Analytical method for detecting alkaline sphingomyelinase and kit for use in such method

Abstract

An analytical fluorometric method and a kit for use in such method are disclosed for assessing the presence of alkaline sphingomyelinase (SMase) in the stools of a patient in need of such an assessment since alkaline SMase is a marker of serious pathological states, such as colon cancer.

1 . A method for detecting alkaline sphingomyelinase in a patient's stools, which comprises the following steps: 
 1) collecting a sample of a patient's stools and drying it up;    2) weighing about 3-4 grams of the dried up sample and suspending it in 20 ml of a homogenization buffer containing 0.25 M sucrose, 0.15 M KC 1, 50 mM KH 2 PO 4  pH 7.4;    3) centrifuging the sample at 4000 rpm at +4° C. for 60 min;    4) recovering the supernatant and centrifuging again for 15 mm. at 4000 rpm at +4° C.;    5) measuring protein content in supernatant with the Pierce Protein Assay with bovine serum albumine as standard using for each sample a range of protein concentration between 32 mg/ml and 40 mg/ml and pipetting 25 μl of each sample into well;    6) adding to each 25 μl sample 65 μl of assay buffer containing 50 mM Tris/HCl, 2 mM EDTA, 0.15 M NaCl pH 9.0 and 10 μl of 29 μM sphingomyelin and in assay buffer adding bile salts (TC, TDC, GC, GCDC) in the concentration of 3 mM;    7) incubating at 37° C. for 1 hr;    8) pipetting 100 μl of each standard lyophilized bacterial sphingomyelinase and 10 μl of sphingomyelin (29 μM), incubating for 1 hr at 37° C. as the samples;    9) after 1 hour, adding 100 μl of reaction buffer containing 50 mM Tris/HCl pH 7.4, 10 mM β-glycerophosphate, 750 μM ATP, 5 mM EDTA, 5 mM EGTA, 100 μM Amplex Red, 8 U/ml alkaline phosphatase, 0.2 U/ml choline oxidase, 2 U/ml horseradish peroxidase;    10) incubating the reactions for 1 hour or longer at 37° C., protected from light;    11) measuring the fluorescence in a fluorescence microplate reader using excitation in the range of 530-560 nm and emission detection at 590 nm;    12) for each point, correcting for background fluorescence by subtracting the values derived from the no-sphingomyelinase control.    
     
     
         2 . The method of  claim 1 , applied to biological fluids.  
     
     
         3 . A kit for detecting alkaline sphingomyelinase in a patient's stools or biological fluid which comprises test tubes separately containing samples of the following reagents: 
 a) sphingomyelin to be hydrolized by alkaline sphingomyelinase present in the stools or biological fluids, to give phosphorylcholine;    b) alkaline phosphatase for catalyzing the hydrolysis of phosphorycholine to choline;    c) choline oxidase for oxidizing choline to hydrogen peroxide;    d) horse-radish peroxidase for assisting reaction of hydrogen peroxide with    e) Ampler Red Reagent (10-acetyl-3,7-dihydroxyphenoxazine) to give the fluorescent compound resorufin whose fluorescence is a marker of the alkaline SMase present in the stools or biological fluids; and    f) lyophilized bacterial sphingomyelinase for use as standard concentrate.    
     
     
         4 . A method for detecting alkaline sphingomyelinase in a biological material from a patient, comprising the steps of 
 1) collecting a sample of the biological material;    2) suspending the sample in an homogenization buffer containing 0.24-0.26 M sucrose, 0.14-0.16 M KCl, 45-55 mM KH 2 PO 4  adjusted to about pH 7.4;    3) centrifuging the sample at least once and recovering the supernatant;    4) measuring the protein content in supernatant;    5) adding to a sample of the supernatant an assay buffer containing 44-55 mM Tris/HCl, 1.9-2.2 mM EDTA, 0.14-0.16 M NaCl pH 8.9-9.1, 28-30 μM sphingomyelin and an assay buffer containing bile salts (TC, TDC, GC, GCDC) at a concentration of 2.9-3.1 mM;    6) incubating the assay mixture at about 37° C. for about 1 hr;    7) mixing a sample from step 6) with 28-31 μM sphingomyelin, and incubating for about 1 hr at about 37° C.;    8) adding reaction buffer containing 45-55 mM Tris/HCl pH 7.3-7.5, 9-11 mM β-glycerophosphate, 745-755 μM ATP, 4-6 mM EDTA, 4-6 mM EGTA, 95-105 μM Amplex Red reagent, 7-9 U/ml alkaline phosphatase, 0.1-0.3 U/ml choline oxidase and 1.5-2.5 U/ml horseradish peroxidase;    9) incubating the reaction mixture for at least 1 hour at about 37° C., protected from light;    10) measuring the fluorescence using excitation in the range  530 - 560  and emission detection at about 590 nm.    
     
     
         5 . A method as claimed in  claim 4 , wherein, for each sample, the fluorescence reading is corrected for background fluorescence by subtracting the values derived from a no-sphingomyelinase control.  
     
     
         6 . A method as claimed in  claim 1  or  5 , wherein the protein content is measured by the Pierce Peotein Assay.  
     
     
         7 . A kit for detecting alkaline sphingomyelinase in a biological sample obtained from a patient comprising: 
 a) sphingomyelin    b) alkaline phosphatase,    c) choline oxidase,    d) horseradish peroxidase,    e) Amplar Red reagent,    f) lyophilized bacterial sphingomyelinase.    
     
     
         8 . A method of detecting alkaline sphingomyelinase substantially as described herein with reference to the figure.  
     
     
         9 . A kit for detecting alkaline sphingomyelinase substantially as described herein with reference to the figure.