Methods and compositions for the detection of bacterial endotoxins

Abstract

The invention provides methods and compositions for the detection and/or quantification of bacterial endotoxins. In particular, provided herein is an inexpensive and reproducible method for producing an improved amebocyte lysate preparation having reduced Factor G activity. Provided also is an endotoxin-specific amebocyte lysate preparation produced by such a method. In addition, the invention provides methods and compositions for enhancing the sensitivity to endotoxins of amebocyte lysate preparations having reducing Factor G activity. In particular, the sensitivity of such amebocyte lysate preparations to endotoxins can be enhanced by the addition of exogenous (1→3) β-D-glucan.

What is claimed is:  
     
         1 . A method of producing an improved amebocyte lysate preparation having reduced Factor G activity, the method comprising the steps of: 
 (a) admixing crude amebocyte lysate with a surfactant in an amount sufficient to produce a solution containing a precipitate; and    (b) separating said precipitate from said solution thereby to produce an amebocyte lysate preparation which is less reactive with (1→3)-β-D glucan than said crude amebocyte lysate.    
     
     
         2 . The method of  claim 1 , wherein said amebocyte lysate preparation is reactive with a bacterial endotoxin.  
     
     
         3 . The method of  claim 1  or  2 , wherein said precipitate contains Factor G.  
     
     
         4 . The method of  claim 1  or  2 , wherein said surfactant is a zwitterionic surfactant.  
     
     
         5 . The method of  claim 4 , wherein said zwitterionic surfactant is a sulfobetaine-type surfactant.  
     
     
         6 . The method of  claim 5 , wherein said sulfobetaine-type surfactant is selected from the group consisting of n-octyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, and n-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate.  
     
     
         7 . The method of  claim 5 , wherein said sulfobetaine-type surfactant is n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate.  
     
     
         8 . The method of  claim 1 , comprising the additional step of removing said surfactant from said solution.  
     
     
         9 . The method of  claim 8 , wherein said surfactant is removed by organic solvent extraction.  
     
     
         10 . The method of  claim 9 , wherein said organic solvent is chloroform.  
     
     
         11 . A method of producing an improved amebocyte lysate preparation having reduced Factor G activity, the method comprising the steps of: 
 (a) admixing crude amebocyte lysate with a zwitterionic surfactant in an amount sufficient to produce a solution containing a precipitate; and    (b) removing said precipitate from said solution thereby to produce an amebocyte lysate preparation which is less reactive with (1→3)-β-D glucan than said crude amebocyte lysate.    
     
     
         12 . The method of  claim 11 , wherein said amebocyte lysate preparation reacts with a bacterial endotoxin.  
     
     
         13 . The method of  claim 11 , wherein said precipitate contains Factor G.  
     
     
         14 . The method of  claim 11 , wherein said zwitterionic surfactant is a sulfobetaine-type surfactant.  
     
     
         15 . The method of  claim 14 , wherein said sulfobetaine-type surfactant is selected from the group consisting of n-octyl- N, N-dimethyl-3-ammonio-1-propanesulfonate, n-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, and n-hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate.  
     
     
         16 . The method of  claim 14 , wherein said sulfobetaine surfactant is n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate.  
     
     
         17 . The method of  claim 11 , comprising the additional step of removing said surfactant from said solution.  
     
     
         18 . The method of  claim 17 , wherein said surfactant is removed by organic solvent extraction.  
     
     
         19 . The method of  claim 18 , wherein said organic solvent is chloroform.  
     
     
         20 . A method of producing an improved amebocyte lysate having reduced Factor G activity, the method comprising the steps of: 
 (a) admixing crude amebocyte lysate with a n-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate surfactant in an amount sufficient to produce a solution containing a precipitate;    (b) separating said precipitate from said solution; and    (c) removing said surfactant from said solution thereby to produce an amebocyte lysate preparation that is reactive with bacterial endotoxin and is less reactive with (1→3)-β-D glucan than said crude amebocyte lysate.    
     
     
         21 . The method of  claim 20 , wherein said surfactant is removed by organic solvent extraction.  
     
     
         22 . The method of  claim 21 , wherein said organic solvent is chloroform.  
     
     
         23 . The method of  claim 2 ,  12 , or  20  comprising the additional step of adding exogenous (1→3)-β-D glucan to said amebocyte lysate preparation in an amount sufficient to enhance the sensitivity of said amebocyte lysate preparation to said endotoxin relative to said amebocyte lysate preparation without said exogenous (1→3)-β-D glucan.  
     
     
         24 . The method of  claim 23 , wherein said exogenous (1→3)-β-D glucan is selected from the group consisting of LAL-RM, curdlan, pachyman, scleratan, leutinan, schizophyllan, coriolan, laminaran, and laminarin.  
     
     
         25 . The method of  claim 23 , wherein said (1→3)-β-D glucan is laminarin.  
     
     
         26 . An amebocyte lysate preparation produced by the method of  claim 1 ,  11 , or  20 .  
     
     
         27 . An amebocyte lysate preparation produced by the method of  claim 23 .  
     
     
         28 . A composition comprising an amebocyte lysate preparation having reduced Factor G activity and exogenously added (1→3)-β-D glucan in an amount sufficient to enhance the sensitivity of said amebocyte lysate preparation to endotoxin relative to said amebocyte lysate preparation without said exogenously added (1→3)-β-D glucan.  
     
     
         29 . The composition of  claim 28 , wherein said (1→3)-β-D glucan is selected from the group consisting of cotton extract, cellulose acetate rinse, curdlan, pachyman, scleratan, leutinan, schizophyllan, coriolan, laminaran, and laminarin.  
     
     
         30 . The composition of  claim 28 , wherein said (1→3)-β-D glucan is laminarin.  
     
     
         31 . In a method of detecting a bacterial endotoxin in a sample, the improvement comprising using the amebocyte lysate preparation of  claim 26 .  
     
     
         32 . In a method of detecting a bacterial endotoxin in a sample, the improvement comprising using the amebocyte lysate preparation of  claim 27 .  
     
     
         33 . In a method of detecting a bacterial endotoxin in a sample, the improvement comprising using the composition of  claim 28  or  30 .