水体环境内毒素活性的鲎法定量检测及影响因素研究

内毒素是革兰氏阴性菌和某些蓝藻细胞壁上的脂多糖复合物,是常见的外源性致热原,具有强免疫刺激能力,与人类多种疾病密切相关.本研究采用最低检测限为0.03 EU.mL-1的动态浊度东方鲎(Tachypleus Amebocyte Lysate,TAL)试剂,对不同水体环境样品的内毒素活性定量检测,分析了pH值和无机盐对TAL试验检测结果的影响.结果表明pH值在6.0～8.4范围内适合进行内毒素检测,过酸条件引起检测结果的抑制,过碱条件引起检测结果的增强,检测前可用无热原Tris-HCl(pH=7.4)溶液调节pH.当NaCl、Na2SO4、CaCl2、MgCl2和KCl浓度小于50 mg.L-1时不会干扰内毒素的检测,当浓度升高至1 000～10 000 mg.L-1时引起抑制效果.浓度为2.5 mg.L-1的FeCl3、Fe2(SO4)3、AlCl3和Al2(SO4)3即可以引起强烈抑制效果,内毒素活性的实测值已经严重偏离实际值.采用动态浊度TAL试验检测超纯水、自来水和景观用水的内毒素活性,结果表明所检测的超纯水的内毒素活性<0.06 EU.mL-1,自来水的内毒素活性为0.46 EU.mL-1,景观用水的内毒素活性为432.68EU·mL-1.

Detection of Endotoxin Activity in Water Environment and Analysis of Influence Factors for TAL Assay

Endotoxins, derived from cell walls of most Gram-negative bacteria and some cyanobacteria,are common pyrogen and highly immunogenic molecules, and related to many diseases. In this paper, a detection method for endotoxin activity in water environment using kinetic-turbid assay of Tachypleus Amebocyte Lysate (TAL) was established, the influence of pH and salts on TAL assay was investigated. Results showed that it was favorable for TAL assay in the pH range of 6.0-8.4, at low pHs, inhibition results were observed and opposite results were obtained at high pHs. The pH should be adjusted by Tris-HCl(pH=7.4)buffer before the endotoxin detection. No significant interference was shown in the detection of water containing NaCl, Na₂SO₄, CaCl₂, MgCl₂ and KCl with a concentration of less than 50 mg ·L^-1, however, the inhibition occurred at the concentration up to 1000-10000 mg ·L^-1. Only 2.5 mg ·L^-1 of FeCl₃, Fe₂(SO₄)₃, AlCl₃ and Al₂(SO₄)₃ caused significant inhibition. Endotoxin activities of ultrapure water, tap water and recreational water were detected by TAL assay, and their endotoxin activities were<0.06 EU ·mL^-1, 0.46 EU ·mL^-1 and 432.68 EU ·mL^-1, respectively.