Environmental endotoxin measurement: interference and sources of variation in the Limulus assay of house dust.
Environmental endotoxin measurement: interference and sources of variation in the Limulus assay of house dust.
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环境内毒素测量：屋尘鲎测定中的干扰和变异来源。
DOI：
10.1080/15428119791012199
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Park,JH
中科院分区：
文献类型：
--
作者：
Milton,DK;Johnson,DK;Park,JH
研究表明，办公室和家庭中的内毒素水平可能与病态建筑综合征或哮喘严重程度增加有关。然而，这些研究中报告的内毒素水平高度不一致，表明不同实验室的测量结果不一致。因此，对室内灰尘中内毒素的鲎试验进行了研究。对测定的干扰是常见的，并且可能产生内毒素估计值，其变化因子> 100，取决于所使用的稀释度。剂量-反应曲线分析允许检测两种类型的干扰：稀释依赖性和稀释无关性。当样品重新测定时，稀释依赖性干扰持续存在，但可通过适当稀释和数据分析获得有效估计值。无法从显示非稀释依赖性干扰的试验中获得有效估计值。然而，通过重复测定，经常克服非稀释依赖性干扰。当使用900的稀释因子时，基于单个样品稀释的估计值平均产生合理的结果。然而，单次稀释的结果（中位变异系数[CV] 40%）的精确度低于完整剂量-反应曲线（中位CV 23%），范围为基于完整剂量-反应数据的估计值的11%至240%。提取方法的比较发现，在磷酸盐-三乙胺缓冲液中超声处理产生的内毒素活性大于在添加皂苷或十二烷基硫酸钠的缓冲液中提取。在4和−20°C下储存的粉尘样品中，内毒素活性可稳定8至10周，但在提取物中不稳定。鲎试剂的批次和动态鲎试验中检测响应的方法对内毒素估计值有显著影响。
Studies suggest that endotoxin levels in offices and homes may be associated with sick building syndrome or increased asthma severity. However, reported endotoxin levels in these studies were highly inconsistent, suggesting incompatible measurements from various laboratories. Therefore, an investigation of Limulus assay for endotoxin in house dust was undertaken. Interference with the assay was common and could produce endotoxin estimates varying by a factor of > 100, depending on the dilution used. Analysis of dose-response curves allowed detection of two types of interference: dilution-dependent and dilution-independent. Dilution-dependent interference persisted when samples were reassayed, but valid estimates could be obtained by appropriate dilution and data analysis. Valid estimates could not be obtained from assays showing dilution-independent interference. However, dilution-independent interference was frequently overcome by repeating the assay. Estimates based on a single sample dilution produced reasonable results on average when a dilution factor of 900 was used. However, the results were less precise from a single dilution (median coefficient of variation [CV] 40%) than from the complete dose-response curve (median CV 23%) and ranged from 11 to 240% of the estimates based on complete dose-response data. A comparison of extraction methods found that sonication in a phosphate-triethylamine buffer produced greater endotoxin activity than extraction in buffer with addition of saponin or sodium dodecyl sulfate. Endotoxin activity was stable for 8 to 10 weeks in dust samples stored at 4 and −20°C but was not stable in extracts. The lot of Limulus amebocyte lysate reagent and method of detecting a response in the kinetic Limulus assay had significant effects on endotoxin estimates.