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.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 60 / 227,573, filed Aug. 25, 2000, and Patent Cooperation Treaty Application No. PCT / US01 / 26259 filed Aug. 24, 2001, which are incorporated entirely by reference.TECHNICAL FIELD OF THE INVENTION

[0002] This invention relates to a method of assaying lysosomal enzymes present in dried samples of bodily fluids and cell tissues. The invention also relates to a diagnostic kit for assaying lysosomal enzymes present in dried samples of bodily fluids and cell tissues.BACKGROUND OF THE INVENTION

[0003] The lysosome is an organelle founded in the cytoplasm of eukaryotic cells, which serves as storage for many hydrolytic enzymes and as a center for degrading and recycling cellular components. This organelle contains several types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases and sulfatases. All enzymes are acid hydrolases. See Lehninger et als. Principles of Bioc...

Examples

example 1

Preparation of Dried Blood Samples

[0099]A drop of blood obtained by venepuncture was spotted on filter paper (Schleicher and Schuell No 903, Keene, N.H., USA) and allowed to dry at room temperature (22° C.) overnight on a flat non-absorbing surface. The dried blood spots on filter paper were stored on plastic bags at 4° C. until analysis.

[0100]A sample for analysis was prepared by punching out a 3 mm-diameter circle (about 5.5 μl of whole blood) with a standard paper punch from the dried blood spots on the filter paper. A sample for analysis was prepared by punching out a 1.5 mm-diameter circle (about 2 μl of whole blood) with a standard paper punch from the dried blood spots on the filter paper.

[0101]This protocol was also performed by utilizing a heelprick finger to obtain the blood.

example 2

Preparation of Dried Chorionic Villae Samples

[0102]A chorionic villae sample (about 10 mg) was sonicated twice by 20 seconds in 50 μl of cold distilled water (Heat Systems-Ultrasonics, Inc., model W225R).

[0103]After removing 10 μl for protein determination, the sonicated cells were spotted on filter paper (Schleicher and Schuell No 903, Keene, N.H., USA) and allowed to dry for 6 hours at room temperature (22° C.) on a flat non-absorbing surface. See Lowry et al., J. Biol. Chem. 193:265-275 (1951). The spotted filter paper was stored on plastic bags at −20° C. until analysis.

[0104]A sample for analysis was prepared by punching out a 3 mm-diameter circle with a standard paper punch from the dried chorionic villae spots on the filter paper.

example 3

Preparation of Dried Cultured Amniocytes Samples

[0105]Cultured amniocytes were suspended in 500 μl of cold phosphate saline buffer (pH 7.4). After centrifugation at 1,200 g for 5 minutes at 4° C. the supernatant was removed by aspiration. The cell pellet was resuspended in 40 μl of cold distilled water and sonicated (Heat Systems-Ultrasonics, Inc., model W225R).

[0106]After removing 10 μl for protein determination, the sonicated cells were spotted on filter paper (Schleicher and Schuell No 903, Keene, N.H., USA) and allowed to dry for 6 hours at room temperature (22° C.) on a flat non-absorbing surface. See Lowry et al., supra. The spotted filter paper was stored on plastic bags at −20° C. until analysis.

[0107]A sample for analysis was prepared by punching out a 3 mm-diameter circle with a standard paper punch from the dried amniocytes spots on the filter paper.

[0001] This application claims the benefit of U.S. Provisional Application No. 60 / 227,573, filed Aug. 25, 2000, and Patent Cooperation Treaty Application No. PCT / US01 / 26259 filed Aug. 24, 2001, which are incorporated entirely by reference.TECHNICAL FIELD OF THE INVENTION

[0002] This invention relates to a method of assaying lysosomal enzymes present in dried samples of bodily fluids and cell tissues. The invention also relates to a diagnostic kit for assaying lysosomal enzymes present in dried samples of bodily fluids and cell tissues.BACKGROUND OF THE INVENTION

[0003] The lysosome is an organelle founded in the cytoplasm of eukaryotic cells, which serves as storage for many hydrolytic enzymes and as a center for degrading and recycling cellular components. This organelle contains several types of hydrolytic enzymes, including proteases, nucleases, glycosidases, lipases, phospholipases, phosphatases and sulfatases. All enzymes are acid hydrolases. See Lehninger et als. Principles of Bioc...

Examples

example 1

Preparation of Dried Blood Samples

[0099]A drop of blood obtained by venepuncture was spotted on filter paper (Schleicher and Schuell No 903, Keene, N.H., USA) and allowed to dry at room temperature (22° C.) overnight on a flat non-absorbing surface. The dried blood spots on filter paper were stored on plastic bags at 4° C. until analysis.

[0100]A sample for analysis was prepared by punching out a 3 mm-diameter circle (about 5.5 μl of whole blood) with a standard paper punch from the dried blood spots on the filter paper. A sample for analysis was prepared by punching out a 1.5 mm-diameter circle (about 2 μl of whole blood) with a standard paper punch from the dried blood spots on the filter paper.

[0101]This protocol was also performed by utilizing a heelprick finger to obtain the blood.

example 2

Preparation of Dried Chorionic Villae Samples

[0102]A chorionic villae sample (about 10 mg) was sonicated twice by 20 seconds in 50 μl of cold distilled water (Heat Systems-Ultrasonics, Inc., model W225R).

[0103]After removing 10 μl for protein determination, the sonicated cells were spotted on filter paper (Schleicher and Schuell No 903, Keene, N.H., USA) and allowed to dry for 6 hours at room temperature (22° C.) on a flat non-absorbing surface. See Lowry et al., J. Biol. Chem. 193:265-275 (1951). The spotted filter paper was stored on plastic bags at −20° C. until analysis.

[0104]A sample for analysis was prepared by punching out a 3 mm-diameter circle with a standard paper punch from the dried chorionic villae spots on the filter paper.

example 3

Preparation of Dried Cultured Amniocytes Samples

[0105]Cultured amniocytes were suspended in 500 μl of cold phosphate saline buffer (pH 7.4). After centrifugation at 1,200 g for 5 minutes at 4° C. the supernatant was removed by aspiration. The cell pellet was resuspended in 40 μl of cold distilled water and sonicated (Heat Systems-Ultrasonics, Inc., model W225R).

[0106]After removing 10 μl for protein determination, the sonicated cells were spotted on filter paper (Schleicher and Schuell No 903, Keene, N.H., USA) and allowed to dry for 6 hours at room temperature (22° C.) on a flat non-absorbing surface. See Lowry et al., supra. The spotted filter paper was stored on plastic bags at −20° C. until analysis.

[0107]A sample for analysis was prepared by punching out a 3 mm-diameter circle with a standard paper punch from the dried amniocytes spots on the filter paper.

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