Peroxidase-amplified assay of sialidase activity toward gangliosides.
Peroxidase-amplified assay of sialidase activity toward gangliosides.
复制标题
过氧化物酶放大唾液酸酶对神经节苷脂的活性测定。
DOI：
10.1016/0003-2697(92)90276-d
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发表时间：
1992
影响因子：
2.9
通讯作者：
Sweeley,CC
中科院分区：
文献类型：
--
作者：
Ogura,K;Ogura,M;Anderson,RL;Sweeley,CC
使用涂在酶免疫测定板孔上的底物神经节苷脂 GD1a 进行唾液酸酶测定。将GD1a与来自霍乱弧菌的唾液酸酶一起孵育后，如下测量产生的神经节苷脂GM1的量：添加与辣根过氧化物酶缀合的霍乱毒素B亚基以特异性结合GM1，然后在比色酶测定中测定结合的过氧化物酶的量。在不存在去污剂的情况下，GM1检测的线性为每孔0至0.5 pmol，并且唾液酸酶检测的灵敏度为每分钟约3 fmol形成的产物。在测定中添加去垢剂 (Triton CF-54) 会降低灵敏度并增加所需底物的量。描述了该测定在检测人皮肤成纤维细胞条件培养基中细胞源性中性（pH 6.5）唾液酸酶活性的应用。
Sialidase assays were carried out with the substrate, ganglioside GD1a, coated onto enzyme immunoassay plate wells. Following the incubation of GD1awith sialidase from V. cholerae, the amount of ganglioside GM1produced was measured as follows: cholera toxin B subunit conjugated to horseradish peroxidase was added to specifically bind to GM1, and then the amount of bound peroxidase was determined in a colorimetric enzymatic assay. In the absence of detergent, linearity for the detection of GM1was 0 to 0.5 pmol per well, and the sensitivity for sialidase detection was about 3 fmol of product formed per minute. The addition of detergent (Triton CF-54) to the assay reduced the sensitivity and increased the amount of substrate required. Application of this assay for the detection of cell-derived neutral (pH 6.5) sialidase activities in the conditioned medium of human skin fibroblasts is described.