Pai Wan Chain, Shien Jui Huang, Liu Hung Jen, Lee Long Huw
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung 403, Taiwan.
Vet Microbiol. 2003 Feb 25;91(4):309-23. doi: 10.1016/s0378-1135(02)00308-5.
Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.
制备了针对在大肠杆菌中合成的禽呼肠孤病毒S1133蛋白σA（eσA）的单克隆抗体（MAb）。对单克隆抗体进行了表征并用于开发诊断测试。在与辣根过氧化物酶偶联后，选择了十种单克隆抗体进行竞争性结合试验。结果表明，这些单克隆抗体确定了eσA的两个表位I和II。使用免疫斑点结合试验来检测变性对表位抗体识别的影响。所有单克隆抗体均与天然形式的eσA结合。在SDS和2-巯基乙醇中煮沸变性后，识别表位I的单克隆抗体的结合完全消除。然而，识别表位II的单克隆抗体的反应性未受影响。分别识别表位I和II的单克隆抗体31和32被选择用于对异源呼肠孤病毒株的交叉反应性研究。结果表明，这两个表位在这些病毒株中高度保守。使用单克隆抗体32和31开发了一种单克隆抗体捕获酶联免疫吸附测定（ELISA）程序，以检测感染禽类的肌腱组织样本和鸡胚成纤维细胞（CEF）细胞培养物中的呼肠孤病毒蛋白σA。接种禽类后最早在接种后2天（PI）就从肌腱标本中检测到禽呼肠孤病毒σA抗原，在接种后7天达到峰值，并一直维持到接种后16天，然后逐渐下降。在禽呼肠孤病毒感染和模拟感染禽类的肌腱样本之间获得了明显的吸光度值差异。在禽呼肠孤病毒感染的CEF和自然感染肉鸡的肌腱组织中也获得了阳性结果。这些结果表明，单克隆抗体捕获ELISA是检测疑似感染禽呼肠孤病毒的鸡中禽呼肠孤病毒的有用方法。
