ICT.
- All horses that tested positive for T. equi using PCR also tested positive using the ICT, further supporting the ICT’s reliability as a serological diagnostic tool.
- The results suggest that Theileria equi is endemic in the Ili, Xinjiang region of China. They also imply that the newly developed ICT is a reliable and efficient tool for diagnosing T. equi infections in horses in this region.
Cite This Article
APA
Song J, Song R, Wang P, Zhang Y, Yan Y, Zhou J, Chahan B, Liao M.
(2020).
Preparation of Monoclonal Antibody Against EMA-1 and Development of Rapid Serological Detection Method for Theileria equi Infection, Xinjiang, China.
J Parasitol, 106(2), 283-290.
https://doi.org/10.1645/19-98 Publication
Researcher Affiliations
- Parasites Laboratory, Veterinary College, Xinjiang Agricultural University, Nongda East 311, Urumqi, Xinjiang, 830052, P. R. China.
- Parasites Laboratory, Veterinary College, Xinjiang Agricultural University, Nongda East 311, Urumqi, Xinjiang, 830052, P. R. China.
- Parasites Laboratory, Veterinary College, Xinjiang Agricultural University, Nongda East 311, Urumqi, Xinjiang, 830052, P. R. China.
- Parasites Laboratory, Veterinary College, Xinjiang Agricultural University, Nongda East 311, Urumqi, Xinjiang, 830052, P. R. China.
- Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, 310058, P. R. China.
- Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, 310058, P. R. China.
- Parasites Laboratory, Veterinary College, Xinjiang Agricultural University, Nongda East 311, Urumqi, Xinjiang, 830052, P. R. China.
- Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, 310058, P. R. China.
MeSH Terms
- Animals
- Antibodies, Monoclonal / immunology
- Antigens, Protozoan / genetics
- Antigens, Protozoan / immunology
- Antigens, Protozoan / isolation & purification
- Blotting, Western
- China
- Chromatography, Affinity
- Electrophoresis, Polyacrylamide Gel
- Enzyme-Linked Immunosorbent Assay
- Erythrocytes / parasitology
- Female
- Fluorescent Antibody Technique, Indirect
- Horse Diseases / blood
- Horse Diseases / diagnosis
- Horse Diseases / parasitology
- Horses
- Hybridomas / cytology
- Immunohistochemistry
- Mice
- Mice, Inbred BALB C
- Polymerase Chain Reaction
- Protozoan Proteins / genetics
- Protozoan Proteins / immunology
- Protozoan Proteins / isolation & purification
- Sensitivity and Specificity
- Spleen / cytology
- Spleen / immunology
- Theileria / immunology
- Theileria / isolation & purification
- Theileriasis / blood
- Theileriasis / diagnosis
- Theileriasis / parasitology
- Tumor Cells, Cultured
Citations
This article has been cited 3 times.- Song R, Zhai X, Fan X, Li Y, Huercha, Ge T, Li C, Li M, He W, Zheng H, Gan L, Zhang Y, Chahan B. Prediction and validation of cross-protective candidate antigen of Hyalomma asiaticum cathepsin L between H. asiaticum and H. anatolicum. Exp Appl Acarol 2022 Feb;86(2):283-298.
- Wang T, Chen X, Yan X, Su Y, Gao W, Liu C, Wang W. Progress in serology and molecular biology of equine parasite diagnosis: sustainable control strategies. Front Vet Sci 2025;12:1663577.
- Cui Y, Cao M, Yu F, Zhao A, Tao D, Zhu T, Zhang Z, Qi M. Molecular detection of piroplasms in domestic donkeys in Xinjiang, China. Vet Med Sci 2024 Jul;10(4):e1468.
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