Luminogenic and nonluminogenic multiplex assay

Abstract

A method to detect the presence or amount of at least one molecule for an enzyme-mediated reaction in a multiplex luminogenic/nonluminogenic assay is provided.

1 . A method to detect the presence or amount of a first enzyme or cofactor for a first enzyme-mediated reaction, comprising:
 a) contacting a sample with a first substrate for the first enzyme, a second substrate for a second enzyme, and optionally a third enzyme, wherein a reaction between the first substrate and first enzyme or a reaction between the third enzyme and a product of a reaction between the first enzyme and the first substrate yields a luminogenic product, wherein the second substrate and/or a product of a reaction between the second substrate and the second enzyme is/are not luminogenic; and   b) detecting the presence or amount of the first enzyme or cofactor for the reaction mediated by the first enzyme.   
     
     
         2 . A method of assaying an enzyme-mediated luminescence reaction to detect the presence or amount of an enzyme or cofactor for a first enzyme-mediated reaction, comprising:
 a) contacting a sample with a first substrate for a first enzyme, a second substrate for a second enzyme, and optionally a third enzyme, wherein a reaction between the first substrate and first enzyme or a reaction between the third enzyme and a product of the reaction between the first enzyme and the first substrate yields a luminogenic product, wherein the second substrate and/or a product of a reaction between the second substrate and the second enzyme is/are not luminogenic; and   b) detecting luminescence.   
     
     
         3 . The method of  claim 1  wherein luminescence is detected. 
     
     
         4 . The method of  claim 1  or  2  wherein luminescence increases in the presence of the first enzyme or cofactor. 
     
     
         5 . The method of  claim 1  or  2  wherein luminescence decreases in the presence of the first enzyme or cofactor. 
     
     
         6 . The method of  claim 1  or  2  further comprising detecting the presence or amount of the second enzyme. 
     
     
         7 . The method of  claim 6  wherein fluorescence is employed to detect the presence or amount of the second enzyme. 
     
     
         8 . The method of  claim 6  wherein the presence or amount of the first enzyme or cofactor and the presence or amount of the second enzyme are detected sequentially. 
     
     
         9 . The method of  claim 6  wherein the presence or amount of the first enzyme or cofactor and the presence or amount of the second enzyme are detected simultaneously. 
     
     
         10 . The method of  claim 6  wherein the presence or amount of the second enzyme is detected calorimetrically. 
     
     
         11 . The method of  claim 6  wherein the presence or amount of the second enzyme is detected by contacting the sample with a fourth enzyme and third substrate for a reaction between the product of the reaction between the second substrate and second enzyme which yields a fluorogenic product. 
     
     
         12 . The method of  claim 1  or  2  further comprising detecting the presence or amount of the nonluminogenic second substrate or the nonluminogenic product of the reaction between the second substrate and the second enzyme. 
     
     
         13 . The method of  claim 1  or  2  wherein the second enzyme does not react substantially with the first substrate. 
     
     
         14 . The method of  claim 1  or  2  wherein the first enzyme does not react substantially with the second substrate. 
     
     
         15 . The method of  claim 1  or  2  wherein the second substrate or the product of the reaction between the second substrate and the second enzyme is fluorescent. 
     
     
         16 . The method of  claim 15  wherein the second substrate or the product of the reaction between the second substrate and the second enzyme comprises ethidium bromide, fluorescein, Cy3, BODIPY, a rhodol, Rox, 5-carboxyfluorescein, 6-carboxyfluorescein, an anthracene, 2-amino-4-methoxynapthalene, a phenalenone, an acridone, fluorinated xanthene derivatives, α-naphtol, β-napthol, 1-hydroxypyrene, coumarin, 7-amino-4-methylcoumarin (AMC), 7-amino-4-trifluoromethylcoumarin (AFC), Texas Red, tetramethylrhodamine, carboxyrhodamine, or rhodamine, cresyl, rhodamine-110 or resorufin. 
     
     
         17 . The method of  claim 1  or  2  wherein one enzyme is a glycosidase, phosphatase, kinase, dehydrogenase, peroxidase, sulfatase, peptidase, or hydrolase. 
     
     
         18 . The method of  claim 1  or  2  wherein one enzyme is a protease. 
     
     
         19 . The method of  claim 18  wherein one enzyme is a caspase. 
     
     
         20 . The method of  claim 19  wherein the caspase includes caspase-3, caspase-7 or caspase-8. 
     
     
         21 . The method of  claim 1  or  2  wherein one of the substrates comprises DEVD, WEHD, LEHD, VEID, VEVD, VEHD, IETD, AEVD, LEXD, VEXD, IEHD, PEHD, ZEVD or LETD. 
     
     
         22 . The method of  claim 1  or  2  wherein one of the substrates comprises X 1 -X 2 -X 3 -D wherein X 1  is Z, Y, D, L, V, I, A, W or P, X 2  is V or E, and X 3  is any amino acid. 
     
     
         23 . The method of  claim 1  or  2  wherein one of the substrates is a substrate for trypsin or tryptase. 
     
     
         24 . The method of  claim 1  or  2  wherein one enzyme cleaves a substrate comprising arginine or lysine. 
     
     
         25 . The method of  claim 1  or  2  wherein the sample is a cell lysate. 
     
     
         26 . The method of  claim 25  wherein the sample is a cellular sample that is treated with a cell death inducing agent prior to lysis. 
     
     
         27 . The method of  claim 1  or  2  wherein the sample comprises intact cells. 
     
     
         28 . The method of  claim 1  or  2  wherein the third enzyme is a luciferase. 
     
     
         29 . The method of  claim 28  wherein the luciferase is a beetle luciferase. 
     
     
         30 . The method of  claim 1  or  2  wherein the second substrate is a substrate for lactate dehydrogenase. 
     
     
         31 . The method of  claim 1  or  2  wherein the sample is contacted with the first substrate before the second substrate. 
     
     
         32 . The method of  claim 1  or  2  wherein the sample is simultaneously contacted with the first and the second substrates. 
     
     
         33 . The method of  claim 1  or  2  wherein the sample is contacted with the second substrate before the first substrate. 
     
     
         34 . The method of  claim 1  or  2  wherein the presence or amount of the cofactor is detected by contacting the sample with the first enzyme.