Motsenbocker M A
Miles Diagnostics, Elkhart, IN 46515.
J Biolumin Chemilumin. 1988 Jan-Mar;2(1):9-16. doi: 10.1002/bio.1170020104.
Conditions for the enhanced horseradish peroxidase (HRP) catalysed reaction between luminol and hydrogen peroxide were optimized to determine detection limits for HRP conjugated to antibody fragment (HRP-Fab) in solution phase. Light output was linear with respect to HRP-Fab concentration but became nonlinear at low HRP-Fab concentrations when an accelerator (enhancer) of the reaction was used. para-Phenylphenol was a more effective enhancer than p-iodophenol at HRP-Fab concentrations below 20 pmol/l. The detection limit for HRP-Fab was 1.2 femtomoles in the absence of p-phenylphenol and 0.08 femtomole in the presence of p-phenylphenol. The acceleration of peroxidase activity at the lowest HRP-Fab concentrations occurred after an incubation time period of up to five minutes. This lag time limited the sensitivity and the mechanism for it was sought. Preincubation experiment results indicated that the lag time phenomenon may involve a reversible alteration in HRP catalytic activity and that enhancer, peroxide, luminol and HRP-Fab had to be incubated together some time before maximum activation could occur.
优化了增强型辣根过氧化物酶（HRP）催化鲁米诺与过氧化氢之间反应的条件，以确定溶液相中与抗体片段偶联的HRP（HRP-Fab）的检测限。发光量与HRP-Fab浓度呈线性关系，但当使用反应促进剂（增强剂）时，在低HRP-Fab浓度下会变为非线性。在HRP-Fab浓度低于20 pmol/l时，对苯二酚是比对碘苯酚更有效的增强剂。在不存在对苯二酚的情况下，HRP-Fab的检测限为1.2飞摩尔，在存在对苯二酚的情况下为0.08飞摩尔。在最低HRP-Fab浓度下，过氧化物酶活性的加速在长达五分钟的孵育时间后出现。这个延迟时间限制了灵敏度，并对其机制进行了探索。预孵育实验结果表明，延迟时间现象可能涉及HRP催化活性的可逆改变，并且增强剂、过氧化物、鲁米诺和HRP-Fab必须一起孵育一段时间才能达到最大激活。
