Method for determining activity of enzymes in metabolically active whole cells

A method of performing an assay for detecting the presence or absence of a disease which is characterized by an abnormal enzyme activity comprising:

• (a) contacting a test, metabolically active whole cell with a cell permeable water soluble assay compound or water soluble physiologically acceptable salt thereof having a fluorogenic indicator group and an unblocked leaving group, said leaving group being selected for cleavage by an enzyme the activity of which changes with the presence of the disease, said indicator group being in a non-fluorescent first state when bonded to said leaving group, and being in a fluorescent second state excitable at a wavelength above 450 nm when said leaving group is cleaved from said indicator group by said enzyme for a period of time at least sufficient for said assay compound to be transferred into said cell and for said leaving group to be cleaved inside said cell from said indicator group by said enzyme, wherein said assay compound has sufficient purity so that fluorescence of impurities in said assay compound is less than autofluorescence of the metabolically active whole cell;
• (b) sensing for said fluorescent second state of the indicator group for the test, metabolically active whole cell to produce test results;
• and
• (c) comparing the test results of said test metabolically active whole cell with reference results obtained from at least one of a diseased reference cell and a non-diseased reference cell thereby detecting the presence or absence of said disease in said test, metabolically active whole cell.

A method for detecting the presence or absence of an abnormality in the activity of an enzyme in a metabolically active whole cell, comprising:

• (a) contacting a plurality of reference, metabolically active whole cells, each having at least one normally functioning enzyme with a medium containing a cell permeable water soluble assay compound having the ability to pass through a cell membrane or a water soluble physiologically acceptable salt thereof having the ability to pass through a cell membrane, said assay compound comprising a fluorogenic indicator group and an unblocked leaving group, said leaving group being selected for cleavage by one of said at least one normally functioning enzyme, said indicator group being in a non-fluorescent first state when bonded to said leaving group, and being in a fluorescent second state excitable at a wavelength above 450 nm when said leaving group is cleaved from said indicator group by the one of said at least one normally functioning enzyme, for a period of time sufficient for said assay compound to be transferred into each of said plurality of reference, metabolically active whole cells for each of the at least one normally functioning enzyme, for each of said plurality of reference, metabolically active whole cells to produce a matrix of reference results and for said leaving group to be cleaved inside of each of said plurality of reference, metabolically active whole cells from said indicator group by the one of said at least one normally functioning enzyme, wherein said assay compound has sufficient purity so that fluorescence of impurities in said assay compound is less than autofluorescence of the metabolically active whole cell;
• (b) sensing for said fluorescent second state of said indicator group for each of the at least one normally functioning enzyme for each said plurality of reference, metabolically active whole cells to produce a matrix of reference results;
• (c) contacting a plurality of test, metabolically active whole cells, each having at least one normally functioning enzyme with said medium for said period of time;
• (d) sensing for said fluorescent second state of said indicator group for each of the at least one normal functioning enzyme for each of said plurality of test, metabolically active whole cells to produce a matrix of test results;
• and
• (e) comparing the matrix of test results of said plurality of test, metabolically active whole cells in said step (d) with the matrix of reference results obtained from said plurality of reference, metabolically active whole cells in said step (b) thereby detecting the presence or absence of said abnormality in the activity of an enzyme in said metabolically active whole cell.

A method of performing an assay for detecting the presence or absence of a disease characterized by an abnormal enzyme activity comprising:

• (a) contacting a test, metabolically active whole cell with at least one cell permeable water soluble assay compound or water soluble physiologically acceptable salt thereof having a fluorogenic indicator group and an unblocked leaving group, said leaving group being selected for cleavage by one of said at least one normally functioning enzyme the activity of which changes with the presence of the disease, said indicator group being in a non-fluorescent first state when bonded to said leaving group, and being in a fluorescent second state excitable at a wavelength above 450 nm when said leaving group is cleaved from said indicator group by the one of said at least one normally functioning enzyme for a period of time at least sufficient for said assay compound to be transferred into said cell and for said leaving group to be cleaved inside said cell from said indicator group by said enzyme wherein said assay compound has sufficient purity so that fluorescence of impurities in said assay compound is less tan autofluorescence of the metabolically active whole cell;
• (b) sensing for said fluorescent second state of the indicator group for the test, metabolically active whole cell to produce a matrix of test results;
• and
• (c) comparing the matrix of test results of said test metabolically active whole cell with a matrix of reference results obtained from at least one of a diseased reference cell and a non-diseased reference cell thereby detecting the presence or absence of said disease in said test, metabolically active whole cell.

A method for measuring the activity of an enzyme in a metabolically active whole cell, comprising:

• (a) contacting a reference, metabolically active whole cell having a normally functioning enzyme with a medium containing a cell permeable water soluble assay compound or a cell permeable water soluble physiologically acceptable salt thereof, said assay compound having a solubility such that when a salt of the compound is dissolved in the aqueous media used in the assay at the concentration used in the assay, the solution has a background color of less than 1000 milliabsorbance units at 340 nm at 25.degree. C. blanked against distilled or deionized water, said assay compound or physiologically acceptable salt thereof having the ability to pass through a cell membrane, said assay compound comprising a fluorogenic indicator group and a leaving group, said leaving group being selected for cleavage by said enzyme, said indicator group being in a non-fluorescent first state when bonded to said leaving group, and being in a fluorescent second state excitable at a wavelength above 450 nm when said leaving group is cleaved from said indicator group by said enzyme, for a period of time sufficient for said assay compound to be transferred into said cell and for said leaving group to be cleaved inside said cell from said indicator group by said enzyme, wherein said assay compound has sufficient purity so that fluorescence of impurities in said assay compound is less than autofluorescence of the metabolically active whole cell;
• (b) sensing for said fluorescent second state of said indicator group for the reference, metabolically active whole cell to produce reference results;
• (c) contacting a test, metabolically active whole cell with said medium for said period of time;
• (d) sensing for said fluorescent second state of said indicator group for the test, metabolically active whole cell to produce test results;
• and
• (e) comparing the reference results of reference test, metabolically active whole cell in said step (b) with the test results obtained from said test metabolically active whole cell in said step (d) thereby measuring the activity of said enzyme in said metabolically active whole cell.

A method for measuring the activity of an enzyme in a metabolically active whole cell, comprising:

• (a) contacting a reference, metabolically active whole cell having a normally functioning enzyme with a medium containing a cell permeable water soluble purified assay compound which is purified to an extent that background noise generated from impurities is less than autofluorescence of a metabolically active whole cell, said compound having the ability to pass through a cell membrane or a water soluble physiologically acceptable salt thereof having the ability to pass through a cell membrane, said assay compound comprising a fluorogenic indicator group and a leaving group, said leaving group being selected for cleavage by said enzyme, said indicator group being in a non-fluorescent first state when bonded to said leaving group, and being in a fluorescent second state excitable at a wavelength above 450 nm when said leaving group is cleaved from said indicator group by said enzyme, for a period of time which is sufficient for said assay compound to be transferred into said cell and for said leaving group to be cleaved inside said cell from said indicator group by said enzyme;
• (b) sensing for said fluorescent second state of said indicator group for the reference, metabolically active whole cell to produce reference results;
• (c) contacting a test, metabolically active whole cell with said medium for said period of time;
• (d) sensing for said fluorescent second state of said indicator group for the test, metabolically active whole cell to produce test results;
• and
• (e) comparing the reference results of reference test, metabolically active whole cell in said step (b) with the test results obtained from said test metabolically active whole cell in said step (d) thereby measuring the activity of said enzyme in said metabolically active whole cell.

A method for measuring the activity of an enzyme in a metabolically active whole cell, comprising:

• (a) contacting a reference, metabolically active whole cell having a normally functioning enzyme with a medium containing a cell permeable water soluble assay compound or a water soluble physiologically acceptable salt thereof, said assay compound or physiologically acceptable salt thereof having the ability to pass through a cell membrane, said assay compound comprising a fluorogenic indicator group and a leaving group, said leaving group being selected for cleavage by said enzyme, said indicator group being in a non-fluorescent first state when bonded to said leaving group, and being in a fluorescent second state excitable at a wavelength above 450 nm when said leaving group is cleaved from said indicator group by said enzyme, for a period of time sufficient for said assay compound to be transferred into said cell and for said leaving group to be cleaved inside said cell from said indicator group by said enzyme, wherein said assay compound has sufficient purity so that fluorescence of impurities in said assay compound is less than autofluorescence of the metabolically active whole cell;
• (b) sensing for said fluorescent second state of said indicator group for the reference, metabolically active whole cell to produce reference results;
• (c) contacting a test, metabolically active whole cell with said medium for said period of time;
• (d) sensing for said fluorescent second state of said indicator group for the test, metabolically active whole cell to produce test results;
• and
• (e) comparing the reference results of reference test, metabolically active whole cell in said step (b) with the test results obtained from said test metabolically active whole cell in said step (d) thereby measuring the activity of said enzyme in said metabolically active whole cell.