Method for assaying the activity of lysosomal enzymes

Abstract

A method, and associated kit, for assaying the activity of lysosomal enzymes present in dried bodily fluids and cell tissue samples, such as α-L-iduronidase, β-D-galactosidase, β-D-glucosidase, chitotriosidase, total α-D-galactosidase and α-D-galactosidase A, hexosaminidase A and B, α-D-mannosidase, β-D-mannosidase, α-L-fucosidase, N-acetyl-α-galactosaminidase, arylsulfatases, sphingomyelinase, β-galactocerebrosidase, iduronate- 2 -sulfatase and β-D-glucuronidase. The method includes: (a) combining with a dried bodily fluid or cell tissue sample containing at least one type of lysosomal enzyme: (1) an eluent, (2) an incubation buffer and (3) a substrate or substrates capable of reacting with the assayed lysosomal enzymes and producing their corresponding enzyme product or products, (b) allowing the dried bodily fluid or cell tissue sample to react with the eluent, incubation buffer and substrate or substrates for an adequate time and temperature, and (c) applying measuring means to the enzyme product to determine the activities of the lysosomal enzymes present.

1 . A method for assaying the activity of a lysosomal enzyme present in a sample of body fluid or cell tissue, comprising the steps of:
 (a) obtaining a sample of body fluid or cell tissue;   (b) dehydrating said sample naturally for a time sufficient to substantially remove fluid from the sample,   (c) forming an incubation media by combining the sample of step (b) with (i) an eluent in an amount sufficient to release assayed lysosomal enzymes from the sample of step (b), (ii) an incubation buffer and (iii) at least one substrate capable of reacting with said lysosomal enzyme and generating at least one enzyme product;   (d) incubating said media under conditions sufficient to generate said enzyme product; and   (e) measuring said enzyme product to determine the activity of said lysosomal enzyme.   
   
   
       2 . The method according to  claim 1 , wherein said sample is exposed at room temperature. 
   
   
       3 . The method according to  claim 1 , wherein said sample is a blood spot. 
   
   
       4 . The method according to  claim 1 , further comprising placing blood on a porous surface material and drying to form a dried blood spot. 
   
   
       5 . The method according to  claim 4 , wherein the porous surface material is filter paper. 
   
   
       6 . The method according to  claim 4 , wherein said dried blood spot is of mammalian origin. 
   
   
       7 . The method according to  claim 6 , wherein said dried blood spot comprises a dried blood spot of sheep, mouse, or human origin. 
   
   
       8 . The method according to  claim 7 , wherein said mammalian origin dried blood spot is human. 
   
   
       9 . The method according to  claim 1 , wherein said eluent, incubation buffer and substrate(s) in step (c) are added sequentially to the sample of step (b) in the order (i), (ii), and (iii). 
   
   
       10 . The method according to  claim 4 , wherein said eluent, incubation buffer and substrate(s) in step (c) are added sequentially to said dried blood spot in the order (i), (ii), and (iii). 
   
   
       11 . The method according to  claim 1 , wherein said eluent comprises water, sodium taurocholate, sodium chloride, citrate-phosphate buffer, sodium acetate, lead acetate, triton X-100, D-saccharic acid-1,4-lactone, or mixtures thereof. 
   
   
       12 . The method according to  claim 1 , wherein said incubation buffer has a pH value of less than 7. 
   
   
       13 . The method according to  claim 12 , wherein said incubation buffer has a pH value of less than 5.5. 
   
   
       14 . The method according to  claim 1 , wherein said incubation buffer comprises sodium formate, sodium citrate, sodium phosphate, sodium acetate, or mixtures thereof. 
   
   
       15 . The method according to  claim 1 , wherein said substrate(s) comprise(s) at least one type of molecule capable of being converted by a lysosomal enzyme. 
   
   
       16 . The method according to  claim 15 , wherein said type of molecules comprise 4-methylumbelliferyl-.alpha.-L-iduronide, 4-methylumbelliferyl-.beta.-D-galactoside, 4-methylumbelliferyl-.beta.-D-glucuronic acid, 4 methylumbelliferyl-.alpha.-D-galactoside, methylumbelliferyl-.beta.-D-galactopyranoside, methylumbelliferyl-.alpha.-D-galctopyranoside, methylumbelliferyl-.beta.-N-N′-N″ triacetylchitotrioside, methylumbelliferyl-sulfate, methylumbelliferyl-2-acetamido-2-deoxy-.beta.-D-glucopyranoside, methylumbelliferyl-N-acetyl-Beta-D-glucopyranoside sulfate, or mixtures thereof. 
   
   
       17 . The method according to  claim 1 , wherein said substrate(s) comprise(s) natural molecules, synthetic molecules, or mixtures thereof. 
   
   
       18 . The method according to  claim 1 , wherein said substrate(s) comprises a fluorophoric moiety. 
   
   
       19 . The method according to  claim 18 , wherein said fluorophoric moiety comprises 4-methyllumbelliferyl. 
   
   
       20 . The method according to  claim 1 , wherein substrate(s) comprise(s) a chromophoric moiety. 
   
   
       21 . The method according to  claim 20 , wherein said chromophoric moiety comprises 4-nitrocatechol or phenolphthalein. 
   
   
       22 . The method according to  claim 1 , wherein said substrate(s) comprises a radioactive moiety. 
   
   
       23 . The method according to  claim 22 , wherein said radioactive moiety comprises .sup.14C sphingomyeline or .sup.3H galactosylceramide. 
   
   
       24 . The method according to  claim 1 , wherein in step (d), said conditions sufficient to generate said enzyme product comprises a time of between 24 and 48 hours. 
   
   
       25 . The method according to  claim 1 , wherein in step (d), said conditions sufficient to generate said enzyme product comprises a temperature of less than 42° C. 
   
   
       26 . The method according to  claim 1 , wherein said incubation in step (d) is halted by non-interfering means prior to performing step (e). 
   
   
       27 . The method according to  claim 26 , wherein said non-interfering means comprises applying heat to the incubation media. 
   
   
       28 . The method according to  claim 27 , wherein said non-interfering means comprises diluting the incubation media. 
   
   
       29 . The method according to  claim 28 , wherein said non-interfering means comprises adding a deproteinizing agent to the incubation media. 
   
   
       30 . The method according to  claim 29 , wherein said non-interfering means comprises adding a stopping buffer to the incubation media. 
   
   
       31 . The method according to  claim 30 , wherein said stopping buffer has a pH value between 7 and 12. 
   
   
       32 . The method according to  claim 31 , wherein said stopping buffer has a pH value between 10 and 11. 
   
   
       33 . The method according to  claim 1 , wherein said measuring means comprises a fluorometer, a calorimeter, a spectrophotometer, or a radioactive counter. 
   
   
       34 . The method according to  claim 1 , further comprising determining the amount of said enzyme product(s) present in said incubation media. 
   
   
       35 . The method according to  claim 4 , wherein said dried blood spot is assayed within 40 days from collection. 
   
   
       36 . The method according to  claim 4 , wherein said dried blood spot is assayed within 20 days from collection. 
   
   
       37 . The method according to  claim 4 , wherein said dried blood spot is assayed within 24 hours from collection. 
   
   
       38 . The method according to  claim 1 , further comprising dehydrating an unprocessed sample. 
   
   
       39 . The method according to  claim 39 , wherein the unprocessed sample is unprocessed whole blood. 
   
   
       40 . The method according to  claim 1 , wherein the lysosomal enzyme is selected from the group consisting of glucosamine-N sulfate, hyaluronidase, steroid sulfatase, phosphotransferase, acid lipase, tryoleil lipase, cholesteryl estrase, acid ceramidase, α-N-acetylgalactosaminidase, shingomyelinase, palmitoyl protein thioesterase, carboxipepitidase, α-glucosaminide-N-acetyltransferase, N-acetylglucosamine-6-sulfatase, cathepsine K, α-L-iduronidase, β-D-galactosidase, β-D-glucuronidase, α-L-fucosidase, α-mannosidase, β-D-mannosidase, Arylsulfatase, β-hexosaminidase, total hexosaminidase, hexosaminidase A, β-D-glucosidase, α-neuramindase, α-D-N-acetylgalactosaminidase, β-galactocerebrosidase, and iduronate-2-sulfatase. 
   
   
       41 . The method according to  claim 1 , wherein the lysosomal enzyme is selected from the group consisting of α-L-iduronidase, β-D-galactosidase, β-D-glucosidase, α-D-galactosidase A, total hexosaminidase, hexosaminidase A, arylsulfatase B, sphingomyelinase, and iduronate-2-sulfatase. 
   
   
       42 . A method for assaying the activity of a lysosomal enzyme present in a dried sample of body fluid, comprising the steps of:
 (a) obtaining a dried sample of body fluid, wherein the sample was prepared by naturally dehydrating the sample for a time sufficient to substantially remove fluid from the sample;   (b) forming an incubation media by combining the dried sample with (i) an eluent in an amount sufficient to release assayed lysosomal enzymes from the dried sample, (ii) an incubation buffer and (iii) at least one substrate capable of reacting with the lysosomal enzyme and generating at least one enzyme product;   (c) incubating the media under conditions sufficient to generate the enzyme product; and   (d) measuring the enzyme product to determine the activity of the lysosomal enzyme.