基于口疮病毒 F1L蛋白单克隆抗体的阻断 ELISA检测方法的建立与应用

Establishment and application of a blocking ELISA based on a monoclonal antibody against the F1L protein of Orf virus for antibody detection

摘要:

背景 传染性脓疱是由口疮病毒感染引起的一种人兽共患病。该病在世界各地羊群中广泛流行，严重影响了养羊业的发展。 目的 建立口疮病毒抗体阻断ELISA方法并对其进行评价。 方法 制备口疮病毒F1L蛋白单克隆抗体，将其偶联辣根过氧化物酶(horseradish peroxidase, HRP)。以杆状病毒表达系统制备的F1L蛋白为检测抗原，以HRP标记的F1L蛋白单克隆抗体为检测抗体，调整优化反应条件，建立口疮病毒抗体阻断ELISA检测方法。使用该方法与间接免疫荧光试验(immunofluorescence assay, IFA)对100份血清样品进行检测，检验该方法的适用性。 结果 HRP标记的F1L蛋白单克隆抗体效价高、性质稳定。F1L蛋白实现可溶性表达，纯化的F1L蛋白纯度高、反应性好。阻断ELISA的最佳反应条件：F1L蛋白的包被浓度和酶标抗体稀释度分别为1.0 μg/mL和1:2 000；临界值A和B分别为37.47%和28.40%，样品阻断率大于37.47%为阳性、小于28.40%为阴性、介于二者之间为可疑；批间变异系数不高于4.49%，批内变异系数不高于6.28%，重复性良好；对传染性脓疱阳性血清的最低检测限为1:128，敏感性比间接IFA高；该方法特异性良好，不与常见其他病原的抗体反应。临床样品检测显示，该方法与间接IFA符合率为97.87%。 结论 建立了基于F1L蛋白的抗体阻断ELISA检测方法，可用于口疮病毒抗体的检测。

Abstract:

Background Contagious ecthyma is a zoonotic disease caused by infection with the Orf virus (ORFV). It is prevalent in sheep and goat flocks worldwide and seriously impacts the development of the sheep and goat farming industry. Objective To establish and evaluate a blocking enzyme-linked immunosorbent assay (ELISA) for detecting ORFV antibodies. Methods A monoclonal antibody (mAb) against the ORFV F1L protein was prepared and conjugated with horseradish peroxidase (HRP). With the F1L protein expressed in a baculovirus system as the detection antigen and the HRP-conjugated F1L mAb as the detection antibody, a blocking ELISA for ORFV antibody detection was established after optimization of the reaction conditions. The applicability of this method was assessed by testing 100 serum samples in parallel with an indirect immunofluorescence assay (IFA). Results The HRP-labeled F1L mAb exhibited a high titer and stable properties. The F1L protein was successfully expressed in the soluble form, with the purified F1L protein showing high purity and good reactivity. The optimal conditions for the blocking ELISA were as follows: coating concentration of F1L protein at 1.0 μg/mL and dilution of the HRP-conjugated antibody at 1:2 000. The cut-off values A and B were determined as 37.47% and 28.40%, respectively. Samples with an inhibition ratio greater than 37.47%, less than 28.40%, and between the two values were considered positive, negative, and doubtful, respectively. The intra-assay coefficients of variation (CV) did not exceed 4.49%, and the intra-assay CV did not exceed 6.28%, indicating good repeatability. The blocking ELISA showed the limit of detection of 1:128 for ORFV-positive serum, demonstrating higher sensitivity than indirect IFA. The method showed good specificity, with no cross-reactivity with antibodies against other common pathogens. Clinical sample testing revealed a 97.87% concordance rate between this method and indirect IFA. Conclusion A blocking ELISA based on the F1L protein is successfully established and can be used for the detection of ORFV antibodies.