Detection method and kit for detecting activity of acidic hydrolases in lysosomes

Abstract

The invention belongs to the technical field of biochemical analysis and detection, in particular to a detection method and a detection kit for acid hydrolase activity in lysosomes. The present invention firstly designs a detection system comprising 5 kinds of mucopolysaccharide acid hydrolase in lysosomes, these five kinds of enzymes are respectively: α-L-iduronidase, iduronate sulfatase, β-N-acetyl half Lactosamine‑6‑sulfatase, β‑galactosidase and arylsulfatase B enzymes. The present invention utilizes the characteristic of fluorescence of 4-methylumbelliferone, uses the new substrate produced by combining the 4-methylumbelliferone group obtained by chemical synthesis with the enzyme substrate, and adopts the acid buffer solution containing the new substrate and mucopolysaccharide The hydrolase reacts, and the free 4-methylumbelliferone fluorescent substance is released after the hydrolase hydrolyzes the substrate. The higher the activity of the hydrolase in the sample, the more the amount of 4-methylumbelliferone released by hydrolyzing the substrate, and the higher the fluorescence intensity generated by the fluorescence detection, so as to realize the quantitative detection of the hydrolase activity. The invention has high detection sensitivity, good selectivity, strong anti-interference ability, good reproducibility and easy operation.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis and detection, and in particular relates to a detection method and a detection kit for acid hydrolase activity in lysosomes. Background technique [0002] Hydrolase is a general term for a class of enzymes that catalyze hydrolysis reactions. It is widely distributed in the human body and plays an important role in human metabolism and biological regulation and other life activities. Lysosomes are vesicular organelles rich in a variety of high-concentration acid hydrolases in cells, and are important organelles responsible for the decomposition and digestion of various substances in cells. A series of hereditary metabolic diseases caused by the loss of lysosomal enzyme activity are collectively referred to as lysosomal diseases. ). The occurrence of lysosomal diseases is due to the defect of one or more enzymes in lysosomes, resulting in the accumulation of their metabolites in tissu...

AI Technical Summary

Problems solved by technology

At present, fluorescence analysis and colorimetric analysis are mostly used in the detection of hydrolase activity at home and abroad. Most of the colorimetric methods use disodium p-nitrophenylphosphate as the color substrate, because the substrate is catalyzed by alkaline phosphatase. Hydrolysis produces a yellow terminal product with a maximum absorption peak at 405nm, thereby realizing the quantitative colorimetric analysis of alkaline phosphatase activity, but this method has disadvantages such as low sensitivity, narrow detection range and weak anti-interference ability, and the bottom The substance has poor stability, is easy to crystallize, and is easy to decompose under light conditions, resulting in "false positive" results

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