A COMPARATIVE-STUDY OF THE ACCURATE MEASUREMENT OF ENDOTOXIN IN LIPOSOME-ENCAPSULATED HEMOGLOBIN
A COMPARATIVE-STUDY OF THE ACCURATE MEASUREMENT OF ENDOTOXIN IN LIPOSOME-ENCAPSULATED HEMOGLOBIN
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DOI：
10.3109/10731199509117949
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发表时间：
1995-01-01
影响因子：
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通讯作者：
RUDOLPH, AS
中科院分区：
文献类型：
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作者：
CLIFF, RO;KWASIBORSKI, V;RUDOLPH, AS
我们已经检查了三种不同的内毒素测定方法，即利用鲎试剂（LAL）测定法准确测定脂质体包封血红蛋白（莱赫）中的内毒素水平，1）凝胶法，2）基于显色光谱的LAL，3）浊度法，该方法根据达到特定浊度所需的时间测定溶液中的内毒素水平。显色法和浊度法都需要对莱赫制剂进行显著稀释，然后才能进行准确测量。我们使用这些方法检测了莱赫溶液中的内毒素水平，并测量了加标已知量内毒素的莱赫、脂质体和血红蛋白样品。三种方法的比较表明，三种方法测定的莱赫中内毒素的绝对值可能存在显著差异。然而，在任何一项试验中，均可准确测定样品中内毒素的加标量。内毒素与莱赫的结合也可能使这些方法的准确性变得复杂。将游离内毒素与莱赫混合，然后离心分离莱赫，这一点很明显。通过暴露于RAW 264.7随后表达肿瘤坏死因子来测量与莱赫结合的内毒素的生物活性。
We have examined three different methods of endotoxin determination utilizing the Limulus Amebocyte Lysate (LAL) assay to accurately determine endotoxin levels in Liposome Encapsulated Hemoglobin (LEH), 1) the gel-clot method, 2) chromogenic spectroscopic-based LAL, and 3) the turbidimetric method which determines endotoxin levels in solutions based on the time needed to reach a specific degree of turbidity. Both the chromogenic and turbidimetric methods require significant dilution of the LEH preparation before accurate measurement can be made. We have tested the levels of endotoxin in LEH solutions using these methods and measured LEH, liposome, and hemoglobin samples spiked with known amounts of endotoxin. A comparison of the three methods shows that the absolute value of endotoxin measured in LEH by the three methods can vary significantly. However, within any one assay the spiked amount of endotoxin in the sample can be accurately measured. The accuracy of these methods may also be complicated by the binding of endotoxin to LEH. This was evident by mixing free endotoxin with LEH followed by centrifugation to separate the LEH. Biological activity of endotoxin bound to LEH was measured by exposure to RAW264.7 followed by the expression of tumor necrosis factor.