CN106290323A

CN106290323A - A kind of stable, uric acid reagent that capacity of resisting disturbance is strong and detection method

Info

Publication number: CN106290323A
Application number: CN201510305396.5A
Authority: CN
Inventors: 谭伯清; 李志明; 甘宜梧; 肖慧; 王绮
Original assignee: Zhangqiu Makall Yi Medical Instrument Ltd Co
Current assignee: Zhangqiu Makall Yi Medical Instrument Ltd Co
Priority date: 2015-06-04
Filing date: 2015-06-04
Publication date: 2017-01-04

Abstract

The present invention relates to uric acid detection technique field, particularly to a kind of uric acid detectable, containing buffer in reagent R1,4-AA, BSA, sucrose, trehalose, peroxidase, ascorbic acid oxidase, Bilirubin oxidase, alkylphenol polyoxyethylene (APEO), preservative；Containing buffer in reagent R2, TOOS uricase_·BSA sucrose_·Trehalose alkylphenol polyoxyethylene (APEO), preservative.Use PIPES(piperazine-Isosorbide-5-Nitrae-two ethyl sulfonic acid) buffer, adds stabilizer BSA, sucrose, trehalose, greatly strengthen the stability of reagent；Use novel surfactant alkylphenol polyoxyethylene (APEO), not only significantly improve the mensuration performance of product, and enhance stability and the capacity of resisting disturbance of reagent.

Description

A kind of stable, uric acid reagent that capacity of resisting disturbance is strong and detection method

Technical field

The present invention relates to uric acid detection technique field, particularly to a kind of uric acid detectable, further relate to use the detection method of this detectable.

Background technology

Uric acid is the end product of purine catabolism.Excreted with urine by kidney.In health adult's body, uric acid content is about 1.1g, and the most about 15% is present in blood, and uric acid in blood is after glomerular filtration, and major part is by reabsorption.Uric acid is one of non-protein nitrogen important component in blood plasma, and when serious kidney damage, in blood, uric acid can significantly raise, and slight impaired time change little.Therefore changes of serum uric acid is the sensitive indicator that diagnosis kidney severe is impaired.

At present, the detection method of uric acid (UA) has phosphotungstic acid reducing process, urinary acidification and HPLC method.Wolframic acid reducing process is the most clinical the more commonly used method, utilizes the uric acid in nonprotein filtrate can be oxidized to allantoin and carbon dioxide by phosphotungstic acid in alkaline environment, and phosphotungstic acid is reduced into tungsten blue (tungsten Blue), according to tungsten blue shade, with 710nm wavelength filter or red filter disc, with the same standard comparing processed, carry out colorimetric, with blank zeroising, its concentration value can be obtained.The method is simple to operate, but can not test by this method when having haemolysis sample, and easily by shortcomings such as other reducing substances interference.Although HPLC detection method is simple, but sample pretreatment is more complicated, and apparatus expensive is difficult to popularize.And present most popular method is uricase-peroxidase conjugate method, this method is sensitive and need not Deproteinization, but is easily affected by the interfering material such as vitamin C and bilirubin.

Given this, the present invention is on the basis of uricase-peroxidase conjugate method, optimizing reaction system, use PIPES buffer, add multiple anti-interference material and stabilizer, significantly improve capacity of resisting disturbance and the stability of reagent, be a kind of more stable, uric acid (UA) reagent that capacity of resisting disturbance is strong.

Summary of the invention

It is an object of the invention to provide a kind of reagent for detecting uric acid (UA) and the method using this reagent detection uric acid content.This test kit uses uricase-peroxidase conjugate method, can effectively detect the content of uric acid, has the advantages such as strong, the good stability of capacity of resisting disturbance.

Ultimate principle:

Uric acid, under uricase effect, generates allantoin and hydrogen peroxide (H₂O₂), recycling Trinder response system, hydrogen peroxide, under peroxidase (POD) is catalyzed, with 4-AA (4-AAP) and N-ethyl-meta-aminotoluene propanesulfonate (TOOS), generates reddish violet pigment.The color of reactant liquor is directly proportional to uric acid concentration.

。

The present invention is obtained through the following steps:

A kind of uric acid detectable, including reagent R1 and reagent R2, the composition of described reagent R1 and reagent R2 is as follows.

Reagent R1 contains:

Buffer_{···············································································}100mmol/L

4-AA_{································································}1.3mmol/L

BSA_{···················································································}1g/L

Sucrose_{···················································································}5g/L

Trehalose_{···············································································}2g/L

Peroxidase_{·······································································}12KU/L

Ascorbic acid oxidase_{·······························································}5KU/L

Bilirubin oxidase_{···································································}8KU/L

Alkylphenol polyoxyethylene (APEO)_{·············································}2g/L

Preservative_{················································································}0.5g/L。

The component of reagent R2 is:

Buffer_{················································································}100mmol/L

TOOS_{··················································································}13mmol/L

Uricase_{·················································································}2KU/L

BSA_{·····················································································}1g/L

Sucrose_{·····················································································}5g/L

Trehalose_{·················································································}2g/L

Alkylphenol polyoxyethylene (APEO)_{···············································}2g/L

Preservative_{··················································································}0.5g/L。

Described uric acid detectable, in reagent R1, buffer is 25 DEG C, and pH is piperazine-Isosorbide-5-Nitrae-two ethanesulfonic acid buffer of 7.6.

Described uric acid detectable, in reagent R2, buffer is 25 DEG C, and pH is piperazine-Isosorbide-5-Nitrae-two ethanesulfonic acid buffer of 7.6.

Described uric acid detectable, described preservative is NaN₃。

The detection method of described uric acid detectable detection uric acid, uses automatic clinical chemistry analyzer to utilize end-point method to be measured, and detection dominant wavelength is 546nm.

Described detection method, the ratio of R1 reagent and R2 reagent is 4:1.

Beneficial effects of the present invention:

1) use new buffer system and stabilizer, significantly improve the stability of reagent；

2) use novel surfactant alkylphenol polyoxyethylene (APEO), not only significantly improve the performance of mensuration, and enhance stability and the capacity of resisting disturbance of reagent；

3) accuracy of reagent and having good stability, low price, 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 stability curve figures.

Detailed description of the invention

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

Embodiment 1 :

The detectable of uric acid, including reagent R1 and reagent R2.

Its R1 consists of:

PIPES(piperazine-Isosorbide-5-Nitrae-two ethyl sulfonic acid) buffer (pH=7.6,25 DEG C)_{····························}100mmol/L

4-AA_{····························································································}1.3mmol/L

BSA_{···············································································································}1g/L

Sucrose_{···············································································································}5g/L

Trehalose_{···········································································································}2g/L

Peroxidase_{···································································································}12KU/L

Ascorbic acid oxidase_{···························································································}5KU/L

Bilirubin oxidase_{·······························································································}8KU/L

Alkylphenol polyoxyethylene (APEO)_{·········································································}2g/L

Liquid BPF aN₃ _{·································································································}0.5g/L。

The component of reagent R2 is:

PIPES(piperazine-Isosorbide-5-Nitrae-two ethyl sulfonic acid) buffer (pH=7.6,25 DEG C)_{··························}100mmol/L

TOOS_{···········································································································}13mmol/L

Uricase_{··········································································································}2KU/L

BSA_{··············································································································}1g/L

Sucrose_{··············································································································}5g/L

Trehalose_{··········································································································}2g/L

The using method of the present embodiment reagent:

The uric acid detectable that the present embodiment describes, uses the automatic clinical chemistry analyzer with double reagent function in use, such as Hitachi 7180 fully-automatic analyzer etc., utilizes end-point method to be measured.Being placed on the reagent position of correspondence according to the ratio of 4:1 by R1 and R2, the correspondence position at specimen disc places distilled water, standard substance and sample, and operation is such as table 1.

Table 1 embodiment 1 reagent test method

Calculate: uric acid content (μ mol /L )=( ∆ A Measure ÷ ∆ A Standard) × C Standard.

Embodiment 2 :

Interference is tested: takes fresh mix serum, is divided into 2 equal portions, then every equal portions is separated into 5 equal portions, add different interfering materials so that it is the concentration in serum reaches the requirement of table 2.The most respectively with embodiment 1 gained reagent, the content of UA in and uric acid (UA) the reagent comparative determination serum simultaneously approved common with market, matched group measurement result with add disturbance material after the measurement result of each group be shown 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 Table 2, test result is not substantially interfered with by embodiment 1 reagent at ascorbic acid≤1704 μm ol/L, bilirubin≤684 μm ol/L, hemoglobin≤10 g/L, triglyceride≤22.6 mmol/L.And matched group reagent is in the presence of above-mentioned concentration interfering material, being substantially interfered with, the interference free performance of this explanation embodiment 1 reagent is far superior to contrast agent.

Table 2 Embodiment reagent interference free performance compares

。

Embodiment 3

Dependency is tested: utilizing embodiment 1 formula reagent preparation, the uric acid reagent box of certain company of the State Food and Drug Administration accreditation common with market to carry out control test, have detected 20 clinical serum samples simultaneously, testing result is as shown in table 3.And obtain the correlation curve (as shown in Figure 1) of two kinds of reagent, and being shown by testing result, the correlation coefficient of two test kits is 0.9992, illustrates that both have great dependency.

Table 3 Embodiment 1 Reagent and market is common and the testing uric acid test kit comparison and detection result that gets the nod

。

Embodiment 4

The stability contrast test of reagent: to the reagent in embodiment 1, uniform subpackage 13 groups, the amount of reagent often organized be R1 be 20mL, R2 be 5mL；And uric acid (UA) test kit taking certain company of 13 groups of common State Food and Drug Administration's accreditations in market compares.Being placed in 2-8 DEG C of refrigerator, (target value is 340 to taking-up on the same day group reagent detection UA quality-control product monthlyμ mol /L), testing result as in figure 2 it is shown, the uric acid (UA) more common than market under 2-8 DEG C of condition of storage of embodiment 1 reagent to measure test kit more stable.

By checking, this reagent is good with similar detectable contrast dependency, and Clinical detection sample results is consistent, it is possible to reach the market application requirement to product, and good in anti-interference performance, is a kind of more stable uric acid (UA) detectable.

Claims

1. a uric acid detectable, it is characterised in that including reagent R1 and reagent R2, the composition of described reagent R1 and reagent R2 is as follows:

1) component of reagent R1 is:

Buffer 100mmol/L

4-AA 1.3mmol/L

BSA···················································································1g/L

Sucrose 5g/L

Trehalose 2g/L

Peroxidase 12KU/L

Ascorbic acid oxidase 5KU/L

Bilirubin oxidase 8KU/L

Alkylphenol polyoxyethylene (APEO) 2g/L

Preservative 0.5g/L

2) component of reagent R2 is:

Buffer 100mmol/L

TOOS·················································································13mmol/L

Uricase 2KU/L

BSA····················································································1g/L

Sucrose 5g/L

Trehalose 2g/L

Alkylphenol polyoxyethylene (APEO) 2g/L

Preservative 0.5g/L

Uric acid detectable the most according to claim 1, it is characterised in that in reagent R1, buffer is 25 DEG C, pH is piperazine-Isosorbide-5-Nitrae-two ethanesulfonic acid buffer of 7.6.

Uric acid detectable the most according to claim 1, it is characterised in that in reagent R2, buffer is 25 DEG C, pH is piperazine-Isosorbide-5-Nitrae-two ethanesulfonic acid buffer of 7.6.

Uric acid detectable the most according to claim 1, it is characterised in that described preservative is NaN₃。

5. the uric acid detectable that a kind uses according to any one of claim 1-4 is to detect the detection method of uric acid, it is characterised in that using automatic clinical chemistry analyzer to utilize end-point method to be measured, detection dominant wavelength is 405nm.

Detection method the most according to claim 5, it is characterised in that the ratio of R1 reagent and R2 reagent is 4:1.