碱性磷酸酶在碱性环境中能够催化磷酸苯二钠生成游离酚，进一步与4-氨基安替比林和铁氰化钾反应生成红色亚醌衍生物，产物在510 nm处具有特征吸收峰，通过吸光值变化即可表征碱性磷酸酶的活性。
标准物质： Phenol①反应过程中加入试剂三后必须立即充分混匀，否则会导致显色不完全；
②粗酶液制备完成后需置于冰上放置，且当天完成活性检测；
③若测定吸光值超出标准吸光值线性范围：高于最高值建议将粗酶液适当稀释后再进行测定，低于最低值建议适当增加样本量或延长反应时间后再进行测定，计算时相应修改；
④试剂三颜色应为黄色，变为蓝绿色则已失效应停止使用；
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