TURBIDIMETRIC METHOD FOR QUANTIFYING SERUM INHIBITION OF LIMULUS AMEBOCYTE LYSATE
TURBIDIMETRIC METHOD FOR QUANTIFYING SERUM INHIBITION OF LIMULUS AMEBOCYTE LYSATE
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DOI：
10.1128/jcm.21.2.211-216.1985
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发表时间：
1985-01-01
影响因子：
9.4
通讯作者：
WARREN, HS
中科院分区：
文献类型：
--
作者：
NOVITSKY, TJ;ROSLANSKY, PF;WARREN, HS
本研究描述了一种通过比浊法鲎变形细胞裂解物测定来量化血清对脂多糖（LPS）活性的抑制的方法。测定在多孔微孔板中进行，并使用微孔板分光光度计测量 380 nm 处的光密度作为浊度。 LPS效力测量为用盐水稀释的LPS的50％最大鲎变形细胞反应(LR50)。通过比较盐水中的 LR50，根据美国参考标准内毒素 EC-5 批次对不同种类细菌的 LPS 进行标准化。发现大肠杆菌 O113 和 O18 以及粘质沙雷氏菌 LPS 的效价与参考标准 EC-5 的效价相同；来自 2 种沙门氏菌的 LPS 效力只有一半。从肺炎克雷伯菌和大肠杆菌粗突变体 J5 中获得的测试效力最弱的 LPS 分别比 EC-5 低 5 倍和 10 倍。作为抑制的量度，将血清中 LPS 的 LR50 与盐水中 LPS 的 LR50 进行比较。与盐水相比，血清抑制 LPS 的效力达 103 至 6400 倍。在盐水中的标准化效力与所测试的各种LPS的血清抑制之间发现正相关性。因此，来自大肠杆菌O113、O18和EC-5以及粘质沙门氏菌的LPS在盐水中表现出最高效力，其被血清抑制最多。同样，测试中效力最弱的大肠杆菌 J5 和肺炎克雷伯菌 LPS 受到的抑制也最少。所测试的所有类型的LPS的抑制程度随着血清浓度的增加而增加。
This study describes a method to quantify the inhibition of lipopolysaccharide (LPS) activity by serum with a turbidimetric Limulus amoebocyte lysate assay. Assays were performed in multiwell microplates, and turbidity was measured as the optical density at 380 nm with a microplate spectrophotometer. LPS potency was measured as the 50% maximal Limulus amoebocyte response (LR50) of LPS diluted with saline. By comparing LR50 in saline, LPS from various species of bacteria were standardized against the U.S. Reference Standard Endotoxin, lot EC-5. The potency of Escherichia coli O113 and O18 and Serratia marcescens LPS was found to be equal to that of the reference standard EC-5; LPS from 2 salmonella species were half as potent. The least potent LPS tested, obtained from Klebsiella pneumoniae and E. coli rough mutant J5, were 5- and 10-fold less potent, respectively, than EC-5. As a measure of inhibition, the LR50 of LPS in serum was compared to the LR50 of LPS in saline. Serum inhibited the potency of LPS 103- to 6400-fold compared with saline. A positive correlation was found between standardized potency in saline and serum inhibition of the various LPS tested. Thus, LPS from E. coli O113, O18 and EC-5 and S. marcescens, which exhibited the highest potency in saline, which inhibited the most by serum. Likewise, E. coli J5 and K. pneumoniae LPS, which were the least potent tested, were the least inhibited. The degree of inhibition of all types of LPS tested increased with increasing serum concentration.