Roth R I, Levin J, Behr S
Department of Laboratory Medicine, University of California School of Medicine, San Francisco.
J Lab Clin Med. 1989 Sep;114(3):306-11.
We have developed a simple modification of the chromogenic Limulus amebocyte lysate test that increases the sensitivity for the detection of bacterial endotoxins. In this assay, free paranitroaniline, cleaved from synthetic chromogenic substrates by proteases that were generated by Limulus lysate after incubation with endotoxin, was then derived. Derivation was with p-dimethylaminocinnamaldehyde in the presence of strong acid, forming a stable Schiff base end product with much greater molar absorbancy than the parent chromogen. Conditions (times and temperatures of incubations, concentrations of reagents) for the augmented chromogenic procedure were optimized. A ten-fold or greater increase in sensitivity for bacterial endotoxin was obtained with the modified assay as compared with the standard chromogenic Limulus test, with unequivocal detection of endotoxin concentrations of less than 100 pg/ml. The greater sensitivity of this modified Limulus test increases its usefulness for a wide range of research applications and clinical investigations.
我们对显色鲎试剂检测法进行了一项简单改进，提高了检测细菌内毒素的灵敏度。在该检测中，将鲎试剂与内毒素孵育后产生的蛋白酶从合成显色底物上裂解下来的游离对硝基苯胺进行衍生化处理。衍生化是在强酸存在下用对二甲氨基肉桂醛进行的，形成一种稳定的席夫碱终产物，其摩尔吸光度比母体色原大得多。对增强显色程序的条件（孵育时间和温度、试剂浓度）进行了优化。与标准显色鲎试剂检测法相比，改进后的检测法对细菌内毒素的灵敏度提高了10倍或更多，能够明确检测出浓度低于100 pg/ml的内毒素。这种改进后的鲎试剂检测法更高的灵敏度增加了其在广泛的研究应用和临床研究中的实用性。
