Reporter enzyme release technology: methods of assaying for the presence of aspartic proteases and other hydrolytic enzyme activities

摘要

Methods of assaying for the presence of enzymatically active hydrolases (i.e., hydrolytic enzymes) in a sample or specimen are disclosed. In particular, a method of detecting candidiasis by assaying for the presence of enzymatically active aspartic protease in a sample is provided. In these methods, a sample or specimen is contacted with a solid support. The solid support with which the sample is contacted has a reporter enzyme (i.e., a signal generating enzyme) immobilized thereon. The reporter enzyme is immobilized on the solid support in a manner such that it is released from the solid support upon action of the enzymatically active hydrolase if the enzymatically active hydrolase is, in fact, present in the sample. The sample after having been contacted with the solid support is combined with an indicator. The indicator is any chemical species which is susceptible to a detectable change, usually a change in color, upon action of the reporter enzyme. A detectable change in the indicator is an indication that the enzymatically active hydrolase is present in the sample. Moreover, the presence of an enzymatically active hydrolase in a sample may indicate the presence of a particular pathogen or disease state such as, for example, candidiasis.;In addition to the methods of assaying for the presence of enzymatically active aspartic protease and other hydrolytic enzymes, a dry, self-contained test device for assaying for the presence of an enzymatically active hydrolase in a sample is disclosed. In particular, a dry, self-contained test device for testing a sample for the presence of candidiasis by assaying for the presence of enzymatically active aspartic protease is also disclosed. These test devices combine a reporter enzyme immobilized on a solid support, an indicator, and all other reagents and components necessary to achieve a detectable indication of the presence or absence of the enzymatically active hydrolase whose presence is being detected in the sample, and preferred embodiments contain positive and negative controls as well.