Biological assay of peptidoglycans

Abstract

The present invention relates to a biological method for assaying peptidoglycans (PGN) in a sample, particularly a sample of glucose polymers. The PGN assay includes: a) treating the glucose polymer sample by sonication, heating, and/or alkalizing; b) placing the treated sample or a dilution thereof in contact with a recombinant cell expressing an exogenous TLR2 (toll-like receptor 2) and a reporter gene directly dependent on the signaling pathway associated with the TLR2. The reporter gene codes for a colored or fluorescent protein or for a protein the activity of which is measurable with or without a substrate; c) measuring the reporter gene signal; and d) determining the amount of PUN in the sample using a standard curve of the correlation between the amount of PGN and the strength of the reporter gene signal.

1 - 14 . (canceled) 
     
     
         15 . A kit for assaying peptidoglycans (PGNs) in a sample of glucose polymers comprising:
 a recombinant cell expressing an exogenous TLR2 receptor (Toll-like Receptor 2) and a reporter gene under the direct dependence of the signaling pathway associated with the TLR2 receptor, said reporter gene coding for a colored or fluorescent protein or for a protein whose activity can be measured with or without a substrate;   either a calibration curve of the correspondence between the amount of PGN and the intensity of the reporter gene signal, or a PGN standard; and   optionally, instructions for use, and a solution for pretreating the sample.   
     
     
         16 . The kit of  claim 15  wherein the PGN standard is derived from a bacterium selected from  Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis  and  Alicyclobacillus acidocaldarius.    
     
     
         17 . The kit of  claim 15 , further comprising an internal standard that is an agonist of TLR2. 
     
     
         17 . The kit of  claim 16 , wherein the agonist of TLR2 is a lipopeptide. 
     
     
         18 . The kit of  claim 17 , wherein said lipopetide is PAM3Cys-Ser-(Lys)4 trihydrochloride. 
     
     
         19 . The kit of  claim 15 , wherein the recombinant cell is a stably transformed HEK-293 cell line. 
     
     
         20 . The kit of  claim 15  further comprising a negative control. 
     
     
         21 . The kit of  claim 20 , wherein the negative control comprises a cell not expressing an innate immunity receptor. 
     
     
         22 . The kit of  claim 21 , wherein the cell not expressing an innate immunity receptor is a HEK-null cell line. 
     
     
         23 . The kit of  claim 15 , wherein the PGN standard is a purified and partially digested PGN. 
     
     
         24 . The kit of  claim 15  further comprising reagents to be used for measuring the response of the reporter gene. 
     
     
         25 . The kit of  claim 15 , wherein the reporter gene codes for an enzyme. 
     
     
         26 . The kit of  claim 25 , wherein the enzyme is luciferase or alkaline phosphatase. 
     
     
         27 . A method of using the kit of  claim 15 , and determining the presence of PGNs.