Assaying apparatus, kit, and method for lipids and associated enzymes

Abstract

Lipid assay method, kit, and apparatus involving exposure of a protein, having a lipid recognition motif that interacts with a target lipid and a competing lipid, to a solution containing the competing lipid, and determining whether the target lipid is present in the solution. The target lipid has a stronger affinity to the lipid recognition motif than does the competing lipid. The lipid recognition motif is preferably a pleckstrin homology (PH) domain, with the target lipid being a phosphoinositide. The assay determines activity of a lipid kinase, the target lipid being a phosphorylation product of a reaction between the lipid kinase and a substrate lipid. The assay can be a cancer screening method for detection of cancer cells, where detection of certain levels of a PI(3,4,5)P3 target lipid is an indicator of a cancer cell.

1 . A lipid assay comprising; 
 a) a target lipid that is a phosphorylation product of a reaction between a phosphoinositide kinase and a substrate phosphoinositide lipid;    b) a predetermined amount of a competing lipid that is labeled by a non-radioactive signal; and    c) a protein having a phosphoinositide lipid recognition motif that interacts with said target lipid and said competing lipid.    
   
   
       2 . The lipid assay of  claim 1 , which further comprises a multi-well assay plate.  
   
   
       3 . The lipid assay of  claim 2 , wherein said multi-well assay plate includes said competing lipid immunobilized in wells of said multi-well assay plate.  
   
   
       4 . The lipid assay of  claim 1 , which further comprises primary and secondary antibodies.  
   
   
       5 . The lipid assay of  claim 1 , wherein said target lipid is a component of a sample selected from the group consisting of a tissue sample, a blood sample, and a serum sample.  
   
   
       6 . The lipid assay of  claim 1 , wherein said assay is selected from the group consisting of an amplified luminescence proximity homogenous assay (ALPHA), a fluorogenic assay, and an enzyme linked immunosorbent assay (ELISA).  
   
   
       7 . The lipid assay of  claim 1 , wherein said target lipid is a phosphoinositide.  
   
   
       8 . The lipid assay of  claim 1 , wherein detection of said signal is automated.  
   
   
       9 . The lipid assay of  claim 1 , wherein said assay comprises a kit.  
   
   
       10 . The lipid assay of  claim 1 , wherein said protein is selected from the group consisting of an anti-PI(3,4,5)P 3  antibody, an anti-PI(3)P antibody, a lipid recognition protein with specificity for PI(3,4,5)P 3 , and a lipid recognition protein with specificity for PI(3)P.  
   
   
       11 . The lipid assay of  claim 1 , wherein said protein contains an affinity tag fusion with a PH domain.  
   
   
       12 . The lipid assay of  claim 1 , wherein detection of said target lipid is indicative of the presence of a cancer cell.  
   
   
       13 . The lipid assay of  claim 1 , wherein said assay determines activity of a lipid kinase, the target lipid being a product of a reaction between said lipid kinase and a substrate lipid.  
   
   
       14 . The lipid assay of  claim 13 , wherein said lipid kinase is PI 3-K and said target lipid is a product of PI 3-K.  
   
   
       15 . The lipid assay of  claim 14 , wherein said product of PI 3-K is PI(3,4,5)P 3  or PI(3)P.  
   
   
       16 . A lipid assay, comprising; 
 a) a target lipid;    b) a competing lipid; and    c) a protein having a lipid recognition motif that interacts with said target lipid and said competing lipid.    
   
   
       17 . The lipid assay of  claim 16 , wherein the competing lipid emits a signal.  
   
   
       18 . The lipid assay of  claim 17 , wherein detection of said signal is automated.  
   
   
       19 . The lipid assay of  claim 16 , wherein said assay comprises a kit.  
   
   
       20 . The lipid assay of  claim 16 , which further comprises primary and secondary antibodies.  
   
   
       21 . The lipid assay of  claim 16 , wherein said assay is selected from the group consisting of an amplified luminescence proximity homogenous assay (ALPHA), a fluorogenic assay, and an enzyme linked immunosorbent assay (ELISA).  
   
   
       22 . The lipid assay of  claim 16 , wherein said target lipid is a component of a sample selected from the group consisting of a tissue sample, a blood sample, and a serum sample.  
   
   
       23 . The lipid assay of  claim 16 , which further comprises a multi-well assay plate.  
   
   
       24 . The lipid assay of  claim 23 , wherein said multi-well assay plate includes said competing lipid immunobilized in wells of said multi-well assay plate.  
   
   
       25 . The lipid assay of  claim 16 , wherein said protein is selected from the group consisting of an anti-PI(3,4,5)P3 antibody, an anti-PI(3)P antibody, a lipid recognition protein with specificity for PI(3,4,5)P3, and a lipid recognition protein with specificity for PI(3)P.  
   
   
       26 . The lipid assay of  claim 16 , wherein said protein contains an affinity tag fusion with a PH domain.  
   
   
       27 . The lipid assay of  claim 16 , wherein said target lipid is a phosphoinositide.  
   
   
       28 . The lipid assay of  claim 16 , wherein detection of said target lipid is indicative of the presence of a cancer cell.  
   
   
       29 . A lipid assay comprising; 
 a) a sample putatively containing 3,4,5-trisphosphate;    b) biotin labeled 3,4,5-trisphosphate; and    c) a protein having a pleckstrin homology domain to a phosphoinositide that interacts with said target lipid and said competing lipid.    
   
   
       30 . A method comprising; exposing a test solution containing an unknown amount of a target lipid, wherein said target lipid is a product of a reaction between a kinase that is added to said test solution and a substrate lipid that is added to said test solution, to a solution containing a protein having a lipid recognition motif that interacts with said target lipid and a predetermined amount of a competing target lipid, thereby allowing for quantification of the kinase activity.  
   
   
       31 . A lipid assay for the detection of a phosphoinositide target lipid comprising; 
 a) a Grp1 protein having a pleckstrin homology domain to a phosphoinositide; and    b) a predetermined amount of a competing lipid that is labeled by a non-radioactive signal; wherein the target lipid has a stronger affinity to the pleckstrin homology domain than does the competing lipid.