ASSAY OF PEROXIDASE ACTIVITY

摘要

Chemiluminescent detection of molecules of synthetic or natural origin such as proteins and nucleic acids (DNA and RNA), as well as other biologic molecules, is increasingly replacing radioactive detection as the method of choice where sensitivity is critical. In such assays, luminescence is customarily achieved by the oxidation of a luminol or isoluminol substrate in the presence of an oxidizing agent such as hydrogen peroxide or hydrogen peroxide source, such as perborate, and a peroxidase catalyst such as horseradish peroxidase. To obtain useful levels of luminescence (e.g., detectable levels) by customary techniques, a luminescent enhancer is also present during oxidation. It has been found in the practice of the present invention that azine enhancers have contained an impurity which reduces the properties of the chemiluminescent assay working solutions. The present invention describes a working solution, a process of using the working solution, and kits containing solutions which can be mixed to form the working solutions comprising a solution which is useful for the chemiluminescent assay of peroxidase activity comprising: a) at least one chemiluminescent cyclic diacylhydrazide, b) at least one azine enhancer, and c) at least one oxidizing agent wherein said azine enhancer comprises less than 0.005 parts mole/mole total basis of azine compounds having a hydrogen atom bonded to a nitrogen atom of the azine ring. It has generally been found that this hydrogen containing azine is a poisoning agent for the performance of the azine enhancer.