CN108226540A - A kind of ellagic acid reagent and its preparation method and activated partial thromboplastin time assay reagent, APTT kits - Google Patents
A kind of ellagic acid reagent and its preparation method and activated partial thromboplastin time assay reagent, APTT kits Download PDFInfo
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- CN108226540A CN108226540A CN201810129827.0A CN201810129827A CN108226540A CN 108226540 A CN108226540 A CN 108226540A CN 201810129827 A CN201810129827 A CN 201810129827A CN 108226540 A CN108226540 A CN 108226540A
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- G—PHYSICS
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- 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
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
Abstract
A kind of ellagic acid reagent and its preparation method and activated partial thromboplastin time assay reagent, APTT kits, are related to reagent for clinical diagnosis field.Preparation method is to add in ellagic acid without in calcium purified water, and quick stirring makes it homodisperse；Add in alkaline buffer or inorganic base；Acidic buffer or organic acid, inorganic acid are rapidly joined in 10 minutes after ellagic acid is added in；Add in metal ion；Amino acid and its derivative are added in, obtains buffer solution；In addition it is added in rabbit cephalin without calcium purified water, mixing emulsifies to obtain emulsification phosphatide；Emulsification phosphatide is added in buffer solution, quickly stirs and evenly mixs, obtains ellagic acid reagent, this is simple for process quick, and is described in detail.Activated partial thromboplastin time assay reagent includes above-mentioned ellagic acid reagent and chlorination calcon, with good stability.APTT kits including above-mentioned activated partial thromboplastin time assay reagent can directly replace import high stability APTT kits.
Description
Technical field
The present invention relates to reagent for clinical diagnosis field, and more particularly to a kind of ellagic acid reagent and its preparation method and activation
Partial thromboplastin time measure reagent, APTT kits.
Background technology
In the field of clinical examination, it is known that study the exception of blooc coagulation factor by measuring the setting time of blood
Deng inspection, wherein, APTT measures one of clinical examination as general blood clotting capability.APTT(activated
Partial thromboplastin time) i.e. activated partial thromboplastin time, blood passes through with having when referring to reflect bleeding
The abnormal contact of negative charge and start endogenous solidification approach function setting time.APTT measure is not only in internal cause
Property coagulation factors shortage or exception screening test in utilize, also utilized in monitoring of heparinotherapy etc..
For existing APTT reagents mostly using rabbit cephalin, it is rufous that the phosphatide is variable in air, there is hygroscopicity, insoluble
In water and acetone, ethyl alcohol is slightly soluble in, is dissolved in chloroform and ether, therefore there are problems of dissolution, if stirring and dissolving can generate not by force
Uniform insoluble matter, it is uncontrollable so as to cause technique, if dissolving by heating phosphatide inactivation may even aoxidized.By retrieving,
The existing patent in part and unspecified phosphating treatment process, there are some for phosphating treatment disclosed in the existing patent of other
Problem, such as：Rabbit brain extraction cephalin distillation water dissolution in the patent of invention of application number CN200710036410.1, this is very
Hardly possible is realized, therefore there are technique misleadings for the patent；Liquid white pottery disclosed in the patent of invention of application number CN200810083554.7
Though native APTT reagents show at its 37 DEG C to stablize at least 30 days, its concrete technology is not disclosed, activator is not in addition
Together, the sensibility of reagent is also different, and the performance of the APTT reagents is not understood.
For Clinical Sensitivity, disease differentiates or the raised purpose of quality, and in recent years, a variety of synthetic phospholipids are used in mixed way
(see, for example, US2011/159597).In addition, the type as synthetic phospholipid, by phosphatidyl-ethanolamine (PE), phosphatidyl courage
3 kinds of alkali (PC) and phosphatidylserine (PS) mix and being contained in reagent becomes mainstream, but synthetic phospholipid type is more, is not easy
It obtains and expensive, in addition each component ratio also more difficult control, equally exists problems of dissolution, and precipitation can partly be caused to generate,
Influence reagent quality.Available reagent can there are ellagic acid and phosphatide sedimentation problem, ellagic acid buffer solution, feedstock processing such as ellagic acids
It can lose activity under alkaline condition, whether phosphatide emulsification is uniform, and proportioning problem of ellagic acid buffer solution and phosphatide etc. is all directly
The quality of domestic reagent is affected, market is caused still to be occupied with giants such as Siemens, think of tower height, fertile sweet smell.
Therefore, a kind of manufacture craft is simple, and stability is high, can directly replace import high stability activated partial thromboplastin
Time (APTT) kit is expected by market.
Invention content
The purpose of the present invention is to provide a kind of ellagic acid reagent and its preparation methods, are emulsified including phosphatide, and ellagic acid is molten
The production technology of solution, each raw material order of addition, APTT time adjustments etc., this is simple for process quick, and is described in detail.
Another object of the present invention is to provide a kind of activated partial thromboplastin time assay reagent, which has good
Good stability can be stablized at least 30 days at 37 DEG C, and reagent is uncapped after at least stablizing at 5 ± 3 DEG C 30 days or more.
Another object of the present invention is to provide a kind of APTT kits, it can directly replace import high stability activated partial
Thromboplastin time (APTT) kit.
The present invention is solved its technical problem and is realized using following technical scheme.
The present invention proposes a kind of preparation method of ellagic acid reagent, includes the following steps：
Ellagic acid is added in without in calcium purified water, quick stirring makes it homodisperse；It is firstly added alkaline buffer or nothing
Machine alkali；Then rapidly join acidic buffer or organic acid, inorganic acid in 10 minutes after ellagic acid is added in, adjust pH to 7.1 ±
0.1；Metal ion is added in later；Amino acid and its derivative are subsequently added into, obtains buffer solution；
It is emulsified in addition, adding in 2~8 DEG C in rabbit cephalin without calcium purified water, and in 2~8 DEG C of mixings, obtains emulsification phosphorus
Fat；
The emulsification phosphatide is added in the buffer solution, quickly stirs and evenly mixs, obtains ellagic acid reagent.
Further, in present pre-ferred embodiments, mixing emulsification is using hollow mixing pipe and hollow
Grind what pipe carried out, specific method is：Grinding pipe is inserted into mixing pipe, 2~8 DEG C of ice water of addition in pipe is ground, while
Rabbit cephalin is added in mixing pipe, then in mixing pipe add in 2~8 DEG C without calcium purified water, ensure at entire emulsion process
In 2~8 DEG C of environment, substance in pipe is ground using grinding pipe.
Further, in present pre-ferred embodiments, the alkaline buffer is sodium acetate, Tris-base, Hepes-
At least one of Na；Inorganic base is at least one of sodium hydroxide and potassium hydroxide.
Further, in present pre-ferred embodiments, the acidic buffer is Tris-hcl, Hepes, biphosphate
At least one of salt；Organic acid is at least one of formic acid, acetic acid, ethanedioic acid；Inorganic acid is hydrochloric acid.
Further, in present pre-ferred embodiments, the metal ion is magnesium ion, in iron ion, manganese ion
It is at least one.
Further, in present pre-ferred embodiments, amino acid and its derivative are alanine, glycine, batanone acid,
At least one of arginine, histidine, glutamic acid and its salt.
Further, in present pre-ferred embodiments, appointing after acidic buffer or organic acid, inorganic acid is added in
Preservative is added in the meaning time, the preservative is at least one of Sodium azide, proclin300, gentamicin.
The present invention proposes a kind of ellagic acid reagent, is made using the preparation method of above-mentioned ellagic acid reagent.
The present invention proposes a kind of activated partial thromboplastin time assay reagent, including above-mentioned ellagic acid reagent and chlorine
Change calcon, the chlorination calcon is the phosphate buffer that calcium chloride is prepared.
The present invention proposes a kind of APTT kits, including above-mentioned activated partial thromboplastin time assay reagent.
The ellagic acid reagent and its preparation method and activated partial thromboplastin time assay reagent of the embodiment of the present invention,
The advantageous effect of APTT kits is：The ellagic acid preparation of reagents method of the embodiment of the present invention is to add in ellagic acid to purify without calcium
It is quick to stir in water, make it homodisperse；It is firstly added alkaline buffer or inorganic base；Then 10 points after ellagic acid is added in
Acidic buffer or organic acid, inorganic acid are rapidly joined in clock, adjusts pH to 7.1 ± 0.1；Metal ion is added in later；Then plus
Enter amino acid and its derivative, obtain buffer solution；In addition, added in rabbit cephalin 2~8 DEG C without calcium purified water, and in 2~8
The emulsification of DEG C mixing, obtains emulsification phosphatide；Emulsification phosphatide is added in buffer solution, quickly stirs and evenly mixs, obtains ellagic acid reagent, the work
Skill is simple and fast, and is described in detail.The activated partial thromboplastin time assay reagent of the embodiment of the present invention includes above-mentioned
Ellagic acid reagent and chlorination calcon, chlorination calcon be the phosphate buffer that calcium chloride is prepared, which has good
Stability, can stablize at least 30 days at 37 DEG C, reagent is uncapped after at least stablizing at 5 ± 3 DEG C 30 days or more.The present invention is implemented
The APTT kits of example include above-mentioned activated partial thromboplastin time assay reagent, can directly replace import high stability APTT
Kit.
Description of the drawings
It in order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range, for those of ordinary skill in the art, without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is a kind of flow chart of the preparation method of ellagic acid reagent provided in an embodiment of the present invention；
Fig. 2 is the correlation scatter plot of different samples in the embodiment of the present invention.
Specific embodiment
Purpose, technical scheme and advantage to make the embodiment of the present invention are clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Below to the ellagic acid reagent and its preparation method and activated partial thromboplastin time assay of the embodiment of the present invention
Reagent, APTT kits are specifically described.
The embodiment of the present invention provides a kind of preparation method of ellagic acid reagent, shown in Figure 1, includes the following steps：
(1) buffer solution is prepared：
Ellagic acid is added in without in calcium purified water, quick stirring makes to uniformly disperse, and revolution is about 500~1000r/min,
Specific rotating speed is not spilt over solution as standard depending on stirring rotator size, such as：Prepare 1000ml ellagic acid reagents, stirring
Rotor is 15*100mm (diameter-length), then revolution 500r/min, and stirring rotator is 8*40mm (diameter-length), then revolution
1000r/min.It, also cannot be complete because ellagic acid solubility is small and purified water pH is about 5.6, therefore even if stirring is accelerated to reach 24 hours
Fully dissolved.Ellagic acid used is without particular/special requirement, and analysis is pure, and the present embodiment preferably uses 0.2~0.5mmol ellagic acids.
Alkaline buffer or inorganic base are firstly added, since ellagic acid quickly dissolves under alkaline condition, add in alkali
It can be completely dissolved within about 2 minutes after property buffer solution or inorganic base.Alkaline buffer used for sodium acetate, Tris-base,
At least one of Hepes-Na；Inorganic base is at least one of sodium hydroxide and potassium hydroxide.The present embodiment is preferably added to
0.3~0.8mmol sodium acetates.
Then acidic buffer or organic acid, inorganic acid are rapidly joined in 10 minutes after ellagic acid is added in, adjusts pH to 7.1
± 0.1, although ellagic acid is soluble in alkali, but the stability in alkaline solution is poor, easily decomposes, therefore is not suitable for wiring solution-forming
Analysis, research work, therefore acidic buffer need to be rapidly joined for a long time are carried out, ensures the stabilization of ellagic acid, and then form
The optimal pHs of APTT reagents.Acidic buffer used is at least one of Tris-hcl, Hepes, dihydric phosphate；Have
Machine acid is at least one of formic acid, acetic acid, ethanedioic acid；Inorganic acid is hydrochloric acid.The present embodiment is preferably added to 0.8~1.3mmol
Tris-hcl。
Metal ion is added in later, and metal ion forms stable chelate with ellagic acid, accelerates ellagic acid activation effect, gold
The addition for belonging to ion directly affects the measured value to form reagent and the correlation with comparison Siemens reagent.Metal ion used
For at least one of magnesium ion, iron ion, manganese ion, the present embodiment is preferably added to 0.02~0.03mmol iron ions.
Amino acid and its derivative are subsequently added into, prevents ellagic acid from precipitating, ensures reagent quality, obtains buffer solution.Used
Amino acid and its derivative are at least one in alanine, glycine, batanone acid, arginine, histidine, glutamic acid and its salt
Kind, the present embodiment is preferably added to arginine monohydrochloride 1.5% and glycine 1.5%.
When preparing buffer solution, can it be added in the random time after adding in acidic buffer or organic acid, inorganic acid
Preservative, preservative are at least one of Sodium azide, proclin300, gentamicin.
(2) emulsification phosphatide is prepared：Added in rabbit cephalin 2~8 DEG C without calcium purified water, and in 2~8 DEG C of ice bath mixings
Emulsification, obtains emulsification phosphatide.The animal phospholipids that the present embodiment uses are derived from rabbit brain, preferably 0.3~0.35 g/l of rabbit brain phosphorus
Fat, the raw materials market are largely on sale.
The mixing emulsification of the present embodiment is carried out using hollow mixing pipe and hollow grinding pipe, and specific method is：
Grinding pipe is inserted into mixing pipe, 2~8 DEG C of ice water are added in pipe is ground, while rabbit cephalin is added in mixing pipe, then
Added in mixing pipe 2~8 DEG C without calcium purified water, ensure that entire emulsion process is in 2~8 DEG C of environment, use grinding pipe
The substance being ground in pipe.The mixing pipe and grinding pipe form the device similar with grinding device, under liquid low temperature environment
By phosphatide quick emulsification, ensure emulsification thoroughly, uniformly, it is quick, using this unique and conveniently emulsifying process, make emulsification
Completely, phospholipid microparticles are uniform, and do not destroy its activity, are configured to uniform liquid emulsion.
(3) emulsification phosphatide is added in special ratios in buffer solution, quickly stirred and evenly mixed, obtained in the homodisperse of yellow
Liquid, i.e. ellagic acid reagent.
The embodiment of the present invention provides a kind of ellagic acid reagent, is the preparation method system using above-mentioned ellagic acid reagent
.
The embodiment of the present invention provides a kind of activated partial thromboplastin time assay reagent, is mainly made of two parts,
That is ellagic acid reagent and chlorination calcon, the appropriate concentration phosphate buffer that chlorination calcon is prepared for calcium chloride, this implementation
Chlorination calcon used in example is formulated by 0.03mmol calcium chloride and 0.5mmol disodium hydrogen phosphates, sodium dihydrogen phosphate, pH
=7.10.The application method of activated partial thromboplastin time assay reagent is：50ul samples are added in blood clotting cup, in 37 DEG C
It is incubated 60S；Above-mentioned APTT measure ellagic acid reagent 50ul are then added thereto, and 60S is incubated in 37 DEG C；It is eventually adding
It states in liquid calcium chloride 50ul to blood clotting cup, setting time will be measured after 240S after sample, APTT reagents, chlorination calcon mixing.
The embodiment of the present invention provides a kind of APTT kits, including activated partial thromboplastin time assay reagent.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of ellagic acid reagents, are made using following preparation method：
(1) 0.4mmol ellagic acids are added in without in calcium purified water, quick stirring makes it homodisperse.
Add in 0.5mmol sodium acetates.
1mmol Tris-hcl are rapidly joined in 10 minutes after ellagic acid is added in, adjust pH to 7.1 ± 0.1.
Add in 0.025mmol iron ions.
Add in arginine monohydrochloride 1.5% and glycine 1.5%.
Add in preservative Sodium azide.
(2) emulsification phosphatide is prepared：Added in 0.32 g/l of rabbit cephalin 5 DEG C without calcium purified water, and in 5 DEG C of ice baths
Mixing emulsifies, and obtains emulsification phosphatide.
(3) emulsification phosphatide is added in special ratios in buffer solution, quickly stirred and evenly mixed, obtained in the homodisperse of yellow
Liquid, i.e. ellagic acid reagent.
The present embodiment also provides a kind of activated partial thromboplastin time assay reagent, including above-mentioned ellagic acid reagent and
Chlorination calcon.
Embodiment 2
The present embodiment provides a kind of ellagic acid reagents, are made using following preparation method：
(1) 0.2mmol ellagic acids are added in without in calcium purified water, quick stirring makes it homodisperse.
Add in 0.4mmol Tris-base.
0.8mmol Hepes are rapidly joined in 10 minutes after ellagic acid is added in, adjust pH to 7.1 ± 0.1.
Add in 0.02mmol magnesium ions.
Add in alanine.
Add in preservative proclin300.
(2) emulsification phosphatide is prepared：3 DEG C of addition mixes without calcium purified water, and in 3 DEG C of ice baths in 0.3 g/l of rabbit cephalin
Even emulsification obtains emulsification phosphatide.
(3) emulsification phosphatide is added in special ratios in buffer solution, quickly stirred and evenly mixed, obtained in the homodisperse of yellow
Liquid, i.e. ellagic acid reagent.
The present embodiment also provides a kind of activated partial thromboplastin time assay reagent, including above-mentioned ellagic acid reagent and
Chlorination calcon.
Embodiment 3
The present embodiment provides a kind of ellagic acid reagents, are made using following preparation method：
(1) 0.5mmol ellagic acids are added in without in calcium purified water, quick stirring makes it homodisperse.
Add in 0.8mmol sodium hydroxides.
About 0.5ml acetic acid is rapidly joined in 10 minutes after ellagic acid is added in, adjusts pH to 7.1 ± 0.1.
Add in 0.03mmol manganese ions.
Add in histidine and glutamic acid.
Add in preservative gentamicin.
(2) emulsification phosphatide is prepared：Added in 0.35 g/l of rabbit cephalin 8 DEG C without calcium purified water, and in 8 DEG C of ice baths
Mixing emulsifies, and obtains emulsification phosphatide.
(3) emulsification phosphatide is added in special ratios in buffer solution, quickly stirred and evenly mixed, obtained in the homodisperse of yellow
Liquid, i.e. ellagic acid reagent.
The present embodiment also provides a kind of activated partial thromboplastin time assay reagent, including above-mentioned ellagic acid reagent and
Chlorination calcon.
Embodiment 4
The present embodiment provides a kind of ellagic acid reagents, are made using following preparation method：
(1) 1.5mmol ellagic acids are added in without in calcium purified water, quick stirring makes it homodisperse.
Add in 2.5mol/L sodium acetates.
1mol/L Tris-hcl are rapidly joined in 10 minutes after ellagic acid is added in, adjust pH to 7.1 ± 0.1.
Add in 0.02mmol iron chloride.
Add in arginine monohydrochloride 1.5% and glycine 1.5%.
Add in preservative gentamicin 0.5g/L.
(2) emulsification phosphatide is prepared：5 DEG C of addition mixes without calcium purified water, and in 5 DEG C of ice baths in 0.32g/L rabbit cephalins
Even emulsification obtains emulsification phosphatide.
(3) emulsification phosphatide is added in special ratios in buffer solution, quickly stirred and evenly mixed, obtained in the homodisperse of yellow
Liquid, i.e. ellagic acid reagent.
Embodiment 5
The present embodiment provides a kind of ellagic acid reagents, and preparation method is roughly the same with the preparation method of embodiment 4, different
Part is：The present embodiment is using 3mmol ellagic acids, 0.04mmol iron chloride.
Embodiment 6
The present embodiment provides a kind of ellagic acid reagents, and preparation method is roughly the same with the preparation method of embodiment 4, different
Part is：The present embodiment is using 6mmol ellagic acids, 0.06mmol iron chloride.
Embodiment 7
The present embodiment provides a kind of ellagic acid reagents, are made using following preparation method：
(1) 3mmol ellagic acids are added in without in calcium purified water, quick stirring makes it homodisperse.
Add in 2.5mol/L sodium acetates.
1mol/L Tris-hcl are rapidly joined in 10 minutes after ellagic acid is added in, adjust pH to 7.1 ± 0.1.
Add in 0.04mmol iron chloride.
Add in arginine monohydrochloride 0.5% and glycine 0.5%.
Add in preservative gentamicin 0.5g/L.
(2) emulsification phosphatide is prepared：5 DEG C of addition mixes without calcium purified water, and in 5 DEG C of ice baths in 0.16g/L rabbit cephalins
Even emulsification obtains emulsification phosphatide.
(3) emulsification phosphatide is added in special ratios in buffer solution, quickly stirred and evenly mixed, obtained in the homodisperse of yellow
Liquid, i.e. ellagic acid reagent.
Embodiment 8
The present embodiment provides a kind of ellagic acid reagents, and preparation method is roughly the same with the preparation method of embodiment 7, different
Part is：The present embodiment uses arginine monohydrochloride 1.5% and glycine 1.5%.
Embodiment 9
The present embodiment provides a kind of ellagic acid reagents, and preparation method is roughly the same with the preparation method of embodiment 7, different
Part is：The present embodiment uses arginine monohydrochloride 3% and glycine 3%.
Different ellagic acid reagents and activated partial thromboplastin time assay reagent are tested below by way of experiment.
1st, be respectively adopted ice bath emulsification, room temperature emulsification, dissolution process phosphatide, specific method are as follows in other ways：
A. ice bath emulsifies：2~8 DEG C of ice water are added in pipe is ground, while rabbit cephalin is added in mixing pipe, are then existed
Added in mixing pipe 2~8 DEG C without calcium purified water, grinding pipe is inserted into mixing pipe, is ground in pipe using grinding pipe
Substance.
B. room temperature emulsifies：In grinding pipe room temperature without calcium purified water, grinding pipe is inserted into mixing pipe, is ground using grinding pipe
Substance in mixing pipe.
C. other modes dissolve：Rabbit cephalin is added in 10ml centrifuge tubes, is then added in without calcium purified water (no temperature limit
It is fixed), rabbit cephalin is pounded little particle as possible with glass bar, then shakes up manually or shakes mixing by other forms.
The emulsification of comparison ice bath, room temperature emulsification, the data of dissolution process phosphatide and Siemens's reagent compare in other ways：
Quality Control measured value 1/2, Quality Control 10 groups of data of repeatability, solution dispersity (homodisperse, whether particle is visible), comparing result is such as
Shown in table 1：
The data comparison result of the different emulsifying manner processing phosphatide of table 1
As can be seen from Table 1, it compared with the mode of room temperature emulsification or other modes dissolution process phosphatide, is emulsified using ice bath
Phosphatide character that mode is handled is good, the coefficient of variation is minimum, meets product demand.
2nd, ellagic acid-three groups of metal ion additive amount data, Quality Control 1/2 are done Quality Control with Siemens reagent and are compared, right
Than the results are shown in Table 2：
The data comparison result of 2 ellagic acids of table-metal ion Different adding amount
As can be seen from Table 2, the Different adding amount of ellagic acid-metal ion:Ellagic acid and metal ion shape in embodiment 5
Into optimum proportioning, Quality Control measured value is consistent with import Siemens APTT ellagic acid reagent measured values.
3rd, phosphatide-arginine monohydrochloride-three groups of glycine additive amount data, Quality Control 1/2, with Siemens reagent do Quality Control into
Row comparison, 37 DEG C~30 days stability, 5 ± 3 DEG C of 30 days stability of uncapping, comparing result are as shown in table 3：
The data comparison result of 3 phosphatide of table-arginine monohydrochloride-glycine Different adding amount
As can be seen from Table 3, the Different adding amount of phosphatide-arginine monohydrochloride-glycine：In embodiment 7-9, embodiment
8 Quality Control measured values are consistent with Siemens APTT ellagic acid reagents, by carrying out 37 DEG C of thermostabilizations with Siemens APTT ellagic acids reagent
Property experiment and 2-8 DEG C of experiment of uncapping, embodiment 8 be respectively provided with good stability, and consistent with import reagent stability.
4, it surveys patient's sample with reagent of the present invention and Siemens's reagent surveys patient's sample and (amounts to 50 samples, No. 1-30 is
30 normal samples, No. 31-40 is 10 hepatopathy section samples, and No. 41-50 is 10 cardiology department's samples) carry out methodology correlation
Property compare, coefficient R=0.998 (R2=0.996), comparing result is as shown in table 4：
The correlation comparing result of the different samples of table 4
Correlation analysis is carried out to the data of table 4, Fig. 2 is correlation scatter plot, right simultaneously it can be seen from table 4 and Fig. 2
Than 50 clinical blood coagulation samples, by measured value and correlation analysis, ellagic acid reagent of the invention and import Siemens APTT tans
The correlation of flower acid reagent is high.
In conclusion the ellagic acid reagent and its preparation method of the embodiment of the present invention are emulsified including phosphatide, ellagic acid dissolving,
The production technology of each raw material order of addition, APTT time adjustments etc., this is simple for process quick, and is described in detail；The present invention
The activated partial thromboplastin time assay reagent of embodiment is with good stability, can stablize at 37 DEG C at least 30 days, examination
Agent is uncapped after at least stablizing at 5 ± 3 DEG C 30 days or more；The APTT kits of the embodiment of the present invention can directly replace import high steady
Qualitative activated partial thromboplastin time (APTT) kit.
Embodiments described above is part of the embodiment of the present invention, instead of all the embodiments.The reality of the present invention
The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of the selected implementation of the present invention
Example.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without creative efforts
Every other embodiment, shall fall within the protection scope of the present invention.
Claims (10)
1. a kind of preparation method of ellagic acid reagent, which is characterized in that it includes the following steps：
Ellagic acid is added in without in calcium purified water, quick stirring makes it homodisperse；It is firstly added alkaline buffer or inorganic
Alkali；Then rapidly join acidic buffer or organic acid, inorganic acid in 10 minutes after ellagic acid is added in, adjust pH to 7.1 ±
0.1；Metal ion is added in later；Amino acid and its derivative are subsequently added into, obtains buffer solution；
It is emulsified in addition, adding in 2~8 DEG C in rabbit cephalin without calcium purified water, and in 2~8 DEG C of mixings, obtains emulsification phosphatide；
The emulsification phosphatide is added in the buffer solution, quickly stirs and evenly mixs, obtains ellagic acid reagent.
2. the preparation method of ellagic acid reagent according to claim 1, which is characterized in that the mixing emulsification is in using
What empty mixing pipe and hollow grinding pipe carried out, specific method is：Grinding pipe is inserted into mixing pipe, is added in pipe is ground
2~8 DEG C of ice water, while rabbit cephalin is added in mixing pipe, then added in mixing pipe 2~8 DEG C without calcium purified water, protect
It demonstrate,proves entire emulsion process to be in 2~8 DEG C of environment, the substance being ground using grinding pipe in pipe.
3. the preparation method of ellagic acid reagent according to claim 1, which is characterized in that the alkaline buffer is acetic acid
At least one of sodium, Tris-base, Hepes-Na；Inorganic base is at least one of sodium hydroxide and potassium hydroxide.
4. the preparation method of ellagic acid reagent according to claim 1, which is characterized in that the acidic buffer is
At least one of Tris-hcl, Hepes, dihydric phosphate；Organic acid is at least one of formic acid, acetic acid, ethanedioic acid；Nothing
Machine acid is hydrochloric acid.
5. the preparation method of ellagic acid reagent according to claim 1, which is characterized in that the metal ion for magnesium from
At least one of son, iron ion, manganese ion.
6. the preparation method of ellagic acid reagent according to claim 1, which is characterized in that amino acid and its derivative are third
At least one of propylhomoserin, glycine, batanone acid, arginine, histidine, glutamic acid and its salt.
7. the preparation method of ellagic acid reagent according to claim 1, which is characterized in that adding in acidic buffer or having
Preservative is added in random time after machine acid, inorganic acid, the preservative is Sodium azide, proclin300, gentamicin
At least one of.
8. a kind of ellagic acid reagent, which is characterized in that it is using the ellagic acid examination as described in any one of claim 1 to 7
The preparation method of agent is made.
9. a kind of activated partial thromboplastin time assay reagent, which is characterized in that it includes tan flower as claimed in claim 8
Acid reagent and chlorination calcon, the chlorination calcon are the phosphate buffer that calcium chloride is prepared.
10. a kind of APTT kits, which is characterized in that it includes activated partial thromboplastin time as claimed in claim 9
Measure reagent.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810129827.0A CN108226540B (en) | 2018-02-08 | 2018-02-08 | Ellagic acid reagent, preparation method thereof, activated partial thromboplastin time determination reagent and APTT kit |
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| CN201810129827.0A CN108226540B (en) | 2018-02-08 | 2018-02-08 | Ellagic acid reagent, preparation method thereof, activated partial thromboplastin time determination reagent and APTT kit |
Publications (2)
| Publication Number | Publication Date |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110108890A (en) * | 2019-05-05 | 2019-08-09 | 深圳优迪生物技术有限公司 | Activated partial thromboplastin time assay reagent and its application |
| CN110887970A (en) * | 2019-11-29 | 2020-03-17 | 北京赛科希德科技股份有限公司 | Extraction buffer solution, rabbit brain extract, PT detection reagent and PT detection kit |
| CN113009161A (en) * | 2021-02-09 | 2021-06-22 | 桂林优利特医疗电子有限公司 | Detection kit for activated partial thromboplastin time and preparation method thereof |
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