CN106556704A - Liver function glycylproline dipeptidyl aminopeptidase determines kit and preparation method - Google Patents
Liver function glycylproline dipeptidyl aminopeptidase determines kit and preparation method Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
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Abstract
The invention discloses a kind of liver function glycylproline dipeptidyl aminopeptidase determines kit and preparation method, its kit includes reagent A and reagent B, wherein：Reagent A includes the raw material of following weight portion：20 25 parts of buffer solution, 16 19 parts of glycylglycine, 14 18 parts of Sodium azide, 8 12 parts of stabilizer, 30 35 parts of deionized water；Reagent B includes the raw material of following weight portion：21 25 parts of glycyl proline paranitroanilinum p-methyl benzenesulfonic acid, 15 19 parts of Sodium azide, 19 23 parts of buffer solution, 11 15 parts of stabilizer, 22 26 parts of ethylene glycol, 69 parts of surfactant, 31 34 parts of deionized water；The buffer solution includes the raw material of following weight portion：15 20 parts of trishydroxymethylaminomethane, 20 25 parts of deionized water, 18 22 parts of hydrochloric acid；Its preparation method is comprised the following steps：Trishydroxymethylaminomethane and deionized water are mixed.The present invention possesses excellent stability, it is possible to increase the preci-sion and accuracy of measure, disclosure satisfy that more strict requirements, expands use range, prepares simple, easy to use, low cost.
Description
Technical field
The present invention relates to medical science and technological field of biochemistry, more particularly to a kind of liver function glycyl proline dipeptides ammonia
Base peptide enzymatic determination kit and preparation method.
Background technology
Early in 1966, Hopsu-Havu and Glenner had found, the biochemistry of mammal peptase and histochemistry grind
In studying carefully, color source substrate glycyl-prolyl-beta-naphthylamine can be hydrolyzed by some enzyme preparations, and they successfully divide from these enzyme preparations
This enzyme that can hydrolyze Gly-Pro- β-NA has been separated out, and its hydrolysate has been proved for glycyl-proline and beta-naphthylamine；For
Seek in mammalian tissues with the presence or absence of this enzyme, they are studied with the liver of mouse as sample first, are found
Really there is this enzyme；Then, the Kidney sections of degreasing mouse are incubated altogether with Gly-Pro- β-NA and azo dyes, are found little
There is the naphthylamines that hydrolysis is produced in the nephridial tissue of mouse, show all there is this dipeptides amino peptide in the liver and kidney of mouse
Enzyme, i.e. glycylproline dipeptidyl aminopeptidase；Glycylproline dipeptidyl aminopeptidase is not only present in mouse, is also existed
In pig, ox, people and various animal tissues；Glycylproline dipeptidyl aminopeptidase can specifically hydrolyzing N-end second
For the peptide bond of prolyl；Synthesize different x- prolyl paranitroanilinum (xPro-p-NA) with different aminoacids, pH=8.7's
Under the conditions of, human serum glycylproline dipeptidyl aminopeptidase the hydrolysis rate size of these artificial substrates is followed successively by Gly-,
Ala-, Lys-, Arg-, Glu- and Asp-Pro-p-NA；Illustrate that substrate -terminal amino acid residue is different, affect glycyl dried meat
Propylhomoserin dipeptidyl aminopeptidase activity and the Km values to substrate.
Health adult's serum glycylproline dipeptidyl aminopeptidase level is more constant, and neonate is substantially less than grown up,
From the rapid stage of development of baby due to 20 years old, serum glycylproline dipeptidyl aminopeptidase content gradually rises；It is primary
In property In Sera of Patients With Hepatocarcinoma glycylproline dipeptidyl aminopeptidase activity be significantly higher than normal person and chronic hepatitis, cirrhosis,
The patient of cholelithiasis, obstructive jaundice, but glycyl proline dipeptides amino in serious hepatitis, alcoholic hepatitis patients serum
The elevated-levels of peptidase activity are more than hepatocarcinoma patient；The work of glycylproline dipeptidyl aminopeptidase in hepatic hemangioma patients serum
Property is normal；The activity reduction of glycylproline dipeptidyl aminopeptidase in Serum Obtained From Advance Gastric Cancer, during other benign enterogastric diseases,
In serum, the activity of glycylproline dipeptidyl aminopeptidase also slightly reduces this.
The patent document of Application No. 201510973109.8 discloses a kind of glycylproline dipeptidyl aminopeptidase inspection
Test agent box, compared with conventional kit, stability and the range of linearity will have well the kit than conventional detection kit
Beneficial to reagent popularization and application clinically；But, its stability does not meet strict requirements, causes use range to have
Limit, and preci-sion and accuracy is relatively low, therefore, we have proposed a kind of liver function glycylproline dipeptidyl aminopeptidase and survey
Determining kit and preparation method is used to solve the above problems.
The content of the invention
Based on the technical problem that background technology is present, the present invention proposes liver function glycylproline dipeptidyl aminopeptidase
Determine kit and preparation method.
A kind of liver function glycylproline dipeptidyl aminopeptidase proposed by the present invention determines kit, including reagent A and
Reagent B, wherein：
Reagent A includes the raw material of following weight portion：Buffer solution 20-25 parts, glycylglycine 16-19 parts, Sodium azide 14-18 parts, surely
Determine agent 8-12 parts, deionized water 30-35 part；
Reagent B includes the raw material of following weight portion：Glycyl proline paranitroanilinum p-methyl benzenesulfonic acid 21-25 parts, Sodium azide
15-19 parts, buffer solution 19-23 parts, stabilizer 11-15 parts, ethylene glycol 22-26 parts, surfactant 6-9 parts, deionization
Water 31-34 parts.
Preferably, the buffer solution includes the raw material of following weight portion：Trishydroxymethylaminomethane 15-20 parts, deionization
Water 20-25 parts, hydrochloric acid 18-22 parts；Its preparation method is comprised the following steps：Trishydroxymethylaminomethane and deionized water are mixed
Close, after stirring, add hydrochloric acid, regulation pH value is 7.5-9.5, and buffer solution is obtained.
Preferably, reagent A includes the raw material of following weight portion：Buffer solution 21-24 parts, glycylglycine 17-18 parts, Sodium azide
15-17 parts, stabilizer 9-10 parts, deionized water 31-34 part.
Preferably, reagent B includes the raw material of following weight portion：Glycyl proline paranitroanilinum p-methyl benzenesulfonic acid 22-
24 parts, Sodium azide 16-18 parts, buffer solution 20-22 parts, stabilizer 12-14 parts, ethylene glycol 23-25 parts, surfactant
7-8 parts, deionized water 32-33 part.
Preferably, the stabilizer include amido crash suck one or more in diketone, sodium chloride and mannitol of combination,
And surfactant includes one or more in sulfonic acid, sodium sulfate of polyethenoxy ether of fatty alcohol and Nonyl pheno of group
Close.
The invention allows for a kind of liver function glycylproline dipeptidyl aminopeptidase determines the preparation method of kit,
Comprise the following steps：
S1：Buffer solution and glycylglycine are mixed, after stirring at 55-60 DEG C, Sodium azide, stabilizer and deionization is added
Water, after being then well mixed at 40-45 DEG C, is obtained reagent A；
S2：Glycyl proline paranitroanilinum p-methyl benzenesulfonic acid is diluted with buffer solution, be well mixed rear shaken at room temperature absorption
2.5-3.5h, is then centrifuged, and is removed supernatant, is subsequently added into Sodium azide, stabilizer, ethylene glycol, surfactant and goes
Ionized water, after being well mixed, is obtained reagent B at 50-60 DEG C；
S3：Packing reagent A and reagent B；
S4：Finished product is assembled into, liver function glycylproline dipeptidyl aminopeptidase is obtained and is determined kit.
Preferably, in the S1, buffer solution and glycylglycine are mixed, after stirring at 56-59 DEG C, adds nitrine
Sodium, stabilizer and deionized water, after being then well mixed at 41-44 DEG C, are obtained reagent A.
Preferably, in the S2, glycyl proline paranitroanilinum p-methyl benzenesulfonic acid is diluted with buffer solution, be well mixed
Afterwards shaken at room temperature absorption 2.7-3.3h, be then centrifuged, remove supernatant, be subsequently added into Sodium azide, stabilizer, ethylene glycol,
Surfactant and deionized water, after being well mixed, are obtained reagent B at 52-58 DEG C.
In the present invention, the liver function glycylproline dipeptidyl aminopeptidase determine kit and preparation method by with
Buffer solution and glycylglycine are reagent A obtained in major ingredient, with glycyl proline paranitroanilinum p-methyl benzenesulfonic acid and buffer solution are
Reagent B obtained in major ingredient causes kit to have good stability, and preci-sion and accuracy is higher, by adding Sodium azide
Preci-sion and accuracy can be improved, is capable of the dispersion stabilization of Contrast agent by adding stabilizer and surfactant, this
Invention possesses excellent stability, it is possible to increase the preci-sion and accuracy of measure, disclosure satisfy that more strict requirements, expands
Use range, prepares simple, easy to use, low cost.
Specific embodiment
The present invention is further explained with reference to specific embodiment.
Embodiment one
The present embodiment proposes a kind of liver function glycylproline dipeptidyl aminopeptidase and determines kit, including reagent A and examination
Agent B, wherein：
Reagent A includes the raw material of following weight portion：20 parts of buffer solution, 16 parts of glycylglycine, 14 parts of Sodium azide, 8 parts of stabilizer,
30 parts of deionized water；
Reagent B includes the raw material of following weight portion：21 parts of glycyl proline paranitroanilinum p-methyl benzenesulfonic acid, Sodium azide 15
Part, 19 parts of buffer solution, 11 parts of stabilizer, 22 parts of ethylene glycol, 6 parts of surfactant, 31 parts of deionized water.
Its preparation method is comprised the following steps：
S1：Buffer solution and glycylglycine are mixed, after stirring at 55 DEG C, Sodium azide, stabilizer and deionized water is added, so
After being well mixed at 40 DEG C afterwards, reagent A is obtained；
S2：Glycyl proline paranitroanilinum p-methyl benzenesulfonic acid is diluted with buffer solution, be well mixed rear shaken at room temperature absorption
2.5h, is then centrifuged, and is removed supernatant, is subsequently added into Sodium azide, stabilizer, ethylene glycol, surfactant and deionization
Water, after being well mixed, is obtained reagent B at 50 DEG C；
S3：Packing reagent A and reagent B；
S4：Finished product is assembled into, liver function glycylproline dipeptidyl aminopeptidase is obtained and is determined kit.
Buffer solution includes the raw material of following weight portion：15 parts of trishydroxymethylaminomethane, 20 parts of deionized water, hydrochloric acid
18 parts；Its preparation method is comprised the following steps：Trishydroxymethylaminomethane and deionized water are mixed, after stirring, is added
Hydrochloric acid, it is 7.5 to adjust pH value, and buffer solution is obtained.
Embodiment two
The present embodiment proposes a kind of liver function glycylproline dipeptidyl aminopeptidase and determines kit, including reagent A and examination
Agent B, wherein：
Reagent A includes the raw material of following weight portion：21 parts of buffer solution, 17 parts of glycylglycine, 15 parts of Sodium azide, 9 parts of stabilizer,
31 parts of deionized water；
Reagent B includes the raw material of following weight portion：22 parts of glycyl proline paranitroanilinum p-methyl benzenesulfonic acid, Sodium azide 16
Part, 20 parts of buffer solution, 12 parts of stabilizer, 23 parts of ethylene glycol, 7 parts of surfactant, 32 parts of deionized water.
Its preparation method is comprised the following steps：
S1：Buffer solution and glycylglycine are mixed, after stirring at 56 DEG C, Sodium azide, stabilizer and deionized water is added, so
After being well mixed at 41 DEG C afterwards, reagent A is obtained；
S2：Glycyl proline paranitroanilinum p-methyl benzenesulfonic acid is diluted with buffer solution, be well mixed rear shaken at room temperature absorption
2.7h, is then centrifuged, and is removed supernatant, is subsequently added into Sodium azide, stabilizer, ethylene glycol, surfactant and deionization
Water, after being well mixed, is obtained reagent B at 52 DEG C；
S3：Packing reagent A and reagent B；
S4：Finished product is assembled into, liver function glycylproline dipeptidyl aminopeptidase is obtained and is determined kit.
Buffer solution includes the raw material of following weight portion：16 parts of trishydroxymethylaminomethane, 21 parts of deionized water, hydrochloric acid
19 parts；Its preparation method is comprised the following steps：Trishydroxymethylaminomethane and deionized water are mixed, after stirring, is added
Hydrochloric acid, it is 8 to adjust pH value, and buffer solution is obtained.
Embodiment three
The present embodiment proposes a kind of liver function glycylproline dipeptidyl aminopeptidase and determines kit, including reagent A and examination
Agent B, wherein：
Reagent A includes the raw material of following weight portion：24 parts of buffer solution, 18 parts of glycylglycine, 17 parts of Sodium azide, stabilizer 11
Part, 34 parts of deionized water；
Reagent B includes the raw material of following weight portion：24 parts of glycyl proline paranitroanilinum p-methyl benzenesulfonic acid, Sodium azide 18
Part, 22 parts of buffer solution, 14 parts of stabilizer, 25 parts of ethylene glycol, 8 parts of surfactant, 33 parts of deionized water.
Its preparation method is comprised the following steps：
S1：Buffer solution and glycylglycine are mixed, after stirring at 59 DEG C, Sodium azide, stabilizer and deionized water is added, so
After being well mixed at 44 DEG C afterwards, reagent A is obtained；
S2：Glycyl proline paranitroanilinum p-methyl benzenesulfonic acid is diluted with buffer solution, be well mixed rear shaken at room temperature absorption
3.3h, is then centrifuged, and is removed supernatant, is subsequently added into Sodium azide, stabilizer, ethylene glycol, surfactant and deionization
Water, after being well mixed, is obtained reagent B at 58 DEG C；
S3：Packing reagent A and reagent B；
S4：Finished product is assembled into, liver function glycylproline dipeptidyl aminopeptidase is obtained and is determined kit.
Buffer solution includes the raw material of following weight portion：19 parts of trishydroxymethylaminomethane, 24 parts of deionized water, hydrochloric acid
21 parts；Its preparation method is comprised the following steps：Trishydroxymethylaminomethane and deionized water are mixed, after stirring, is added
Hydrochloric acid, it is 8.5 to adjust pH value, and buffer solution is obtained.
Example IV
The present embodiment proposes a kind of liver function glycylproline dipeptidyl aminopeptidase and determines kit, including reagent A and examination
Agent B, wherein：
Reagent A includes the raw material of following weight portion：25 parts of buffer solution, 19 parts of glycylglycine, 18 parts of Sodium azide, stabilizer 12
Part, 35 parts of deionized water；
Reagent B includes the raw material of following weight portion：25 parts of glycyl proline paranitroanilinum p-methyl benzenesulfonic acid, Sodium azide 19
Part, 23 parts of buffer solution, 15 parts of stabilizer, 26 parts of ethylene glycol, 9 parts of surfactant, 34 parts of deionized water.
Its preparation method is comprised the following steps：
S1：Buffer solution and glycylglycine are mixed, after stirring at 60 DEG C, Sodium azide, stabilizer and deionized water is added, so
After being well mixed at 45 DEG C afterwards, reagent A is obtained；
S2：Glycyl proline paranitroanilinum p-methyl benzenesulfonic acid is diluted with buffer solution, be well mixed rear shaken at room temperature absorption
3.5h, is then centrifuged, and is removed supernatant, is subsequently added into Sodium azide, stabilizer, ethylene glycol, surfactant and deionization
Water, after being well mixed, is obtained reagent B at 60 DEG C；
S3：Packing reagent A and reagent B；
S4：Finished product is assembled into, liver function glycylproline dipeptidyl aminopeptidase is obtained and is determined kit.
Buffer solution includes the raw material of following weight portion：20 parts of trishydroxymethylaminomethane, 25 parts of deionized water, hydrochloric acid
22 parts；Its preparation method is comprised the following steps：Trishydroxymethylaminomethane and deionized water are mixed, after stirring, is added
Hydrochloric acid, it is 9.5 to adjust pH value, and buffer solution is obtained.
Kit obtained in embodiment one to four and conventional kit are tested, test result is as follows：
| Embodiment one | Embodiment two | Embodiment three | Example IV | Conventional kit | |
| Precision | Batch in CV≤4.5%, batch between phase To extreme difference≤6.5% | CV≤5.3% in batch, batch between it is relative Extreme difference≤7.9% | CV≤5.7% in batch, batch between it is relative Extreme difference≤7.4% | CV≤4.8% in batch, batch between it is relative Extreme difference≤6.9% | Batch in CV≤6.5%, batch between antipode Difference≤8.5% |
| The degree of accuracy | Relative deviation≤9% | Relative deviation≤12% | Relative deviation≤11% | Relative deviation≤10% | Relative deviation≤15% |
As seen from the above table, the precision of kit obtained in embodiment one to four, the degree of accuracy and stability compare conventional kit
Precision, the degree of accuracy and stability is respectively provided with and significantly improves.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, should all be included within the scope of the present invention.
Claims (8)
1. a kind of liver function glycylproline dipeptidyl aminopeptidase determines kit, it is characterised in that including reagent A and reagent
B, wherein：
Reagent A includes the raw material of following weight portion：Buffer solution 20-25 parts, glycylglycine 16-19 parts, Sodium azide 14-18 parts, surely
Determine agent 8-12 parts, deionized water 30-35 part；
Reagent B includes the raw material of following weight portion：Glycyl proline paranitroanilinum p-methyl benzenesulfonic acid 21-25 parts, Sodium azide
15-19 parts, buffer solution 19-23 parts, stabilizer 11-15 parts, ethylene glycol 22-26 parts, surfactant 6-9 parts, deionization
Water 31-34 parts.
2. a kind of liver function glycylproline dipeptidyl aminopeptidase according to claim 1 determines kit, its feature
It is that the buffer solution includes the raw material of following weight portion：Trishydroxymethylaminomethane 15-20 parts, deionized water 20-25
Part, hydrochloric acid 18-22 parts；Its preparation method is comprised the following steps：Trishydroxymethylaminomethane and deionized water are mixed, stirring
After uniform, hydrochloric acid is added, regulation pH value is 7.5-9.5, and buffer solution is obtained.
3. a kind of liver function glycylproline dipeptidyl aminopeptidase according to claim 1 determines kit, its feature
It is that reagent A includes the raw material of following weight portion：Buffer solution 21-24 parts, glycylglycine 17-18 parts, Sodium azide 15-17 parts,
Stabilizer 9-10 parts, deionized water 31-34 part.
4. a kind of liver function glycylproline dipeptidyl aminopeptidase according to claim 1 determines kit, its feature
It is that reagent B includes the raw material of following weight portion：Glycyl proline paranitroanilinum p-methyl benzenesulfonic acid 22-24 parts, nitrine
Sodium 16-18 parts, buffer solution 20-22 parts, stabilizer 12-14 parts, ethylene glycol 23-25 parts, surfactant 7-8 parts, go from
Sub- water 32-33 parts.
5. a kind of liver function glycylproline dipeptidyl aminopeptidase according to claim 1 determines kit, its feature
Be, the stabilizer include amido crash suck one or more in diketone, sodium chloride and mannitol of combination, and surface active
Agent includes one or more in sulfonic acid, sodium sulfate of polyethenoxy ether of fatty alcohol and Nonyl pheno of combination.
6. a kind of liver function glycylproline dipeptidyl aminopeptidase determines the preparation method of kit, it is characterised in that include
Following steps：
S1：Buffer solution and glycylglycine are mixed, after stirring at 55-60 DEG C, Sodium azide, stabilizer and deionization is added
Water, after being then well mixed at 40-45 DEG C, is obtained reagent A；
S2：Glycyl proline paranitroanilinum p-methyl benzenesulfonic acid is diluted with buffer solution, be well mixed rear shaken at room temperature absorption
2.5-3.5h, is then centrifuged, and is removed supernatant, is subsequently added into Sodium azide, stabilizer, ethylene glycol, surfactant and goes
Ionized water, after being well mixed, is obtained reagent B at 50-60 DEG C；
S3：Packing reagent A and reagent B；
S4：Finished product is assembled into, liver function glycylproline dipeptidyl aminopeptidase is obtained and is determined kit.
7. a kind of liver function glycylproline dipeptidyl aminopeptidase according to claim 6 determines the preparation side of kit
Method, it is characterised in that in the S1, buffer solution and glycylglycine are mixed, after stirring at 56-59 DEG C, addition Sodium azide,
Stabilizer and deionized water, after being then well mixed at 41-44 DEG C, are obtained reagent A.
8. a kind of liver function glycylproline dipeptidyl aminopeptidase according to claim 6 determines the preparation side of kit
Method, it is characterised in that in the S2, dilutes glycyl proline paranitroanilinum p-methyl benzenesulfonic acid with buffer solution, is well mixed
Afterwards shaken at room temperature absorption 2.7-3.3h, be then centrifuged, remove supernatant, be subsequently added into Sodium azide, stabilizer, ethylene glycol,
Surfactant and deionized water, after being well mixed, are obtained reagent B at 52-58 DEG C.
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| CN111172234A (en) * | 2020-01-03 | 2020-05-19 | 浙江夸克生物科技有限公司 | Glycylproline dipeptide aminopeptidase determination kit |
| CN111500672A (en) * | 2020-03-20 | 2020-08-07 | 中科梅奥(山东)医学检验有限公司 | Glycylproline dipeptide aminopeptidase determination kit with high analysis sensitivity and preparation method and application thereof |
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| CN111172234A (en) * | 2020-01-03 | 2020-05-19 | 浙江夸克生物科技有限公司 | Glycylproline dipeptide aminopeptidase determination kit |
| CN111172234B (en) * | 2020-01-03 | 2023-05-16 | 浙江夸克生物科技有限公司 | Glycylproline dipeptide aminopeptidase assay kit |
| CN111500672A (en) * | 2020-03-20 | 2020-08-07 | 中科梅奥(山东)医学检验有限公司 | Glycylproline dipeptide aminopeptidase determination kit with high analysis sensitivity and preparation method and application thereof |
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