Estimation of serum antibody to subgingival species using checkerboard immunoblotting
Estimation of serum antibody to subgingival species using checkerboard immunoblotting
复制标题
DOI：
10.1111/j.1399-302x.1997.tb00395.x
复制
发表时间：
1997-10-01
影响因子：
--
通讯作者：
Taubman, MA
中科院分区：
文献类型：
--
作者：
Sakellari, D;Socransky, SS;Taubman, MA
测量针对龈下细菌种类的血清抗体已有助于区分可能的牙周病原体和评估宿主对龈下细菌种类的免疫反应。开发了一种免疫测定系统，用于测量单个硝酸纤维素膜上多个血清样本中多种龈下物种的血清抗体水平。该测定的主要步骤如下： I) 细菌制剂中的抗原与硝酸纤维素膜上平行泳道中的蛋白 A 结合； 2) 在与抗原泳道垂直的泳道中孵育已知浓度的人免疫球蛋白以及来自患者的各种稀释度的血清； 3) 将膜与过氧化物酶缀合的第二抗体一起孵育； 4)增强化学发光检测阳性反应。发射的光被拍摄在照片上，其中阳性反应在抗原与适当抗体的交叉点处显示为正方形。使用激光密度计对抗体进行定量，以将未知样品的信号强度与同一膜上捕获的已知量的人免疫球蛋白产生的信号强度进行比较。该测定允许同时筛选和/或定量多达 45 个血清样本中针对多达 45 种细菌的抗体。一次测定中重复变异系数的平均值和标准误差平均为 7.3 +/- 2.3%。在不同日期运行的测定针对一系列龈下物种的血清抗体的变异系数平均为 10.1 +/- 2.1%。棋盘免疫印迹是一种简单而快速的免疫测定法，可对多种血清样本中多种龈下物种或抗原的抗体进行定量和/或筛选。
Measurement of serum antibody to subgingival bacterial species has been useful in discriminating possible periodontal pathogens and in assessing the host's immune response to subgingival species. An immunoassay system was developed to measure the level of serum antibody to multiple subgingival species in multiple serum samples on a single nitrocellulose membrane. The principle steps of the assay are the following: I) binding of antigens from bacterial preparations and protein A in parallel lanes on nitrocellulose membranes; 2) incubation of known concentrations of human immunoglobulin as well as various dilutions of serum from patients in lanes perpendicular to the antigen lanes; 3) incubation of the membrane with a peroxidase-conjugated second antibody; 4) detection of positive reactions by enhanced chemiluminescence. Emitted light was captured on a photographic him in which the positive reactions appeared as squares at the intersections of antigens with appropriate antibody. Antibody was quantified using a laser densitometer to compare the signal intensity of unknown samples with the ones generated by known amounts of human immunoglobulin captured on the same membrane. The assay permitted simultaneous screening and/or quantification of antibody in as many as 45 serum samples against up to 45 bacterial species. The mean and standard error of the coefficients of variation for replicates within an assay averaged 7.3 +/- 2.3%. Coefficients of Variation of the assay run on different days for serum antibody to a range of subgingival species averaged 10.1 +/- 2.1%. Checkerboard immunoblotting is a simple and rapid immunoassay to permit quantification and/or screening of antibody to multiple subgingival species or antigens in multiple serum samples.