Westcott J Y, Chang S, Balazy M, Stene D O, Pradelles P, Maclouf J, Voelkel N F, Murphy R C
Prostaglandins. 1986 Dec;32(6):857-73. doi: 10.1016/0090-6980(86)90095-x.
The recent availability of fast and sensitive radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures to measure icosanoids has led to utilization of these techniques by many investigators. A major concern has been that techniques based on immunoreactivity may lack specificity, in particular if complex biologic fluids or tissue extracts are evaluated. The purpose of this investigation was the comparison of icosanoid measurements obtained either with EIA or RIA with those obtained by gas chromatography/mass spectrometry (GC/MS). Rats were injected with Salmonella enteritidis endotoxin, killed at various times after the injection and the lung extract assayed for 6-keto-PGF1 alpha, 5-HETE and LTC4. By EIA lung tissue was found to contain large quantities of 6-keto-PGF1 alpha after endotoxin stimulation. Comparisons made between EIA and GC/MS analysis showed good correlation between 6-keto-PGF1 alpha amounts in lung as determined by each technique. It was also determined that little purification of lung extract was needed to obtain reliable quantitation of 6-keto-PGF1 alpha, probably due to the specificity of the antibody and the large quantity of this prostaglandin produced. Crudely purified (Sep-Pak) lung extracts gave 5-HETE levels by RIA which were highly correlated with GC/MS values, but RIA values were 70% higher than those obtained by GC/MS. The presence of other components in lung extract which cross react with this 5-HETE antibody was probably responsible for the higher values obtained by RIA. LTC4 was measured by immunoassay in crude lung extracts, as well as after Sep-Pak purification and HPLC purification. LTC4 levels were identical in unpurified lung extract and after Sep-Pak purification, but decreased substantially after HPLC purification. Thus, by validating the icosanoid immunoassays, we have found that they can give accurate and reproducible results in lung tissue, although LTC4 and 5-HETE must be purified prior to analysis.
近期可用于测量类二十烷酸的快速灵敏放射免疫分析（RIA）和酶免疫分析（EIA）方法，已促使许多研究人员采用这些技术。一个主要问题是基于免疫反应性的技术可能缺乏特异性，尤其是在评估复杂生物体液或组织提取物时。本研究的目的是比较用EIA或RIA获得的类二十烷酸测量值与通过气相色谱/质谱联用（GC/MS）获得的测量值。给大鼠注射肠炎沙门氏菌内毒素，在注射后的不同时间处死大鼠，并对肺提取物进行6-酮-前列腺素F1α、5-羟二十碳四烯酸（5-HETE）和白三烯C4（LTC4）的检测。通过EIA发现，内毒素刺激后肺组织含有大量的6-酮-前列腺素F1α。EIA和GC/MS分析之间的比较表明，两种技术测定的肺中6-酮-前列腺素F1α含量之间具有良好的相关性。还确定，获得6-酮-前列腺素F1α的可靠定量所需的肺提取物纯化很少，这可能是由于抗体的特异性以及所产生的这种前列腺素的大量存在。经粗纯化（Sep-Pak）的肺提取物通过RIA测得的5-HETE水平与GC/MS值高度相关，但RIA值比GC/MS获得的值高70%。肺提取物中与该5-HETE抗体发生交叉反应的其他成分的存在，可能是RIA获得较高值的原因。通过免疫分析在未纯化的肺提取物以及Sep-Pak纯化和高效液相色谱（HPLC）纯化后测量LTC4。未纯化的肺提取物和Sep-Pak纯化后的LTC4水平相同，但HPLC纯化后显著降低。因此，通过验证类二十烷酸免疫分析，我们发现它们在肺组织中可以给出准确且可重复的结果，尽管LTC4和5-HETE在分析前必须进行纯化。
