CN108690868A - A kind of adenosine deaminase assay kit and its assay method - Google Patents
A kind of adenosine deaminase assay kit and its assay method Download PDFInfo
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Abstract
The present invention provides a kind of adenosine deaminase assay kit, including reagent R1 and reagent R2, the reagent R1 include following component and its concentration:TRIS, HCL, xanthine oxidase, purine nucleoside phosphorylase, 4-AA, peroxidase, Asparagus cochinchinensis ammonia acid sodium;The reagent R2 includes following component and its concentration:TRIS, HCL, adenosine, N- ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt, glycerine.The invention belongs to technical field of biological, seminal plasma fructose detection kit ingredient provided by the invention is simpler, and stability is good, and rapid reaction, cost is relatively low, and the accurate detection to adenosine deaminase may be implemented, the testing result no significant difference with commercialized product.
Description
Technical field
The invention belongs to technical field of biological more particularly to a kind of adenosine deaminase assay kit and its measurement sides
Method.
Background technology
Adenosine deaminase (ADA) is a kind of important enzyme in purine nucleotide cycle, can be catalyzed adenosine and be changed into
Inosine, then hypoxanthine is generated through Phosphorylating Nucleosides enzyme effect, end product of metabolism is uric acid, in nucleic acid metabolism
It has great significance.ADA be distributed widely in the small intestinal mucosa of human body, spleen, liver, kidney, etc. in tissues and cell.In serum
ADA is mainly derived from liver, belongs to the cytoplasm enzyme of liver cell.When liver cell is impaired, downright bad or membrane permeability increases, equal energy
Cause serum ADA is active to increase.Therefore ADA can be used as hepatic sclerosis auxiliary diagnosis, disease progression to monitor preferable biochemistry and refer to
Mark.In addition, ADA is also frequently as the diagnosis index of tuberculous pleurisy, inflammatory pleural effusion and typhoid etc..
Existing ADA activity determination methods include Ammonia Gas Sensor Electrode Method, fluorescence method, high performance liquid chromatography and enzyme coupling method
Deng.Due to enzyme coupling method specificity and it is reproducible, be not necessarily to special instruments and equipment, become the common detection method of ADA.Enzyme is even
Connection method is coupled, and it is real generally to react joint by purine nucleoside phosphorylase (PNP), xanthine oxidase (XOD) and Trider
The active detections of existing ADA.ADA Activity Assay Kits are more, and agent formulations and measurement effect are not quite similar, but generally existing is fast
The poor problem of the stability of purine nucleoside phosphorylase and xanthine oxidase is easy during preservation because enzyme inactivation causes to survey
It is unreliable to determine result.Chinese patent application CN 101514358 discloses a kind of raising investigating adenosine deamiase by coupling enzymatic reaction
The method of reagent stability is included in basic reagent and Ca-EDTA, iron ethylenediaminetetraacetate, sodium molybdate, molybdenum is added
The step of sour ammonium, sodium glutamate, bovine serum albumin(BSA) or superoxide dismutase are as stabilizer can improve the stabilization of reagent
Property, but the dosage of stabilizer is larger, may influence the quick progress of reaction, or even detection is caused to interfere.
Chinese patent application CN 102586397 discloses a kind of enzymatic detection adenosine deaminase kit, by 1 He of reagent
Reagent 2 forms, and reagent 1 includes buffer solution, toos, Triton x-100, sodium benzoate, FeCl3, PNP, Xot, Peo, glycerine
Aldehyde, natrium arsenicum;Reagent 2 includes disodium hydrogen phosphate, KCl, mannitol, proclin300, adenosine, 4- amino for than woods;By
Accelerating agent is added in reagent 1 to accelerate the reaction of adenosine deaminase, the stability that mannitol improves reagent is added in reagent 2,
But reagent is more complex, and stability is still not ideal enough.
Therefore it provides a kind of ingredient is relatively simple, stable reagent, rapid reaction adenosine deaminase assay kit there is weight
Want meaning.
Invention content
To solve problems of the prior art (ingredient is more complex, reagent stability is not ideal enough etc.), the present invention carries
For a kind of adenosine deaminase assay kit, agent formulations are simpler, and stability is good, and rapid reaction, cost is relatively low, may be implemented
Accurate detection to adenosine deaminase, the testing result no significant difference with commercialized product.
The purpose of the present invention will further illustrate by the following detailed description.
The present invention provides a kind of adenosine deaminase assay kit, including reagent R1 and reagent R2, the reagent R1 include
Following component and its concentration:5~20g/L of TRIS, 15~25g/L of 6N HCL, 1~5KU/L of xanthine oxidase, purine nucleosides
1~5KU/L of phosphorylase, 50~80mg/L of 4-AA, 2~10KU/L of peroxidase, Asparagus cochinchinensis ammonia acid sodium 30
~80g/L;The reagent R2 includes following component and its concentration:5~20g/L of TRIS, 15~25g/L of 6N HCL, adenosine 1~
0.1~5g/L of 10g/L, N- ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt, 180~300ml/L of glycerine.
The correlated response formula of testing principle of the present invention is as follows:
The detection of adenosine deaminase, reaction are realized by the generating rate of the dynamic monitoring quinone derivative at wavelength 550nm
Object generating rate is directly proportional to adenosine deaminase in sample (ADA) activity.TRIS is the abbreviation of trishydroxymethylaminomethane;N refers to
Equivalent concentration, 6N HCl, that is, 6mol/LHCl.
Asparagus cochinchinensis ammonia acid sodium has excellent aobvious fresh, antisepsis, is mainly used in food service industry;In addition it applies also for
In pharmaceuticals industry, the raw material as heart disease class drug, liver function-promoter, micro fatigue crack agent and amino acid transfusion etc..XOD belongs to
The more complicated multimeric protein in flavoprotein oxidases, substrate catalytic mechanism is complicated, factors affecting stability compared with
It is more.The present invention is remarkably improved adenosine deaminase measurement it has been unexpectedly found that adding a small amount of Asparagus cochinchinensis ammonia acid sodium in reagent R1
The stability of reagent, asparatate are polyol, and hydrophilic protective layer is contributed to form in reagent R1 systems so that
XOD keeps good stability in water phase, reduces cost, has widened the application range of Asparagus cochinchinensis ammonia acid sodium.
In addition, by adding a certain amount of glycerine in reagent R2, the Precipitation of adenosine is can avoid, is conducive to steady in a long-term
It keeps.
Preferably, the reagent R1 includes following component and its concentration:10~15g/L of TRIS, 18~22g/ of 6N HCL
L, 2~4KU/L of xanthine oxidase, 2~4KU/L of purine nucleoside phosphorylase, 55~65mg/L of 4-AA, peroxide
3~8KU/L of compound enzyme, 40~60g/L of Asparagus cochinchinensis ammonia acid sodium;The reagent R2 includes following component and its concentration:TRIS 10~
15g/L, 18~22g/L of 6N HCL, 2~6g/L of adenosine, N- ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt
0.5~2g/L, 200~300ml/L of glycerine.
It is highly preferred that the reagent R1 includes following component and its concentration:TRIS 12g/L, 6N HCL 20g/L, Huang are fast
Purine oxidizing ferment 3KU/L, purine nucleoside phosphorylase 3KU/L, 4-AA 60mg/L, peroxidase 6KU/L, Tianmen
Winter propylhomoserin sodium 50g/L;The reagent R2 includes following component and its concentration:TRIS 12g/L, 6N HCL 20g/L, adenosine 5g/
L, N- ethyls-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt 0.5g/L, glycerine 200ml/L.
Preferably, the adenosine deaminase assay kit further includes preservative;The reagent R2 further includes preservative.
Preferably, the preservative is potassium sorbate, a concentration of 1g/L.
Preferably, the adenosine deaminase assay kit further includes human serum standard items.
In addition, the present invention also provides the assay method of adenosine deaminase assay kit, include the following steps:Detach serum
Sample, reagent R1 is added into blood serum sample, and after being incubated 3~6min, reagent R2, mixing, by measuring quinone is then added in mixing
Derivative rate of change of absorbance at 550nm measures ADA activity.
Compared with prior art, the beneficial effects of the invention are as follows:The present invention in adenosine deaminase measure reagent by adding
Add a small amount of Asparagus cochinchinensis ammonia acid sodium that can significantly increase the enzyme activity stability of XOD in measure reagent, is surveyed to improve adenosine deaminase
The stability for determining reagent R1 improves the stability of reagent R2 by adding a certain amount of glycerine, can avoid the precipitation analysis of adenosine
Go out, realize the stable detection of adenosine deaminase, in 2~8 degree of conditions long shelf-life up to 18 months, will not be to the active surveys of ADA
Surely it interferes, the quick progress of reaction will not be influenced, reduce cost, the testing result with existing commercialized product is without apparent
Difference.
Description of the drawings
The response curve figure of Fig. 1 formulas one.
The response curve figure of Fig. 2 formulas two.
The response curve figure of Fig. 3 formulas five.
Specific implementation mode
The present invention is described in further details with reference to the accompanying drawings and examples.
In the present invention, involved component is conventional commercial product, or can be obtained by ordinary skill in the art means
.
One adenosine deaminase assay kit of embodiment (formula two)
Adenosine deaminase assay kit includes reagent R1 and reagent R2, the reagent R1 include following component and its dense
Degree:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, xanthine oxidase 3KU/L, purine nucleoside phosphorylase
3KU/L, 4-AA 60mg/L, peroxidase 6KU/L, Asparagus cochinchinensis ammonia acid sodium 50g/L;The reagent R2 includes such as
Lower component and its concentration:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, adenosine 5g/L, N- ethyl-N- (2- hydroxyls
Base -3- sulfopropyls) -3- methylaniline sodium salts 0.5g/L, glycerine 200ml/L.
Two adenosine deaminase assay kit of embodiment
Adenosine deaminase assay kit includes reagent R1 and reagent R2, the reagent R1 include following component and its dense
Degree:TRIS 15g/L, 6N HCL 18g/L, potassium sorbate 1g/L, xanthine oxidase 2KU/L, purine nucleoside phosphorylase
2KU/L, 4-AA 55mg/L, peroxidase 3KU/L, Asparagus cochinchinensis ammonia acid sodium 40g/L;The reagent R2 includes such as
Lower component and its concentration:TRIS 15g/L, 6N HCL 18g/L, potassium sorbate 1g/L, adenosine 3g/L, N- ethyl-N- (2- hydroxyls
Base -3- sulfopropyls) -3- methylaniline sodium salts 1g/L, glycerine 300ml/L.
Three adenosine deaminase assay kit of embodiment
Adenosine deaminase assay kit includes reagent R1 and reagent R2, the reagent R1 include following component and its dense
Degree:TRIS 10g/L, 6N HCL 22g/L, potassium sorbate 1g/L, xanthine oxidase 4KU/L, purine nucleoside phosphorylase
4KU/L, 4-AA 65mg/L, peroxidase 8KU/L, Asparagus cochinchinensis ammonia acid sodium 60g/L;The reagent R2 includes such as
Lower component and its concentration:TRIS 10g/L, 6N HCL 22g/L, potassium sorbate 1g/L, adenosine 6g/L, N- ethyl-N- (2- hydroxyls
Base -3- sulfopropyls) -3- methylaniline sodium salts 2g/L, glycerine 200ml/L.
One adenosine deaminase assay kit of comparative example (formula one)
Adenosine deaminase assay kit includes reagent R1 and reagent R2, the reagent R1 include following component and its dense
Degree:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, xanthine oxidase 3KU/L, purine nucleoside phosphorylase
3KU/L, 4-AA 60mg/L, peroxidase 6KU/L;The reagent R2 includes following component and its concentration:
TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, adenosine 5g/L, N- ethyl-N- (2- hydroxyl -3- sulfopropyls) -3- first
Base aniline sodium salt 0.5g/L, glycerine 200ml/L.
Formula one with formula two difference lies in:Without Asparagus cochinchinensis ammonia acid sodium.
Two adenosine deaminase assay kit of comparative example
Adenosine deaminase assay kit includes reagent R1 and reagent R2, the reagent R1 include following component and its dense
Degree:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, xanthine oxidase 3KU/L, purine nucleoside phosphorylase
3KU/L, 4-AA 60mg/L, peroxidase 6KU/L, sodium glutamate 50g/L;The reagent R2 includes such as the following group
Point and its concentration:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, adenosine 5g/L, N- ethyl-N- (2- hydroxyls -3-
Sulfopropyl) -3- methylaniline sodium salts 0.5g/L, glycerine 200ml/L.
Comparative example two with formula two difference lies in:Asparagus cochinchinensis ammonia acid sodium is free of in reagent R1, contains sodium glutamate.
Three adenosine deaminase assay kit of comparative example
Adenosine deaminase assay kit includes reagent R1 and reagent R2, the reagent R1 include following component and its dense
Degree:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, xanthine oxidase 3KU/L, purine nucleoside phosphorylase
3KU/L, 4-AA 60mg/L, peroxidase 6KU/L, Asparagus cochinchinensis ammonia acid sodium 50g/L;The reagent R2 includes such as
Lower component and its concentration:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, adenosine 5g/L, N- ethyl-N- (2- hydroxyls
Base -3- sulfopropyls) -3- methylaniline sodium salts 0.5g/L.
Comparative example three with formula two difference lies in:Glycerine is free of in reagent R2.
The sample detection comparison of the different detection reagents of test example one
Experiment reagent:Above-mentioned comparative example one (formula one) and embodiment one (formula two) and Guangzhou section side biotechnology
The ADA assay kits (hereinafter referred to as commercially available group) of Co., Ltd's production.Above-mentioned experiment reagent is used for adenosine deamination respectively
40 blood serum samples are calibrated with Olympus AU400 Biochemical Analyzers while being measured in the detection of enzyme simultaneously, and the results are shown in Table 1.
The different detection reagents of table 1 measure the result (unit of 40 blood serum samples:U/L)
Through statistics, commercially available group of measurement result and one measurement result correlation coefficient r=0.9998 of formula, commercially available group of measurement result
With formula two measurement result correlation coefficient r=0.9998, formula one measurement result with formula two measurement result correlation coefficient rs=
0.9999, illustrate that correlation is good, 50g/L Asparagus cochinchinensis ammonia acid sodiums are added in reagent R1 to interfere testing result, match
Side one and formula two and the product no significant difference through State Food and Drug Administration's approval listing, as a result accurately and reliably.
The accelerated stability of the different detection reagents of test example two is investigated
37 degree of accelerated shelf life tests are carried out to formula one, formula two and comparative example two, to investigate the stability of its component,
As a result as shown in table 2 and table 3.
Sample detection result (the unit of the formula of table 2 one and two accelerated destructive test of formula after 1 week:U/L)
Sample detection result (unit of table 3 comparative example, the two accelerated destructive test after 1 week:U/L)
37 degree of accelerated shelf life tests:Refer to and reagent is mounted in bottle and is sealed, is placed on inside 37 degree of water baths, into
Row accelerated shelf life test, 1 week time of 37 degree of destructive tests are equivalent to (2~8 degree) of general storage temperature and preserve 1 year.
As known from Table 2, when not accelerating, 40 sample detection result average values for being formulated one are 45.6, are formulated two detection
As a result average value is 45.5, substantially quite with formula one;After 37 degree of accelerated shelf life tests 1 week, it is formulated one testing result
Average value is 23.9, and the testing result average value for being formulated two is 44.7.As known from Table 3, when not accelerating, 40 samples of comparative example two
Product testing result average value is 45.3, and after 37 degree of accelerated shelf life tests 1 week, the testing result average value of comparative example two is
40.4。
Through statistics:When protective agent not being added in formula one, measured value declines much after acceleration, and fall reaches
47.6%;It is 10.8% that sodium glutamate is added in comparative example two as measured value fall is accelerated after protective agent;And in formula two
Middle addition Asparagus cochinchinensis ammonia acid sodium is only 1.8% as measured value fall is accelerated after protective agent.Fall=(do not accelerate
Mean value after the degree of value -37 accelerates 1 week)/do not accelerate mean value.As it can be seen that Asparagus cochinchinensis ammonia acid sodium is added can significantly increase gland as protective agent
The stability of guanosine deaminase measure reagent;Although sodium glutamate can also play certain enhancing stabilizing effect, obviously it is not so good as
The Asparagus cochinchinensis ammonia acid sodium of the present invention.
In addition, kit (formula two) provided by the invention carries out 40 samples after 2~8 degree of conditions preserve 18 months
The average result of detection only declines 2.9% compared with preserving 0 month average result, belongs to zone of reasonableness, and it is accurate to remain to realize
Detection.
One reagent measured value of formula after accelerating 1 week for 37 degree of verification, which declines more, to be caused since which raw material inactivates,
Carry out following experiment:It is added respectively in the one R1 reagents of formula after 37 degree accelerate 1 week and mainly forms PNP and XOD, is added
Enter amount is respectively initial concentration 50%, then the results are shown in Table 4 to reagent 4 for detection reagent 1 respectively.
4 reagent 1 of table to reagent 4 sample detection result (unit:U/L)
| Sample serial number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Reagent 1 | 16.2 | 5 | 21 | 90.4 | 2.2 | 101.1 | 74.6 | 43.2 | 86.5 | 11.1 |
| Reagent 2 | 8.8 | 2.4 | 10.5 | 45.1 | 1.1 | 50.4 | 36.1 | 26.3 | 38.5 | 5.1 |
| Reagent 3 | 8.4 | 2.3 | 10.2 | 44.9 | 1 | 50.2 | 36.5 | 26.2 | 37.9 | 5.5 |
| Reagent 4 | 15.9 | 5.1 | 21.1 | 90.2 | 2.1 | 101.2 | 74.5 | 42.9 | 86.3 | 10.9 |
| Sample serial number | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| Reagent 1 | 97.6 | 49.3 | 31.3 | 12.8 | 25.3 | 88.7 | 2.3 | 9.3 | 50.9 | 109.3 |
| Reagent 2 | 43.2 | 24.3 | 16.7 | 6.1 | 11.3 | 44.1 | 1.1 | 4.5 | 24.9 | 51.1 |
| Reagent 3 | 43.4 | 24.9 | 16.7 | 6.3 | 11.4 | 43.3 | 0.9 | 4.3 | 25.1 | 49.9 |
| Reagent 4 | 97.4 | 49.1 | 31.3 | 12.5 | 24.9 | 88.5 | 2.1 | 9.1 | 50.4 | 109.1 |
| Sample serial number | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |
| Reagent 1 | 17.1 | 5 | 34 | 29.2 | 74 | 48.6 | 113.3 | 77.8 | 9.3 | 53.5 |
| Reagent 2 | 8.5 | 2.1 | 14.9 | 14.6 | 37.1 | 24.6 | 56.9 | 33.9 | 4.1 | 26.5 |
| Reagent 3 | 8.7 | 2.3 | 14.8 | 14.9 | 36.9 | 25.1 | 57.1 | 33.6 | 4.4 | 26.4 |
| Reagent 4 | 16.8 | 4.8 | 33.6 | 28.9 | 73.5 | 47.8 | 112.7 | 77.4 | 9.1 | 53.2 |
| Sample serial number | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | 40 |
| Reagent 1 | 30 | 19.6 | 3.2 | 27.8 | 105.9 | 58.1 | 15 | 81.9 | 3.5 | 67.2 |
| Reagent 2 | 15.2 | 11.1 | 1.1 | 13.1 | 47.5 | 24.1 | 7.4 | 35.1 | 1.2 | 33.5 |
| Reagent 3 | 15.9 | 11.3 | 1.2 | 13.3 | 47.8 | 23.9 | 7.6 | 35.4 | 1.1 | 32.9 |
| Reagent 4 | 29.4 | 19.1 | 2.9 | 37.4 | 105.5 | 57.7 | 14.7 | 81.4 | 3.2 | 66.5 |
Reagent 1:R1 and R2 is not accelerated;
Reagent 2:37 degree of R1 accelerates 1 week, and R2 is not accelerated;
Reagent 3:37 degree of R1 accelerates 1 week, and is added into PNP 1.5KU/L in R1, and R2 is not accelerated;
Reagent 4:37 degree of R1 accelerates 1 week, and is added into XOD1.5KU/L in R1, and R2 is not accelerated.
Through statistics, as known from Table 4:1. reagent 2 (not adding PNP and XOD) through R1 accelerate 1 week after after measured value have with reagent 1
Notable difference;2. reagent 3 (individually adding PNP) measured value after R1 accelerates 1 week has notable difference with reagent 1, with 2 measured value of reagent
It is almost the same, illustrate that PNP is not inactivated in reagent R1;3. reagent 4 (individually adding XOD) measured value and examination after R1 accelerates 1 week
1 no significant difference of agent has clear improvement compared with 2 measured value of reagent, illustrates that XOD enzymes have apparent inactivation in reagent R1.
Further, the R1 reagents of formula one and the R1 reagents of formula two are detected respectively when not accelerating to accelerate 1 week with 37 degree
XOD enzyme activities, each detection 20 times, as a result as shown in table 5 and table 6.
XOD enzyme activity (the units of the R1 reagents detection of the formula of table 5 one:KU/L)
XOD enzyme activity (the units of the R1 reagents detection of the formula of table 6 two:KU/L)
From table 5 and table 6 as can be seen that being formulated one accelerated 1 week, XOD enzymes inactivation is serious, and inactivation ratio reaches
48.8%, and it is only 1.3% to be formulated two since a certain amount of Asparagus cochinchinensis ammonia acid sodium is added as XOD enzymes inactivation after protective agent, is said
Monosodium L-aspartate will have preferable protective effect to XOD enzymes tomorrow.
Dosage of the three stabilizer Asparagus cochinchinensis ammonia acid sodium of test example in reagent R1 is investigated
Formula one is as follows:R1:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, xanthine oxidase 3KU/L,
Purine nucleoside phosphorylase 3KU/L, 4-AA 60mg/L, peroxidase 6KU/L;R2:TRIS 12g/L,6N
HCL 20g/L, potassium sorbate 1g/L, adenosine 5g/L, N- ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt
0.5g/L, glycerine 200ml/L.
Formula two is as follows:R1:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, xanthine oxidase 3KU/L,
Purine nucleoside phosphorylase 3KU/L, 4-AA 60mg/L, peroxidase 6KU/L, Asparagus cochinchinensis ammonia acid sodium 50g/L;
R2:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, adenosine 5g/L, N- ethyl-N- (2- hydroxyl -3- sulfopropyls) -
3- methylaniline sodium salts 0.5g/L, glycerine 200ml/L.
Formula three is as follows:R1:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, xanthine oxidase 3KU/L,
Purine nucleoside phosphorylase 3KU/L, 4-AA 60mg/L, peroxidase 6KU/L, Asparagus cochinchinensis ammonia acid sodium 20g/L;
R2:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, adenosine 5g/L, N- ethyl-N- (2- hydroxyl -3- sulfopropyls) -
3- methylaniline sodium salts 0.5g/L, glycerine 200ml/L.
Formula four is as follows:R1:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, xanthine oxidase 3KU/L,
Purine nucleoside phosphorylase 3KU/L, 4-AA 60mg/L, peroxidase 6KU/L, Asparagus cochinchinensis ammonia acid sodium 30g/L;
R2:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, adenosine 5g/L, N- ethyl-N- (2- hydroxyl -3- sulfopropyls) -
3- methylaniline sodium salts 0.5g/L, glycerine 200ml/L.
Formula five is as follows:R1:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, xanthine oxidase 3KU/L,
Purine nucleoside phosphorylase 3KU/L, 4-AA 60mg/L, peroxidase 6KU/L, Asparagus cochinchinensis ammonia acid sodium 80g/L;
R2:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, adenosine 5g/L, N- ethyl-N- (2- hydroxyl -3- sulfopropyls) -
3- methylaniline sodium salts 0.5g/L, glycerine 200ml/L.
Formula six is as follows:R1:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, xanthine oxidase 3KU/L,
Purine nucleoside phosphorylase 3KU/L, 4-AA 60mg/L, peroxidase 6KU/L, Asparagus cochinchinensis ammonia acid sodium 100g/
L;R2:TRIS 12g/L, 6N HCL 20g/L, potassium sorbate 1g/L, adenosine 5g/L, N- ethyl-N- (2- hydroxyl -3- sulphurs third
Base) -3- methylaniline sodium salts 0.5g/L, glycerine 200ml/L.
To investigate dosage of the stabilizer Asparagus cochinchinensis ammonia acid sodium in reagent R1, detection formula one to formula six is not adding respectively
XOD enzyme activities when speed accelerates 1 week with 37 degree, the results are shown in Table 7.
XOD enzyme activity (the units when not accelerating to accelerate 1 week with 37 degree to formula six of the formula of table 7 one:KU/L)
As can be seen from Table 7, Asparagus cochinchinensis ammonia acid sodium is not added in formula one as stabilizer, XOD inactivations are serious, inactivate ratio
Example has reached 48.5%, after being separately added into stabilizer Asparagus cochinchinensis ammonia acid sodium 20g/L, 30g/L, 50g/L, 80g/L, 100g/L, XOD
Enzyme inactivates:34.7%, 20.9%, 1.3%, 1.7%, 2.7%.Illustrate stabilizer Asparagus cochinchinensis ammonia acid sodium in reagent R1
Sample-adding amount 20-100g/L when can play certain stabilization, but when 20-30g/L, stablizing effect is less desirable,
When 50g/L-100g/L, stablizing effect is relatively good.
In addition, being detected respectively to the formula response curve of formula one to formula six, formula one is fast to four reaction of formula
Speed, and be formulated five and react slower with formula six, one response curve is formulated as shown in Figure 1, being formulated two response curve such as Fig. 2 institutes
Show, be formulated five response curve it is as shown in Figure 3.Illustrating can be to formula when the addition of Asparagus cochinchinensis ammonia acid sodium reaches 80 or 100g/L
Middle ingredient generates certain inhibition, causes reaction that cannot carry out rapidly.
Therefore, the resultant effect of formula two (addition of Asparagus cochinchinensis ammonia acid sodium is 50g/L) is best.
The stability test of adenosine in four reagent R2 of test example
Respectively using the reagent R2 in embodiment one and comparative example three as sample, the stability of adenosine in reagent R2 is investigated,
As a result as shown in table 8 and table 9.
The stability of reagent R2 in 8 embodiment one of table
| Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| There is precipitation | ||||||||||
| Without precipitation | √ | √ | √ | √ | √ | √ | √ | √ | √ | √ |
The stability of reagent R2 in 9 comparative example three of table
| Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| There is precipitation | √ | √ | √ | √ | √ | √ | √ | √ | √ | √ |
| Without precipitation |
From table 8 and table 9 it is found that a certain amount of glycerine is added in reagent R2 when preparing have important meaning to the stability of adenosine
Justice.
The reagent R2-1 to R2-5 containing not same amount glycerine is prepared respectively, the stability of adenosine is investigated, as a result such as table
Shown in 10.
Reagent R2-1:TRIS 12g/L, 6N HCL20g/L, potassium sorbate 1g/L, adenosine 5g/L, TOOS 0.5g/L.
Reagent R2-2:It is TRIS 12g/L, 6N HCL20g/L, potassium sorbate 1g/L, adenosine 5g/L, TOOS 0.5g/L, sweet
Oily 100ml/L.
Reagent R2-3:It is TRIS 12g/L, 6N HCL20g/L, potassium sorbate 1g/L, adenosine 5g/L, TOOS 0.5g/L, sweet
Oily 150ml/L.
Reagent R2-4:It is TRIS 12g/L, 6N HCL20g/L, potassium sorbate 1g/L, adenosine 5g/L, TOOS 0.5g/L, sweet
Oily 200ml/L.
Reagent R2-5:It is TRIS 12g/L, 6N HCL20g/L, potassium sorbate 1g/L, adenosine 5g/L, TOOS 0.5g/L, sweet
Oily 300ml/L.
The adenosine stability result table of the different glycerine additive amounts of table 10
As known from Table 10, when glycerine not being added in reagent R2, Precipitation after preparation;In reagent R2 be added 100 or
The glycerine of 150ml/L, still there is Precipitation;And the glycerine of 200 or 300ml/L is added in reagent R2, no Precipitation, to adenosine
Stability play an important role.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
The specific implementation of the present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the present invention's
Protection domain.
Claims (7)
1. a kind of adenosine deaminase assay kit, it is characterised in that:Include such as including reagent R1 and reagent R2, the reagent R1
Lower component and its concentration:5~20g/L of TRIS, 15~25g/L of 6N HCL, 1~5KU/L of xanthine oxidase, purine nucleosides phosphorus
1~5KU/L of phosphorylase, 50~80mg/L of 4-AA, 2~10KU/L of peroxidase, Asparagus cochinchinensis ammonia acid sodium 30~
80g/L;The reagent R2 includes following component and its concentration:5~20g/L of TRIS, 15~25g/L of 6N HCL, adenosine 1~
0.1~5g/L of 10g/L, N- ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt, 180~300ml/L of glycerine.
2. adenosine deaminase assay kit according to claim 1, it is characterised in that:The reagent R1 includes such as the following group
Point and its concentration:10~15g/L of TRIS, 18~22g/L of 6N HCL, 2~4KU/L of xanthine oxidase, purine nucleosides phosphoric acid
Change 2~4KU/L of enzyme, 55~65mg/L of 4-AA, 3~8KU/L of peroxidase, 40~60g/ of Asparagus cochinchinensis ammonia acid sodium
L;The reagent R2 includes following component and its concentration:10~15g/L of TRIS, 18~22g/L of 6N HCL, 2~6g/L of adenosine,
0.5~2g/L of N- ethyl-N- (2-Hydroxy-3-sulfopropyl)-3-methylaniline sodium salt, 200~300ml/L of glycerine.
3. adenosine deaminase assay kit according to claim 2, it is characterised in that:The reagent R1 includes such as the following group
Point and its concentration:TRIS 12g/L, 6N HCL 20g/L, xanthine oxidase 3KU/L, purine nucleoside phosphorylase 3KU/L, 4-
Amino-antipyrine 60mg/L, peroxidase 6KU/L, Asparagus cochinchinensis ammonia acid sodium 50g/L;The reagent R2 include following component and
Its concentration:TRIS 12g/L, 6N HCL 20g/L, adenosine 5g/L, N- ethyl-N- (2- hydroxyl -3- sulfopropyls) -3- methylanilines
Sodium salt 0.5g/L, glycerine 200ml/L.
4. adenosine deaminase assay kit according to any one of claim 1 to 3, it is characterised in that:The reagent
R1 further includes preservative;The reagent R2 further includes preservative.
5. adenosine deaminase assay kit according to claim 4, it is characterised in that:The preservative is sorbic acid
Potassium, a concentration of 1g/L.
6. adenosine deaminase assay kit according to any one of claim 1 to 3, it is characterised in that:Further include people
Serum standard panel.
7. the assay method of adenosine deaminase assay kit according to claim 1, it is characterised in that:Including walking as follows
Suddenly:Blood serum sample is detached, reagent R1 is added into blood serum sample, mixing after being incubated 3~6min, is then added reagent R2, mixes
It is even, measure ADA activity by measuring quinone derivative rate of change of absorbance at 550nm.
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Cited By (3)
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| CN109988816A (en) * | 2019-04-12 | 2019-07-09 | 浙江夸克生物科技有限公司 | A kind of adenosine deaminase assay kit |
| CN111662955A (en) * | 2020-07-26 | 2020-09-15 | 武汉中太生物技术有限公司 | Adenosine deaminase assay kit |
| CN111808918A (en) * | 2020-07-26 | 2020-10-23 | 武汉景川诊断技术股份有限公司 | Kit for determining 5' -nucleotidase |
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| CN109988816A (en) * | 2019-04-12 | 2019-07-09 | 浙江夸克生物科技有限公司 | A kind of adenosine deaminase assay kit |
| CN111662955A (en) * | 2020-07-26 | 2020-09-15 | 武汉中太生物技术有限公司 | Adenosine deaminase assay kit |
| CN111808918A (en) * | 2020-07-26 | 2020-10-23 | 武汉景川诊断技术股份有限公司 | Kit for determining 5' -nucleotidase |
| CN111662955B (en) * | 2020-07-26 | 2023-05-02 | 武汉中太生物技术有限公司 | Adenosine deaminase assay kit |
| CN111808918B (en) * | 2020-07-26 | 2024-01-23 | 武汉景川诊断技术股份有限公司 | Kit for determining 5' -nucleotidase |
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