Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5
Optimization and standardization of an enzyme-linked immunosorbent assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5
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胸膜肺炎放线杆菌血清型 5 血清诊断酶联免疫吸附测定方案的优化和标准化
DOI：
10.1128/jcm.30.1.46-53.1992
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发表时间：
1992
影响因子：
9.4
通讯作者：
S. Larivière
中科院分区：
文献类型：
--
作者：
Y. Trottier;P. Wright;S. Larivière
一个间接酶联免疫吸附测定协议已被优化，特别强调给予测定标准化和质量控制。对微孔板的选择、抗原固定、缓冲液组成、血清的最佳筛选稀释度和酶促反应动力学等技术方面进行了研究和评价，以设计提供最大分析灵敏度和特异性的标准方案，并获得最小的板内和板间变异性。在检测的27个平板中，发现Nunc 475-094和269-620免疫平板在高阳性-阴性比和低变异性方面最好。通过使用各种组合物或pH的缓冲液，发现抗原固定无显著差异;然而，镁离子（Mg 2 +; 0.02 M）的存在导致非特异性背景增加两倍。确定血清的最佳筛选稀释度为1：200。发现供试血清的1小时孵育期最佳。通过将底物（H2 O2）和氢供体[2，2 ' -连氮双（3-乙基苯并噻唑啉磺酸）]的浓度分别调节至4和1 mM，获得过氧化物酶的最大酶活性。为了控制板间变异性，采用了计时方案。还通过使用样品放置配置模式控制板内变异性。通过重复检测样品的横截面来确定滑动标尺，以设定板内和板间变异性的验收限度。这些限度用于质量控制程序中，以监测测定性能。所得结果表明，该标准化方案可能是有用的血清诊断胸膜肺炎放线杆菌血清5型。
An indirect enzyme-linked immunosorbent assay protocol has been optimized with special emphasis given to assay standardization and quality control. Technical aspects such as choice of a microplate, antigen immobilization, buffer composition, optimal screening dilution of sera, and kinetics of the enzymatic reaction were studied and evaluated in order to design a standard protocol offering maximal analytical sensitivity and specificity, as well as to obtain minimal within- and between-plate variability. Among the 27 plates tested, the Nunc 475-094 and 269-620 immunoplates were found to be the best in terms of high positive-to-negative ratio and low variability. No significant differences in antigen immobilization were found by using buffers of various compositions or pHs; however, the presence of magnesium ions (Mg2+; 0.02 M) resulted in a twofold increase in nonspecific background. An optimal screening dilution of sera was established at 1:200. A 1-h incubation period for test serum was found to be optimal. Maximum enzymatic activity for peroxidase was obtained by adjusting both substrate (H2O2) and hydrogen donor [2,2' -azinobis(3-ethylbenz-thiazoline sulfonic acid)] concentrations to 4 and 1 mM, respectively. To control between-plate variability, a timing protocol was adopted. Within-plate variability was also controlled by using a sample placement configuration pattern. Sliding scales were determined by repeated testing of a cross section of samples to set acceptance limits for both within- and between-plate variability. These limits were used in a quality control program to monitor assay performance. The results obtained suggest that this standardized protocol might be useful in the serodiagnosis of Actinobacillus pleuropneumoniae serotype 5.
镁在粗大肠杆菌 J5 菌株和淋病奈瑟菌脂多糖酶联免疫吸附测定中的作用。
DOI：
10.1093/infdis/142.4.532
发表时间：
1980
期刊：
The Journal of infectious diseases
影响因子：
--
作者：
ItoJr,JI;Wunderlich,AC;Lyons,J;Davis,CE;Guiney,DG;Braude,AI
通讯作者：
Braude,AI