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...

AI Technical Summary

Benefits of technology

This method allows for clear differentiation between healthy and diseased individuals, including carriers, and enables reliable enzyme activity measurement in samples stored for years, even under adverse conditions, facilitating early diagnosis and reduced dosage treatment.

Problems solved by technology

In the absence of hexosaminidase A, the GM2 ganglioside lipid accumulates abnormally in nervous cells causing progressive and irreparable damage to the brain.

There is not therapy available for these disorders at the present.

Other LSDs, like for instance, late stage GM1 gangliosidosis, demand risky and costly therapeutical procedures such as bone marrow transplants.

However, the application of these techniques to LSD detection has been restricted by the following limitations:1) In hybridization assays the test is limited to known mutations of the lysosomal enzyme under study.

For sequencing assays it is unfeasible to detect carrier individuals.

The lysosomal enzyme activity assays known in the art not based in DNA technology have also several restrictions in their application.

One of their principal limitations is the minimum sample volume (usually between 5 to 10 ml of blood) necessary for testing.

This volume is too high to allow infants to stand neonatal screening for these disorders.

The consequences of this limitation are substantial.

Another inconvenience of the enzymatic activity assays is that the sources of samples more commonly utilized for these tests (whole blood, plasma and serum) must be stored under controlled conditions and only for limited periods of time (no more than 3 days).

These restrictions limit considerably the time that may lapse between the sample collection and the enzymatic activity assay.

Two additional limitations of the known lysosomal enzyme activity assays are their complexity and unavailability.

Leukocyte isolation and purification from blood is a very specialized and tedious laboratory procedure.

In addition, these assays are not performed in routinary clinical testing.

These conditions restrict considerably the access of the general population to this type of tests.

However, this method does not provide a conclusive LSD diagnosis.

The method is unable to identify the specific deficient lysosomal enzyme.

In practice this method is complemented with other conventional enzyme activity assays for LSDs diagnosis because of its unreliability.

However, this method is not used in routine clinical practice, especially for newborns.

This procedure is extremely uncomfortable to patients, especially infants, and sample collection is difficult.

In addition, a relative high volume of tears is required for testing.

It is frequently necessary to repeat sampling when using his method because the amount of sample collected is insufficient for assay.

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