建立并应用一种 iELISA 检测方法用于检测猫弓形虫顶膜抗原 1（AMA1）抗体。
Establishment and application of an iELISA detection method for measuring apical membrane antigen 1 (AMA1) antibodies of Toxoplasma gondii in cats.
机构信息
School of Basic Medical Sciences and Forensic Medicine, Hangzhou Medical College, Hangzhou, 310013, China.
Engineering Research Center of Novel Vaccine of Zhejiang Province, Hangzhou Medical College, Hangzhou, China.
出版信息
BMC Vet Res. 2023 Nov 3;19(1):229. doi: 10.1186/s12917-023-03775-1.
BACKGROUND
Diseases caused by Toxoplasma gondii (T. gondii) have introduced serious threats to public health. There is an urgent need to develop a rapid detection method for T. gondii infection in cats, which are definitive hosts. Recombinant apical membrane antigen 1 (rAMA1) was produced in a prokaryotic expression system and used as the detection antigen. The aim of this study was to evaluate and optimize a reliable indirect enzyme-linked immunosorbent assay (iELISA) method based on rAMA1 for the detection of antibodies against T. gondii in cats.
RESULTS
The rAMA1-iELISA method was developed and optimized by the chessboard titration method. There were no cross-reactions between T. gondii-positive cat serum and positive serum for other pathogens, indicating that rAMA1-iELISA could only detect T. gondii in most cases. The lowest detection limit of rAMA1-iELISA was 1:3200 (dilution of positive serum), and the CV of repeated tests within batches and between batches were confirmed to be less than 10%. The results of 247 cat serum samples detected by rAMA1-iELISA (kappa value = 0.622, p < 0.001) were in substantial agreement with commercial ELISA. The ROC curve analysis revealed the higher overall check accuracy of rAMA1-iELISA (sensitivity = 91.7%, specificity = 93.6%, AUC = 0.956, 95% CI 0.905 to 1.000) than GRA7-based iELISA (sensitivity = 91.7%, specificity = 85.5%, AUC = 0.936, 95% CI 0.892 to 0.980). Moreover, the positive rate of rAMA1-iELISA (6.5%, 16/247) was higher than that of GRA7-based iELISA (3.6%, 9/247) and that of commercial ELISA kit (4.9%, 12/247).
CONCLUSION
The iELISA method with good specificity, sensitivity, and reproducibility was established and can be used for large-scale detection of T. gondii infection in clinical cat samples.
背景
弓形虫（Toxoplasma gondii）引起的疾病对公共卫生造成了严重威胁。因此，迫切需要开发一种用于检测猫（终末宿主）弓形虫感染的快速检测方法。本研究旨在评估和优化一种基于重组顶膜抗原 1（rAMA1）的间接酶联免疫吸附试验（iELISA）方法，用于检测猫血清中的抗弓形虫抗体。
方法
采用棋盘滴定法对 rAMA1-iELISA 方法进行了开发和优化。结果显示，rAMA1-iELISA 与其他病原体的阳性血清无交叉反应，表明该方法只能在大多数情况下检测到弓形虫。rAMA1-iELISA 的最低检测限为 1:3200（稀释的阳性血清），批内和批间重复试验的 CV 均小于 10%。rAMA1-iELISA 检测的 247 份猫血清样本的结果（kappa 值=0.622，p<0.001）与商业 ELISA 具有高度一致性。ROC 曲线分析显示，rAMA1-iELISA 的整体检测准确性较高（敏感性=91.7%，特异性=93.6%，AUC=0.956，95%CI 0.905 至 1.000），高于 GRA7 基于 iELISA（敏感性=91.7%，特异性=85.5%，AUC=0.936，95%CI 0.892 至 0.980）。此外，rAMA1-iELISA 的阳性率（6.5%，16/247）高于 GRA7 基于 iELISA（3.6%，9/247）和商业 ELISA 试剂盒（4.9%，12/247）。
结论
本研究建立了一种具有良好特异性、敏感性和可重复性的 iELISA 方法，可用于临床猫样本中弓形虫感染的大规模检测。
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