Liquid-stable 5 '-nucleotidase calibrator, detection kit and application thereof

A nucleotidase and calibrator technology, which is applied in the determination/inspection of microorganisms, measuring devices, instruments, etc., can solve the problems of interference, insufficient validity period, poor stability of purine nucleoside phosphorylase and xanthine oxidase, etc. , to achieve the effect of strong anti-interference ability, increased accuracy, and good clinical practicability

Pending Publication Date: 2021-11-02
DIASYS DIAGNOSTIC SYST SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has poor stability of the used purine nucleoside phosphorylase and xanthine oxidase, and there is also the phenomenon that ALP in the serum will interfere with the overall reaction
On the other hand, the validity period of the existing 5'-nucleotidase calibrator and its detection kit is generally 12 months, which is relatively not long enough.

Method used

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  • Liquid-stable 5 '-nucleotidase calibrator, detection kit and application thereof
  • Liquid-stable 5 '-nucleotidase calibrator, detection kit and application thereof
  • Liquid-stable 5 '-nucleotidase calibrator, detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the screening of liquid stable 5'-nucleotidase calibrator stabilizer

[0050] 1.1. Preparation of liquid stable 5'-nucleotidase solution:

[0051] Table 1

[0052]

[0053] The following substances 1) to 4) are used as stabilizers for 5'-nucleotidase solutions

[0054] 1) 0.5%, 1.0%, 2.0% Prionex

[0055] 2) 1.0%, 5.0%, 10.0% glycerin

[0056] 3) 5.0%, 10.0%, 20.0% trehalose

[0057] 4) 0.5%, 1.0%, 2.0% bovine serum albumin (BSA)

[0058] 1.2. The assay method of 5'-nucleotidase is as follows:

[0059] The test conditions for 5'-nucleotidase are as follows:

[0060] Table 2

[0061] wavelength 546nm Cuvette diameter 1.0cm temperature 37℃ analysis type rate method detection Subtract reagent blank

[0062] The reagents and samples used are as follows:

[0063] R1 (reagent 1) (see Table 4)

[0064] R2 (reagent 2) (see Table 5)

[0065] S (calibrator or sample) (see Table 1 or 3)

[0066] The operation ...

Embodiment 2

[0073] Embodiment 2: the preparation of liquid stable 5'-nucleotidase calibrator

[0074] 1.1. Preparation of 5'-nucleotidase calibrator:

[0075] table 3

[0076]

[0077] 1.2, The assay method of 5'-nucleotidase is the same as Example 1 of the present invention.

[0078] 1.3 Thermal stability of 5'-nucleotidase liquid calibrator:

[0079] Store the prepared liquid 5'-nucleotidase calibrator at 37°C, take it out after 0, 4, 7, 14, 21, and 28 days and use a commercially available 5'-nucleotidase detection kit for detection In the test, the measured value at each time point is compared with 0 day at 37°C, and each measured value is recorded. The specific results are as follows: image 3 shown.

[0080] image 3 The results show that the loss of enzyme activity of the liquid 5'-nucleotidase calibrator at 37°C for 28 days is less than ±3%, and the thermal stability is good.

Embodiment 3

[0081] Embodiment 3: Optimization of 5'-nucleotidase detection kit anti-ALP interference performance

[0082] 1.1 Preparation of 5'-nucleotidase detection kit

[0083] Table 4

[0084]

[0085]

[0086] table 5

[0087]

[0088] 1.2, The assay method of 5'-nucleotidase is the same as Example 1 of the present invention.

[0089] 1.3. Preparation of alkaline phosphatase (ALP) interference samples:

[0090] (1) Take 60 μL of pure ALP product with a fixed value (known concentration is 15000U / L) and add it to 540 μL of clinical serum, mix well and use it as an interference sample;

[0091] (2) Draw 60 μL of normal saline (0.9% NaCl) and add it to 540 μL of clinical serum, mix well and use it as a blank sample;

[0092] (3) Mix according to the following ratio, and obtain the ALP interference sample after mixing. The specific dilution ratio operation method is shown in the table below:

[0093] Table 6

[0094] ALP concentration (U / L) Blank sample (μL) I...

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Abstract

The invention discloses a liquid stable 5 '-nucleotidase calibrator. The calibrator comprises the following components: a buffer solution, a stabilizer, a preservative and 5'-nucleotidase. The invention also discloses a 5 '-nucleotidase detection kit. The kit comprises a 5'-nucleotidase R1 reagent and a 5 '-nucleotidase R2 reagent. The 5 '-nucleotidase R1 reagent is an enzyme reaction system and consists of a first buffer solution, a stabilizer, a first preservative, 4-aminoantipyrine, an enzyme activator, purine nucleoside phosphorylase (PNP), xanthine oxidase (XOD) and peroxidase (POD); the 5 '-nucleotidase R2 reagent is a chromogenic system and is composed of a second buffer solution, a second preservative, inosine-5'-disodium phosphate, beta-sodium glycerophosphate and a Trinder chromogenic substrate. The invention also discloses an application of the 5 '-nucleotidase detection kit in determination of 5'-nucleotidase.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnosis, and relates to a liquid stable 5'-nucleotidase calibrator and detection kit and applications thereof. Background technique [0002] 5'-Nucleotidase (5'-Nucleotidase, 5'-NT) is a specific phosphatase that catalyzes the hydrolysis of phosphate bonds in nucleotide molecules. Epidermal or endothelial barrier function and lymphocyte adhesion, recirculation, immune response and other aspects play an important role. As a glycoprotein, human serum 5'-nucleotidase is composed of two subunits with a molecular weight of 70000D and is a dimer with a hydrophobic structure. The optimum pH is 6.6-7.0, subject to Mg 2+ or Mn 2+ Activated by Ni 2+ inhibition. [0003] 5'-Nucleotidases are widely present in the cell membranes of tissues, and in addition to the liver and gallbladder, they are also commonly found in the brain, heart, lung, small intestine, and renal tubules. However, the 5'-NT relea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/44C12Q1/28C12Q1/26C12Q1/48
CPCC12Q1/44C12Q1/28C12Q1/26C12Q1/48C12Q2326/96G01N2333/91142G01N2333/9029
Inventor 吕良杰张杰张艳邹艳芳
Owner DIASYS DIAGNOSTIC SYST SHANGHAI
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