Mattsby-Baltzer I, Lindgren K, Lindholm B, Edebo L
Department of Clinical Bacteriology, University of Göteborg, Sweden.
Infect Immun. 1991 Feb;59(2):689-95. doi: 10.1128/iai.59.2.689-695.1991.
The endotoxin activities of gram-negative bacteria and their lipopolysaccharides (LPS) have been quantitated by a chromogenic Limulus amocbocyte lysate (CLAL) assay. When bacterial cell exposing various cell surface structures were compared, the highest Limulus activities were found in R strains of Escherichia coli and Salmonella typhimurium mutants. E. coli with K antigens did not differ from K-negative strains. By measuring beta-hydroxymyristic acid (3-OH tetradecanoic acid, beta-OHC14:0), it was possible to compare the CLAL activities of LPS bound to bacterial cells, LPS shed into the culture medium, and purified LPS. After 16 h of growth, the cell-free culture supernatants of three E. coli O1K1 strains and S. typhimurium showed CLAL activities 14.3 to 20.3 times higher than did the corresponding bacterial cell suspensions in relation to their beta-OHC14:0 contents. Four other E. coli strains (O serotypes O14, O24, and O75) and the S. typhimurium 395 R mutants MR5 and MR6 showed CLAL values 2.8 to 7.9 times higher in their culture supernatants. LPS of E. coli O1K1 and S. typhimurium had lower CLAL activities than the culture supernatants (1/10 and 1/4, respectively). Although the beta-OHC14:0 concentrations of the culture supernatants were approximately half those of the corresponding bacterial cells, all had CLAL values that were 2 to 21 times higher. The bacterial cell suspension, culture supernatant, and purified LPS of S. typhimurium MS were compared by CLAL assay and a quantitative enzyme-linked immunosorbent assay based on monoclonal antibodies to the O5 antigen. Endotoxin shed into the culture medium was the most CLAL-active form of LPS, while purified LPS was the most antigen-active form. The results emphasize the importance of appropriate standards when quantifying endotoxin in various states. In conclusion, E. coli and S. typhimurium bacteria shed significant amounts of endotoxin into the surrounding medium during growth. This form of LPS is more CLAL active than the cell-bound or purified LPS.
革兰氏阴性菌及其脂多糖（LPS）的内毒素活性已通过显色鲎试剂法（CLAL）进行定量。当比较暴露于各种细胞表面结构的细菌细胞时，发现大肠杆菌的R菌株和鼠伤寒沙门氏菌突变体具有最高的鲎试剂活性。带有K抗原的大肠杆菌与K阴性菌株没有差异。通过测量β-羟基肉豆蔻酸（3-羟基十四烷酸，β-OHC14:0），可以比较与细菌细胞结合的LPS、释放到培养基中的LPS和纯化的LPS的CLAL活性。生长16小时后，三种大肠杆菌O1K1菌株和鼠伤寒沙门氏菌的无细胞培养上清液的CLAL活性比相应细菌细胞悬液的CLAL活性高14.3至20.3倍，这与它们的β-OHC14:0含量有关。其他四种大肠杆菌菌株（O血清型O14、O24和O75）以及鼠伤寒沙门氏菌395 R突变体MR5和MR6的培养上清液的CLAL值高2.8至7.9倍。大肠杆菌O1K1和鼠伤寒沙门氏菌的LPS的CLAL活性低于培养上清液（分别为1/10和1/4）。尽管培养上清液的β-OHC14:0浓度约为相应细菌细胞的一半，但所有培养上清液的CLAL值都高2至21倍。通过CLAL测定法和基于针对O5抗原的单克隆抗体的定量酶联免疫吸附测定法，对鼠伤寒沙门氏菌MS的细菌细胞悬液、培养上清液和纯化的LPS进行了比较。释放到培养基中的内毒素是LPS中CLAL活性最高的形式，而纯化的LPS是抗原活性最高的形式。结果强调了在定量各种状态下的内毒素时使用适当标准的重要性。总之，大肠杆菌和鼠伤寒沙门氏菌在生长过程中会向周围培养基中释放大量内毒素。这种形式的LPS比细胞结合型或纯化型LPS具有更高的CLAL活性。
