一种快速制备敏感鲎试剂的方法。1 .水蛭LAL和血淋巴的批间和批内差异评价。

M. Tvede, L. Baek
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引用次数: 6

摘要

本文描述了一种改进的制备鲎试剂(LAL)的方法,并与另外两种众所周知的方法进行了比较。在5例鲎试剂中,研究了三种不同的出血程序和三种不同的细胞破裂方法。通过在出血过程中避免使用抗凝剂n -乙基马来酰亚胺(NEM)、优化机械细胞破裂方法和使用无热原条件,LAL的敏感性得以提高。一般情况下,LAL与每毫升10(-15)克脂多糖(LPS)反应。为了探讨不同鲎试剂之间LAL反应性差异的可能解释,在一项实验中,我们对6只鲎试剂进行了放血,并测定了LAL和无细胞血淋巴中的pH和蛋白质、Ca2+、Mg2+、Cu2+、Na+和K+的浓度。LAL在批间和批内存在差异,但LAL的敏感性与LAL和无细胞血淋巴中上述成分的含量没有相关性。在出血过程中使用NEM和在匀浆细胞中加入不同浓度的NEM的实验表明,NEM抑制了LAL对LPS的反应性。结果表明,改进的无NEM出血法和最佳机械细胞破裂法生产LAL的方法快速、简便,并能生产出一种非常灵敏的鲎试剂。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
A rapid method to produce a sensitive Limulus amoebocyte lysate (LAL). I. Evaluation of inter and intra batch differences in LAL and hemolymph from Limulus polyphemus.
An improved method for preparing Limulus amoebocyte lysate (LAL) is described and compared with two other well-known procedures. In five Limuli, three different bleeding procedures and three different cell rupture methods were studied. Increased sensitivity of LAL was accomplished by avoidance of the anticoagulant N-ethylmaleimid (NEM) in the bleeding procedure, optimal methods for mechanical cell rupture and use of pyrogen free conditions. Reaction of LAL with as little as 10(-15) gram Lipopolysaccharide (LPS) per ml was generally obtained. To evaluate a possible explanation for the differences in reactivity of LAL between different Limuli, found in one experiment, six Limuli were bled, and the pH and the concentration of protein, Ca2+, Mg2+, Cu2+, Na+ and K+ were determined in LAL and cell-free hemolymph. Inter and intra batch variations were found in LAL, but there was no correlation between the sensitivity of LAL and the content of the above mentioned constituents in LAL and cell-free hemolymph. Experiments with use of NEM in the bleeding procedure and addition of NEM to homogenized cells in different concentrations, showed that NEM inhibits the reactivity of LAL to LPS. It is concluded that the modified method of producing LAL by bleeding without NEM and by using optimal methods for mechanical cell rupture is quick, simple and produces a very sensitive reagent for the Limulus test.
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