Methods for detection of lysosomal storage disease
a lysosomal and storage disease technology, applied in the field of methods for detection of lysosomal storage disease, can solve problems such as damage to both somatic organs and the central nervous system, and achieve the effects of monitoring the efficacy of a particular treatment, and reducing acid glucocerebrosidase activity
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example 1
Synthesis of GLA and GAA Substrate and Internal Standards
[0200]The methods used to synthesize the internal standards and substrates for the GLA and GAA assays are essentially the same as those described by Li et al. (2004, Clinical Chemistry 50:1785-96). The methodology is briefly summarized as follows:
[0201]General Methods. Thin layer chromatography (TLC) is carried out on silica plates (Merck, 60F254), and flash column chromatography is carried out with silica gel (Merck, 230-400 Mesh). Preparative HPLC is carried out can be monitored with a UV detector (□=254 nm). Dry CH2Cl2 can be obtained by distillation from CaH2 under Ar, and other dry solvents are obtained from Aldrich (Sure-Seal). As noted below, reactions are carried out in a round bottom flask (RBF) or in a vial with a Teflon septum-lined screw cap. 1H-NMR spectra are obtained on a Bruker DPX200 spectrometer (200 MHz) unless otherwise noted.
[0202]Acetic acid 4-nitro-phenyl ester (1): Acetic anhydride (50 ml) is added to a...
example 2
Enzyme Screening Assays
[0216]The following example describes the specific protocol used to perform the enzyme assays (ASM, first ABG, GAA, GLA, and GALC assay) of the present invention.
[0217]Prior to beginning the DBS extraction, the assay mixtures should be warmed to room temperature and vortexed briefly. If needed, the ABG and GALC assay mixtures can be warmed in hot (40-45° C.) water for 5 minutes if solutions are not clear (the GALC cocktail may remain slightly cloudy).
DBS Extraction Method
[0218]DBS were obtained from adult, adolescent, and newborn patients that had been previously diagnosed as having one of the lysosomal storage diseases described herein based on other diagnostic tests (referred to generally as “test samples”). Test samples were obtained from patients confirmed as having one of ASM, ABG, GAA, GLA, GALC, or MPS1 deficiencies. A 3 mm hole punch was used to punch smaller samples from the DBS. It is possible to punch 6 or 7 times from one DBS, however, punches shou...
example 3
Second ABG Assay
[0236]The second ABG DBS assay measures the ABG-catalyzed cleavage of the fluorogenic substrate 4-MU-β-Glu by detecting the product 4-MU in a fluorometer.
Reagent Preparation
[0237]The following preparations are adequate for 20 plates. If possible, reagents should be made in batches large enough to cover an entire study. This is particularly important for the Buffered Extractant.
Substrate Stock Solution, 1 M
[0238]2.29 mL DMSO was added to 774.26 mg of 4-MU-β-Glu in a 15 mL screw cap tube. The mixture was thawed at RT until dissolved completely. The tube may be thawed briefly in a 37° C. if necessary. The solution was well vortexed and then 110 μL aliquots were placed in 1.5 mL microtubes. Aliquots of this solution can be thawed and refrozen several times, but should not be left thawed for more than two hours, and should be protected from light and moisture.
Buffered Extractant: 0.30 M Citrate Phosphate with 1% Sodium Taurodeoxycholate and 1% Triton X-100, pH 5.2
[0239]A ...
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