Detection reagent for N-acetyl-beta-D-glucosaminidase with high analytical sensitivity

Abstract

The invention discloses a detection reagent for N-acetyl-beta-D-glucosaminidase with high analytical sensitivity. The reagent disclosed by the invention comprises a reagent R1 and a reagent R2; the reagent takes an artificially-synthesized product 5-(4-(3-methyl-styrene)-rhodanine-3-rhodanine-acetic acid amino-N-acetamino-beta-D-glucoside as a reaction substrate, so that the sensitivity of the reagent is improved; the reagent adopts two kinds of buffer solutions, i.e., an MES (2-(N-morpholine)ethyl sulfonic acid) buffer solution and an AMP (2-amino-2-methyl-1-propanol) buffer solution, so that the catalytic action of an NAG enzyme can be promoted while buffer capacity is guaranteed, a strongly-alkaline environment is effectively improved, and the efficiency of color development is guaranteed; ingredients are reasonably matched and used, so that the overall sensitivity, specificity and accuracy of the reagent are relatively high, and the further popularized use in market is facilitated.

Description

technical field [0001] The invention relates to a urine N-acetyl-β-D-glucosaminidase (NAG) detection reagent with high analytical sensitivity. Background technique [0002] N-acetyl-β-D-glucosaminidase (NAG) is a hydrolytic enzyme present in the lysosome of cells. NAG in urine mainly comes from renal proximal tubule epithelial cells, but the urine content of healthy people is very small. The increase of urinary NAG activity level is a sensitive indicator of early kidney injury and diabetic microangiopathy. NAG activity can be used for early diagnosis of tubulointerstitial nephritis, urinary tract infection, diabetic nephrotic syndrome, hypertensive nephropathy, rejection after kidney transplantation and nephrotic syndrome. The increase of NAG activity in the above diseases is earlier than other corresponding indicators, which is conducive to early detection and timely treatment of diseases. NAG activity can also be used for population screening and occupational disease inv...

AI Technical Summary

Problems solved by technology

This method is simple and fast, but the repeatability is not good and the error is large

Immunoassay, this type of method is established based on the different immunogenicity of isoenzymes, including immunoprecipitation, immunocapture, enzyme-linked immunosorbent assay and other related methods. These methods have strong specificity and accurate detection results, but most of them operate It is relatively complicated and costly, and it is not convenient for large-scale clinical promotion. At present, the colorimetric endpoint method is mostly used clinically. The basic principle of this method is that the NAG substrate generates related products under the catalysis of NAG. The substance will change color in a strong alkaline environment, and the degree of change is proportional to the NAG activity in the sample, so as to detect the activity of NAG

Among them, there are many kinds of colorimetric methods, and the more traditional one is the PNP-NAG method. This method uses p-nitrophenyl-β-D-glucosamine (PNP-NAG) as a substrate and reacts with enzymes at 37°C for a period of time. , NAG in urine catalyzes the hydrolysis of the substrate to release PNP, then terminates the reaction with lye and develops the color, measures the absorbance at a wavelength of 405nm, and calculates the NAG activity according to the working curve or molar absorptivity coefficient. The disadvantage of the PNP-NAG method is that a sample blank must be set. And the operation is time-consuming, not suitable for large-scale automated analysis

In 1983, Noto proposed the MCP-NAG method using m-cresolsulfonphthalein-N-acetyl-β-D-glucosamine (MCP-NAG) as the substrate. This method can avoid the interference of urochromogen, and it is not necessary to make a blank , but the substrate cost is very high; in 1961; Leaback proposed to use non-fluorescent 4-methylumbelliferone-N-acetyl-β-D-glucosaminidone (4MU-NAG) as the substrate, the method has high sensitivity and is comparable to The PNP method has good correlation and is not interfered by urine color. There are domestic reagents, but they need a fluorescence photometer, which requires relatively high requirements for instruments and operators. Most of the instruments need imported instruments, so this undoubtedly increases the cost of using the department.

Therefore, according to the problems of low analytical sensitivity in the current detection method, or the substrate is too expensive, or the requirements for instruments and personnel are too high, a kind of NAG detection reagent with high analytical sensitivity is specially invented. The reagent is artificially synthesized 5-( 4-(3-methyl-styrene)-rhodanine-3-acetate amino-N-acetylamino-β-D-glucoside was used as a substrate, the reaction system was optimized, and activators and surfactants were added to improve The analytical sensitivity of the product, the cost of the substrate of the present invention is relatively low, suitable for the use of fully automatic biochemical analysis instruments, very conducive to clinical use, and even the use and promotion of ordinary hospitals

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