Taesuji Machimaporn, Rattanamas Khate, Kulthonggate Usakorn, Mamom Thanongsak, Ruenphet Sakchai
Master of Science Program in Animal Biotechnology, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, Thailand.
Clinic for Horse, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, Thailand.
Vet World. 2022 Dec;15(12):2760-2763. doi: 10.14202/vetworld.2022.2760-2763. Epub 2022 Dec 5.
The immune responses of animals infected with African horse sickness (AHS) virus are determined by enzyme-linked immunosorbent assay (ELISA), complement fixation, and virus neutralization test. During the outbreaks of AHS in Thailand, the immune response after vaccination has been monitored using commercial test kits such as blocking ELISA, which are expensive imported products unavailable commercially in Thailand. This study aimed to assess the sensitivity and specificity of anti-AHS virus antibodies using dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine.
A total of 186 horse sera, namely, 93 AHS-unvaccinated samples and 93 AHS-vaccinated samples, were used in this study. All sera underwent antibodies detection using commercial blocking ELISA and in-house dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine. The numbers of true positive, false positive, true negative, and false negative results in the dot blotting were compared with those in blocking ELISA and the sensitivity and specificity of dot blotting were assessed.
For the monovalent antigen, there were 78, 19, 74, and 15 true positive, false positive, true negative, and false negative results, respectively, while for the polyvalent antigen, the corresponding numbers were 84, 34, 58, and 9. Meanwhile, the diagnostic sensitivity and specificity for monovalent antigen were 83.87% and 79.57%, respectively, but 90.32% and 62.37% for polyvalent antigen.
Dot blotting for AHS antibodies detection using vaccine antigen showed high sensitivity and rather a high specificity compared with the findings with the commercial ELISA test kit. In countries where commercial ELISA test kits are not available and when the size of a serum sample is small, dot blotting could become a good alternative test given its advantages, including its simplicity, rapidity, and convenience. To the best of our knowledge, these findings are the first report on the use of dot blotting for detecting AHS antibodies in horses. In conclusion, monovalent antigen-based dot blotting could be used as a reliable alternative serodiagnostic test for monitoring AHS humoral immune response, especially in vaccinated horses.
感染非洲马瘟（AHS）病毒的动物的免疫反应通过酶联免疫吸附测定（ELISA）、补体结合试验和病毒中和试验来确定。在泰国AHS疫情爆发期间，使用如阻断ELISA等商业检测试剂盒监测疫苗接种后的免疫反应，这些试剂盒是昂贵的进口产品，在泰国无法商业获取。本研究旨在基于减毒活AHS疫苗的单价和多价毒株，通过斑点印迹法评估抗AHS病毒抗体的敏感性和特异性。
本研究共使用了186份马血清，即93份未接种AHS疫苗的样本和93份接种AHS疫苗的样本。所有血清均采用商业阻断ELISA以及基于减毒活AHS疫苗单价和多价毒株的自制斑点印迹法进行抗体检测。将斑点印迹法中的真阳性、假阳性、真阴性和假阴性结果数量与阻断ELISA中的结果进行比较，并评估斑点印迹法的敏感性和特异性。
对于单价抗原，真阳性、假阳性、真阴性和假阴性结果分别为78、19、74和15例，而对于多价抗原，相应数量分别为84、34、58和9例。同时，单价抗原的诊断敏感性和特异性分别为83.87%和79.57%，但多价抗原的诊断敏感性和特异性分别为90.32%和62.37%。
与商业ELISA检测试剂盒的结果相比，使用疫苗抗原的AHS抗体斑点印迹法显示出高敏感性和较高的特异性。在无法获得商业ELISA检测试剂盒且血清样本量较小的国家，由于其具有简单、快速和方便等优点，斑点印迹法可能成为一种很好的替代检测方法。据我们所知，这些发现是关于使用斑点印迹法检测马中AHS抗体的首次报告。总之，基于单价抗原的斑点印迹法可作为一种可靠的替代血清学诊断试验，用于监测AHS体液免疫反应，尤其是在接种疫苗的马匹中。
