Klipstein F A, Lee C S, Engert R F
Infect Immun. 1976 Oct;14(4):1004-10. doi: 10.1128/iai.14.4.1004-1010.1976.
Assay of Escherichia coli enterotoxins by in vitro perfusion in rats was evaluated by examining the effects of variously prepared fractions of heat-labile (LT) and heat-stable (ST) toxins on water transport in this system. The assay was found to respond equally well, in a dose-related manner, to both LT and ST; it was sufficiently sensitive to detect the toxigenic effect of concentrations as small as 1 ng/ml. With the assay, it was found that LT is produced in cultures grown under aerobic, but not anaerobic, conditions; in contrast, ST is elaborated in stationary aerobic and anaerobic broth cultures but not in those grown under agitated aerobic conditions. Both toxins can be precipitated by either ammonium sulfate or acetone. The two toxin forms were completely separated from each other by sequential ultrafiltration. LT alone (thermolabile after exposure to 100 degrees C for 30 min) was retained by a PM-30 membrane, and ST alone was present in UM-2 retentates; ST was retained more effectively by a UM-05 membrane, with a 1,000-fold increase in activity over that of the UM-2 retentate. Washed ultrafiltration retentates containing either LT or ST derived from the proper culture conditions all induced water secretion at concentrations of 100 ng or less per ml. These results indicate that in vivo perfusion in rats is a sensitive, duplicable assay for both the LT and ST forms of E. coli enterotoxin.
通过检测热不稳定毒素（LT）和热稳定毒素（ST）的不同制备组分对大鼠体外灌注系统中水转运的影响，对大肠杆菌肠毒素的体外灌注检测方法进行了评估。结果发现，该检测方法对LT和ST均有良好的剂量相关反应；其灵敏度足以检测低至1 ng/ml浓度的产毒效应。通过该检测方法发现，LT是在需氧而非厌氧条件下培养的细菌中产生的；相反，ST在需氧和厌氧的静止肉汤培养物中产生，但在需氧搅拌培养条件下不产生。两种毒素均可被硫酸铵或丙酮沉淀。通过连续超滤可将两种毒素形式完全分离。仅LT（暴露于100℃ 30分钟后不耐热）被PM - 30膜截留，而仅ST存在于UM - 2截留物中；ST被UM - 05膜更有效地截留，其活性比UM - 2截留物提高了1000倍。含有源自适当培养条件的LT或ST的洗涤超滤截留物在浓度为每毫升100 ng或更低时均能诱导水分泌。这些结果表明，大鼠体内灌注是检测大肠杆菌肠毒素LT和ST形式的一种灵敏、可重复的检测方法。
