Employment of a phenoxy-substituted acridinium ester as a long-lived chemiluminescent indicator of glucose oxidase activity and its application in an alkaline phosphatase amplification cascade immunoassay.

This paper describes the employment of a novel phenoxy-substituted acridinium ester (di-ortho-bromophenyl-AE) as a chemiluminescent endpoint indicator for ligand binding assays. The reactivity of this compound is such that it is capable of generating a high-intensity chemiluminescent signal at neutral pH. Under these conditions, when present in excess, it has been used as an indicator of hydrogen peroxide generation by the action of glucose oxidase (GOx, EC 1.1.3.4) on glucose substrate. The resulting chemiluminescent signal is a long-lived glow. The magnitude of the chemiluminescent signal is directly proportional to the quantity of GOx present and has been used to measure GOx with a sensitivity of 1.8 x 10(-16) mol. In addition, this ability to monitor GOx activity has been utilized in an alkaline phosphatase (ALP, EC 3.1.3.1) amplification cascade assay. Here ALP catalyzes the formation of FAD from a prosthetogenic substrate FADP. FAD, a cofactor for a number of oxidase enzymes, then converts inactive apo-GOx to holo-GOx, the activity of which is monitored by the chemiluminescent endpoint and facilitates detection of ALP over the range 10(-15) to 4.1 x 10(-19) mol. The clinical utility of this system has been demonstrated by application to the assay of human thyrotrophin (TSH, sensitivity 0.005 mU/liter).