State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
Biosens Bioelectron. 2012 May 15;35(1):390-393. doi: 10.1016/j.bios.2012.03.027. Epub 2012 Mar 30.
In this paper, a simple, easily-operated and enzyme-amplified fluorescence immunoassay method using magnetic particles for the detection of antibody against Actinobacillus pleuropneumoniae (APP) has been presented. The A protein of APP Repeats-in-Toxin IV (ApxIVA) with high specificity to the APP species was immobilized onto the magnetic bead surfaces. Horseradish peroxidase (HRP), which can catalyze the substrate 4-hydroxyphenylacetic acid (p-HPA), generating fluorescent bi-p, p'-hydroxyphenylacetic acid (DBDA), was selected as an enzymatic-amplified tracer. The ApxIVA antibody was detected for the presence of APP infection by measuring the fluorescence intensity of DBDA. Under optimal conditions, the calibration plot obtained for standard positive serum was approximately linear within the dilution range 1:160-1:5120. The limit of detection (LOD) for the assay was 1:10240, considerably lower than that of ApxIVA-ELISA (1:320) (S/N=3). A series of repeatability measurements of using 1:320-fold diluted standard positive serum gave reproducible results with a relative standard deviation (RSD) of 4.8% (n=11). The ability of the immunosensor to analyze clinical samples was tested on porcine sera. The immunosensor yielded an efficiency of 89.7%, sensitivity of 90.9% and specificity of 89.3% compared with ApxIVA-ELISA.
本文提出了一种简单、易操作且具有酶放大功能的基于磁性粒子的荧光免疫分析方法，用于检测抗胸膜肺炎放线杆菌（APP）的抗体。APP 重复毒素 IV（ApxIVA）的 A 蛋白具有很高的 APP 种特异性，被固定在磁性珠表面。辣根过氧化物酶（HRP）可以催化 4-羟苯基乙酸（p-HPA）作为酶放大示踪物，生成荧光双 p-羟苯基乙酸（DBDA）。通过测量 DBDA 的荧光强度，可以检测出 ApxIVA 抗体是否存在 APP 感染。在最佳条件下，标准阳性血清的校准曲线在 1:160-1:5120 的稀释范围内呈近似线性。该测定的检测限（LOD）为 1:10240，明显低于 ApxIVA-ELISA（1:320）（S/N=3）的检测限。使用 1:320 倍稀释的标准阳性血清进行一系列重复性测量，得到了具有 4.8%（n=11）相对标准偏差（RSD）的可重复结果。该免疫传感器分析临床样本的能力在猪血清上进行了测试。与 ApxIVA-ELISA 相比，该免疫传感器的效率为 89.7%，灵敏度为 90.9%，特异性为 89.3%。
