A kind of stable, Serum Adenosine Deaminase detectable that capacity of resisting disturbance is strong and detectionMethodTechnical field
The present invention relates to Serum Adenosine Deaminase detection technique field, particularly to a kind of Serum Adenosine Deaminase detection examinationAgent, further relates to use the detection method of this detectable.
Background technology
ADA Adenosine deaminase (ADA) is the important enzyme in human body fast cry of certain animals nucleotide metabolism, is distributed widely in human body and respectively organizesIn, wherein the highest with content in thymus, spleen and other lymphoid tissues, and liver, lung, kidney and skeletal muscle equal size are relatively low；In serumADA essentially from liver, be the sensitive indicator of reflection hepatic injury, can be as one of liver function routine examination project, according to shouldEnzymatic activity increases or reduces reflection hepatocyte injury and recovery extent；Chronic hemolysis erythrocyte ADA activity is notable to be raised；SwollenIn tumor patients serum and tissue, ADA activity all raises；Patients with Tuberculous Meningitis CSF-ADA is significantly raised.
At present, the assay method of ADA Adenosine deaminase has Podbielniak development process, Ammonia Gas Sensor Electrode Method, enzyme process, enzyme coupling method etc.；RippleFamily name's development process reagent is simple, easily operated, but this method is easily affected by exogenous ammonium radical ion；Although Ammonia Gas Sensor Electrode Method is anti-dryAbility of disturbing is relatively strong, but is difficult to control to response signal and the response time of electrode, is unsuitable for the mensuration of clinical batch samples；Enzyme processSensitiveer, can realize automated analysis, but repeatability and accuracy are poor；Enzyme coupling method result is accurate, simple and practical, but is easily subject toThe material such as ascorbic acid and bilirubin disturbs.
In consideration of it, the present invention is on the basis of enzyme coupling method, optimizing reaction system, add Bilirubin oxidase and Vitamin CAcid oxidase, can be prevented effectively from the interference of bilirubin and ascorbic acid, is greatly enhanced the capacity of resisting disturbance of reagent, and usesNew Trinder reacts chromogen substance HTBA(3-hydroxyl-2,4,6-Triiodobenzoic acids), significantly enhance reagent stability andAccuracy；Additionally, by adding stabilizer, such as glycerol, polyethylene glycol 6000, mannitol, trehalose, BSA, ethylene glycol etc. canIt is effectively improved the stability of reagent；And the addition of preferred new non-ionic surfactants alkyl polyglucoside (APG) can prevent insteadAnswer system muddy, strengthen the stability of substrate, improve the capacity of resisting disturbance of reagent；This reagent operation is easy quickly, it is adaptable to fromDynamic fractional analysis, is a kind of more stable, Serum Adenosine Deaminase (ADA) reagent that capacity of resisting disturbance is strong.
Summary of the invention
It is an object of the invention to provide a kind of for detecting the reagent of Serum Adenosine Deaminase (ADA) and using this reagent to examineThe method surveying Serum Adenosine Deaminase activity.This test kit uses enzyme coupling method, can effectively detect Serum Adenosine DeaminaseActivity, the advantages such as capacity of resisting disturbance is strong, good stability.
Ultimate principle:
ADA(ADA Adenosine deaminase) catalytic substrate adenosine deamination generation inosine, inosine is again through PNP(purineNucleoside phosphorylase) effect generation hypoxanthine.Hypoxanthine is at XOD(xanthine oxidase) under effect, generate uric acid and mistakeHydrogen oxide (H2O2).Hydrogen peroxide (H2O2), can be with 3-hydroxyl-2 in the presence of peroxidase, 4,6-triiodo-benzenesFormic acid (HTBA) and 4-amino ammonia produce coloured awake compounds for pyrrole quinoline (4-AA) reaction, the generation speed of coloured awake compoundsRate and ADA(ADA Adenosine deaminase) activity relevant.By measuring the climbing speed of absorbance, the most coloured awake class at 550nm wavelengthCompound generating rate measures the activity of ADA Adenosine deaminase.
The present invention is obtained through the following steps:
A kind of Serum Adenosine Deaminase detectable, including reagent R1 and reagent R2, the composition of described reagent R1 and reagent R2 is such asUnder:
Reagent R1 contains
Buffer··························100mmol/L,
4-amino ammonia replaces pyrrole quinoline··················5mmol/L,
PNP(purine nucleoside phosphorylase)···········1.5KU/L,
XOD(xanthine oxidase)···············3.1KU/L,
POD(peroxidase)·················5KU/L,
Bilirubin oxidase····················3KU/L,
Ascorbic acid oxidase··················3.5KU/L,
Glycerol··························4ml/L,
Polyethylene glycol 6000····················5g/L,
Ethylene glycol··························6ml/L,
Mannitol··························25g/L,
Trehalose··························15g/L,
BSA·····························1g/L,
Alkyl polyglucoside (APG)···················1g/L,
Preservative··························0.5g/L；
2) component of reagent R2 is:
Buffer··························100mmol/L,
Adenosine····························14mmol/L,
HTBA(3-hydroxyl-2,4,6-Triiodobenzoic acid)···5mmol/L,
Glycerol··························4ml/L,
Polyethylene glycol 6000····················5g/L,
Ethylene glycol··························6ml/L,
Mannitol··························25g/L,
Trehalose··························15g/L,
BSA·····························1g/L,
Alkyl polyglucoside (APG)···················1g/L,
Preservative··························0.5g/L。
Described Serum Adenosine Deaminase detectable, in reagent R1, buffer is 25 DEG C, and pH is the HEPES buffering of 8.0Liquid.
Described Serum Adenosine Deaminase detectable, in reagent R2, buffer is 25 DEG C, and pH is the Tris buffering of 4.0Liquid.
Described Serum Adenosine Deaminase detectable, described preservative is NaN3.
Described Serum Adenosine Deaminase detectable detects the detection method of Serum Adenosine Deaminase content, uses completeAutomatic biochemistry analyzer utilizes performance rate method to be measured, and detection dominant wavelength is 550nm.
Described detection method, the ratio of R1 reagent and R2 reagent is 2:1.
Beneficial effects of the present invention:
1) add Bilirubin oxidase and ascorbic acid oxidase, the interference of bilirubin and ascorbic acid can be prevented effectively from, greatlyThe capacity of resisting disturbance of big Contrast agent；
2) new Trinder is used to react chromogen substance HTBA(3-hydroxyl-2,4,6-Triiodobenzoic acids), significantly enhance reagentStability and accuracy；
3) addition of new non-ionic surfactants alkyl polyglucoside (APG) can prevent reaction system muddy, strengthens the steady of substrateQualitative, improve the capacity of resisting disturbance of reagent；
4) optimizing reaction system to add glycerol, polyethylene glycol 6000, mannitol, trehalose, BSA, ethylene glycol etc. multiple surelyDetermine agent, the stability of reagent can be significantly improved；
5) accuracy of reagent and having good stability, strong interference immunity, 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,
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 Serum Adenosine Deaminase that is common with market and that get the nod measures test kit comparison and detection knotReally.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described.
Embodiment 1
The detectable of Serum Adenosine Deaminase, including reagent R1 and reagent R2:
1) the consisting of of its R1:
HEPES(4-hydroxyethyl piperazine ethanesulfonic acid) buffer (pH=8.0,25 DEG C)··100mmol/L,
4-amino ammonia replaces pyrrole quinoline·································5mmol/L,
PNP(purine nucleoside phosphorylase)··························1.5KU/L,
XOD(xanthine oxidase)······························3.1KU/L,
POD(peroxidase)································5KU/L,
Bilirubin oxidase···································3KU/L,
Ascorbic acid oxidase·································3.5KU/L,
Glycerol·········································4ml/L,
Polyethylene glycol 6000···································5g/L,
Ethylene glycol·········································6ml/L,
Mannitol·········································25g/L,
Trehalose·········································15g/L,
BSA············································1g/L,
Alkyl polyglucoside (APG)··································1g/L,
Liquid BPF aN3·····································0.5g/L；
2) component of reagent R2 is:
Tris(trishydroxymethylaminomethane) buffer (pH=4.0,25 DEG C)·····100mmol/L,
Adenosine···········································14mmol/L,
HTBA(3-hydroxyl-2,4,6-Triiodobenzoic acid)··················5mmol/L,
Glycerol·········································4ml/L,
Polyethylene glycol 6000···································5g/L,
Ethylene glycol·········································6ml/L,
Mannitol·········································25g/L,
Trehalose·········································15g/L,
BSA············································1g/L,
Alkyl polyglucoside (APG)··································1g/L,
Liquid BPF aN3·····································0.5g/L；
3) using method of the present embodiment reagent:
The Serum Adenosine Deaminase detectable that the present embodiment describes, uses in use and has the full-automatic raw of double reagent functionFractional analysis instrument, such as Hitachi 7180 fully-automatic analyzer etc., utilizes performance rate method to be measured；R1 and R2 is put according to the ratio of 2:1Putting on the reagent position of correspondence, the correspondence position at specimen disc places distilled water, standard substance and sample, and operation is such as Fig. 3.
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, adds different doingDisturb material so that it is the concentration in serum reaches the requirement of Fig. 4；The most respectively with embodiment 1 gained reagent, common with market alsoThe activity of ADA, matched group measurement result and addition in Serum Adenosine Deaminase (ADA) reagent of accreditation comparative determination serum simultaneouslyAfter disturbance material, the measurement result of each group is shown in Fig. 4；Relative deviation (%)=(mensuration average-check sample of interference sampleMensuration average) mensuration average × 100% of/check sample；
As seen from Figure 4, embodiment 1 reagent is in total bilirubin≤30mg/dL, triglyceride≤3000mg/dL, hemoglobinWhen≤800mg/dL, ascorbic acid≤220mg/dL, test result is not substantially interfered with, and matched group reagent is in above-mentioned concentrationIn the presence of interfering material, being substantially interfered with, this explanation is by optimizing reaction buffer system, Bilirubin oxidase and ascorbic acidAfter oxidase, and new non-ionic surfactants alkyl polyglucoside (APG), the interference free performance of embodiment 1 reagent significantly improves,It is far superior to contrast agent.
Embodiment 3
Dependency is tested: utilize embodiment 1 formula reagent preparation, the State Food and Drug Administration accreditation common with marketADA Adenosine deaminase (ADA) test kit of certain company carry out control test, have detected 20 clinical serum samples, detection knot simultaneouslyFruit as it is shown in figure 5, and obtain the correlation curve (as shown in Figure 1) of two kinds of reagent, shown by testing result, two reagentThe correlation coefficient of box is 0.9996, illustrates that both have great dependency.
Embodiment 4
The stability contrast test of reagent: to the reagent in embodiment 1, uniform subpackage 13 groups, amount of reagent R1 often organized is 20mL,R2 is 10mL；And take the ADA Adenosine deaminase of certain company of 13 groups of common State Food and Drug Administration's accreditations in market(ADA) test kit compares；It is placed in 2-8 DEG C of refrigerator, taking-up on the same day group reagent detection ADA quality-control product (target monthlyValue is for 35.7U/L), testing result is as in figure 2 it is shown, the adenosine more common than market under 2-8 DEG C of condition of storage of embodiment 1 reagent takes offIt is more stable that ammonia enzyme (ADA) measures test kit；
By checking, this reagent is good with similar detectable contrast dependency, and Clinical detection sample results is consistent, it is possible to reach cityThe field application requirement to product, and good in anti-interference performance, be a kind of more stable, good ADA Adenosine deaminase (ADA) detectionReagent.