CN105238847A

CN105238847A - Stable adenosine deaminase reagent high in anti-interference capability and detection method

Info

Publication number: CN105238847A
Application number: CN201510588862.5A
Authority: CN
Inventors: 甘宜梧; 史秀明; 谢清华; 谭柏清; 王绮; 赵新
Original assignee: Biobase Biodustry Shandong Co Ltd
Current assignee: Biobase Biodustry Shandong Co Ltd
Priority date: 2015-09-16
Filing date: 2015-09-16
Publication date: 2016-01-13

Abstract

The invention relates to the technical field of adenosine deaminase detection, in particular to an adenosine deaminase detection reagent. A reagent R1 comprises a buffer solution, 4-aminoantipyrine, BSA, cane sugar, trehalose, peroxidase, ascorbic acid oxidase, bilirubin oxidase and preservatives; a reagent R2 comprises a buffer solution, adenosine, EHSPT, BSA, cane sugar and preservatives. The adenosine deaminase detection reagent has the advantages that the phosphate buffer solution is adopted, the stabilizer BSA, the cane sugar and the trehalose are added, and accordingly, stability of the reagent is improved greatly; determination performance is improved remarkably, and stability and anti-interference capability of the reagent are enhanced.

Description

A kind of stable, adenosine deaminase reagent that immunity from interference is strong and detection method

Technical field

The present invention relates to adenosine detection technique field, particularly a kind of adenosine deaminase detection reagent, also relate to the detection method using this detection reagent.

Background technology

Adenosine deaminase (ADA) is a kind of purine nucleotides katabolism enzyme of having important relationship active in Cellular Immunity, and late 1950s, zymetology field is introduced in ADA determination of activity, at present for the diagnosis of various diseases.ADA can produce irreversible deamination reaction by specific catalytic adenosine.ADA activity reduces will affect nucleosides synthesis, make lymphoid progenitor cell be converted into lymphoblast and the reduction of plasmacytic ability, thus will cause immunologically competent cell to reduce, hypofunction.In body fluid ADA active oneself become the important clinical index that the Diagnosis and differential diaggnosis of the diseases such as malignant tumour, tuberculosis, central nervous system disease, hepatopathy and typhoid fever, observation of curative effect and the state of an illness follow.Serum ADA activity raises, be common in hepatitis, obstructive jaundice, prostate gland and bladder cancer that liver cirrhosis, hemochromatosis, tumour cause, hemolytic anemia, typhoid fever, gout, tuberculosis and heart failure etc.Measure the research to the diagnosis of some disease, differential diagnosis, treatment and immunologic function of blood, ADA in body fluid and isozyme level thereof and be more and more subject to clinical attention.

At present, the measuring method of the ADA activity of bibliographical information has Ammonia Gas Sensor Electrode Method, fluorescent method, chemoluminescence method, isotope method, high performance liquid chromatography etc.These method plant and instrument require high, and analysis cost is high, are difficult to popularize application in an all-round way.Measuring common method in current body fluid is ammonia reagent colorimetry.This method is simple to operate, is easy to promote, but can not be used for the mensuration of batch sample.

Given this, the present invention generates on the basis of ammonia and inosine reaction in the hydrolysis of ADA catalysis adenosine, coupling three enzymatic reactions successively again, establishing one can be used for manual operations, and the four enzyme couplings that can be used for again automatic biochemistry analyzer measure the novel method of ADA activity.

Summary of the invention

The object of this invention is to provide one for detecting the reagent of adenosine deaminase (ADA) and using this reagent to detect the method for ADA content.This test kit adopts four enzyme coupling methods, effectively can detect the content of ADA, and immunity from interference is strong, the advantages such as good stability.

Ultimate principle: adenosine, under ADA effect, generates inosine and ammonia,

ADA

Adenosine+H ₂o inosine+NH ₃

Inosine, under purine nucleoside phosphorylase (PNP) catalysis, generates xanthoglobulin and ribose 1-phosphoric acid with phosphorus reaction

PNP

Inosine+Pi xanthoglobulin+ribose 1-phosphoric acid

Xanthoglobulin generates uric acid and H at XOD (XOD) catalyzed oxidation ₂o ₂:

XOD

Xanthoglobulin+2H ₂o+O ₂ uric acid+2H ₂o ₂

Coupling is by the reaction of Catalyzed Synthesis By Peroxidase again

POD

2H ₂o ₂+ 4-AA+EHSPT 4H ₂o+ quinone dyestuff

ADA activity can be recorded at the rate of change (Δ A/min) of 546nm place absorbancy by measuring red naphtoquinone compounds.

The present invention is obtained by following steps:

A kind of ADA examines sour test agent, and comprise reagent R1 and reagent R2, described reagent R1's and reagent R2 is composed as follows:

Contain in reagent R1

Damping fluid 100mmol/L

4-AA _{·············································································}4mmol/L

PNP _{···············································································}0.3U/mL

XOD _{···········································································}0.4U/mL

POD _{············································································}0.1U/mL

Sanitas 0.5g/L;

The component of reagent R2 is:

Damping fluid 100mmol/L

Adenosine 11mmol/L

EHSPT _{····································································}4mmol/L

The phosphate buffer soln of pH7.4 all used by above reagent.

Above ADA detection reagent, described sanitas is NaN ₃.

Described ADA detection reagent detects the detection method of ADA, and use automatic clinical chemistry analyzer to utilize rate method to measure, detection predominant wavelength is 546nm.

Described detection method, the ratio of R1 reagent and R2 reagent is 2:1.

Beneficial effect of the present invention:

1) adopt new buffer system and stablizer, significantly improve the stability of reagent;

2) adopt four enzyme coupling methods, not only significantly improve the performance measured, and enhance immunity from interference, suitable batch sample automatical analysis;

3) reagent accuracy and have good stability, easy to use, can meet clinical needs completely.

Accompanying drawing explanation

Fig. 1 is the correlation curve figure of two kinds of reagent,

Fig. 2 is two kinds of reagent effect phase beta stability line figure.

Fig. 3 is embodiment 1 reagent test method,

Fig. 4 is that embodiment reagent interference free performance compares,

Fig. 5 is that embodiment 1 reagent and market are common and the adenosine deoxygenase got the nod measures test kit comparison and detection result.

Embodiment

Below in conjunction with specific embodiment, the present invention is further described:

embodiment 1

The detection reagent of adenosine deaminase, bag reagent R1 and reagent R2:

1) the consisting of of its R1:

Damping fluid 100mmol/L

4-AA _{···············································································}4mmol/L

PNP _{················································································}0.3U/mL

XOD _{················································································}0.4U/mL

POD _{··················································································}0.1U/mL

BSA _{·····················································································}1g/L，

Sucrose 5g/L,

Trehalose 2g/L,

Peroxidase 12KU/L,

Vitamin C oxidase 5KU/L,

Bilirubin oxidase 8KU/L,

Liquid BPF aN ₃0.5g/L;

2) component of reagent R2 is:

Damping fluid 100mmol/L

Adenosine 11mmol/L

EHSPT _{··················································································}4mmol/L

BSA _{···························································································}1g/L，

Sucrose 5g/L,

Trehalose 2g/L,

Liquid BPF aN ₃0.5g/L.

3) using method of the present embodiment reagent:

The ADA detection reagent that the present embodiment describes, adopts the automatic clinical chemistry analyzer with double reagent function in use, as Hitachi 7180 fully-automatic analyzer etc., utilizes rate method to measure.Be placed on corresponding reagent position according to the ratio of 2:1 by R1 and R2, place distilled water, standard substance and sample at the correspondence position of sample disc, operation is as figure tri-.

embodiment 2

Interference is tested: get fresh mix serum, be divided into 2 equal portions, then every equal portions are divided into 5 equal portions again, add different interfering substances, makes its concentration in serum reach the requirement of table 2.Then use embodiment 1 gained reagent respectively, and the adenosine deaminase approved (ADA) reagent common with market is the content of ADA in comparative determination serum simultaneously, control group measurement result with add disturbance material after the measurement result respectively organized in table 2.Mensuration average × 100% of relative deviation (%)=(the mensuration average of the mensuration average-check sample of interference sample)/check sample.

As can be seen from figure tetra-, in xitix≤1704, μm ol/L, bilirubin≤684 μm ol/L, oxyphorase≤10g/L, triglyceride level≤22.6mmol/L obviously do not disturb test result embodiment 1 reagent.And control group reagent is when above-mentioned concentration interfering substance exists, be subject to obvious interference, this illustrates that the interference free performance of embodiment 1 reagent is far superior to contrast agent.

embodiment 3

Dependency is tested: utilize embodiment 1 formulated reagent, the ADA test kit of certain company that the State Food and Drug Administration common with market is approved carries out control test, and have detected 20 clinical serum samples, detected result as shown in Figure 5 simultaneously.And obtain the correlation curve (as shown in Figure 1) of two kinds of reagent, shown by detected result, the relation conefficient of two test kits is 0.9992, and describing both has great dependency.

embodiment 4

The stability simultaneous test of reagent: to the reagent in embodiment 1, even packing 13 groups, the amount of reagent often organized is R1 be 20mL, R2 is 10mL; And adenosine deaminase (ADA) test kit getting certain company of the common State Food and Drug Administration's accreditation in 13 groups of market compares.Be placed in 2-8 DEG C of refrigerator, a taking-up on the same day group reagent monthly detects ADA quality control product, and as shown in Figure 2, it is more stable that the adenosine deaminase (ADA) more common than market under 2-8 DEG C of condition of storage of embodiment 1 reagent measures test kit to detected result.

By checking, it is good that this reagent and similar detection reagent contrast dependency, and clinical detection sample results is consistent, can reach the application requiring of market to product, and good in anti-interference performance, be a kind of more stable, good adenosine deaminase (ADA) detection reagent.

Claims

1. an adenosine deaminase detection reagent, it is characterized in that comprising reagent R1 and reagent R2, described reagent R1's and reagent R2 is composed as follows:

1) the consisting of of its R1:

Damping fluid 100mmol/L

Sucrose 5g/L,

Trehalose 2g/L,

Peroxidase 12KU/L,

Vitamin C oxidase 5KU/L,

Bilirubin oxidase 8KU/L,

Liquid BPF aN ₃0.5g/L;

2) component of reagent R2 is:

Damping fluid 100mmol/L

Adenosine 11mmol/L

Sucrose 5g/L,

Trehalose 2g/L,

Liquid BPF aN ₃0.5g/L.

2. adenosine deaminase detection reagent according to claim 1, it is characterized in that in reagent R1, damping fluid is 25 DEG C, pH is the phosphate buffered saline buffer of 7.4.

3. adenosine deaminase detection reagent according to claim 1, it is characterized in that in reagent R2, damping fluid is 25 DEG C, pH is the phosphate buffered saline buffer of 7.4.

4. adenosine deaminase detection reagent according to claim 1, is characterized in that described sanitas is NaN ₃.

5. use the adenosine deaminase detection reagent according to any one of claim 1-4 to detect a detection method for adenosine deaminase, it is characterized in that using automatic clinical chemistry analyzer to utilize rate method to measure, detecting predominant wavelength is 546nm.

6. detection method according to claim 5, is characterized in that the ratio of R1 reagent and R2 reagent is 2:1.