CN108226540B - Ellagic acid reagent, preparation method thereof, activated partial thromboplastin time determination reagent and APTT kit - Google Patents
Ellagic acid reagent, preparation method thereof, activated partial thromboplastin time determination reagent and APTT kit Download PDFInfo
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Abstract
An ellagic acid reagent, a preparation method thereof, an activated partial thromboplastin time determination reagent and an APTT kit, and relates to the field of clinical diagnosis reagents. The preparation method comprises adding ellagic acid into calcium-free purified water, and rapidly stirring to uniformly disperse the ellagic acid; adding an alkaline buffer solution or inorganic base; quickly adding an acidic buffer solution or organic acid and inorganic acid within 10 minutes after adding the ellagic acid; adding metal ions; adding amino acid and derivatives thereof to obtain a buffer solution; in addition, adding calcium-free purified water into the rabbit brain phospholipid, and uniformly mixing and emulsifying to obtain emulsified phospholipid; adding the emulsified phospholipid into the buffer solution, and quickly stirring and uniformly mixing to obtain the ellagic acid reagent. The activated partial thromboplastin time measuring reagent comprises the ellagic acid reagent and the calcium chloride reagent, and has good stability. The APTT kit comprising the activated partial thromboplastin time measuring reagent can directly replace an imported high-stability APTT kit.
Description
Technical Field
The invention relates to the field of clinical diagnosis reagents, and in particular relates to an ellagic acid reagent, a preparation method thereof, an activated partial thromboplastin time determination reagent and an APTT kit.
Background
In the field of clinical examination, examination for examining abnormalities in blood coagulation factors and the like by measuring the coagulation time of blood is known, and among these, APTT measurement is one of clinical examinations for general blood coagulation ability. APTT (activated partial thromboplastin time), which is the activated partial thromboplastin time, is the coagulation time reflecting the function of the intrinsic coagulation pathway of blood that starts by contact with a negatively charged foreign substance during bleeding. The APTT assay is used not only for screening and examining the deficiency or abnormality of intrinsic coagulation factors but also for monitoring heparin therapy.
Most of the existing APTT reagents adopt rabbit brain phospholipid, the phospholipid is easy to turn into reddish brown in the air, has hygroscopicity, is insoluble in water and acetone, is slightly soluble in ethanol, and is soluble in chloroform and diethyl ether, so that the problem of dissolution exists, uneven insoluble substances can be generated if the phospholipid is stirred and dissolved forcibly, the process is uncontrollable, and the phospholipid can be inactivated or even oxidized if the phospholipid is dissolved by heating. By way of search, some of the prior patents do not specify the phospholipid treatment process, and other prior patents disclose problems, such as: the rabbit brain extracted cephalin in the invention patent with the application number of CN200710036410.1 is dissolved by distilled water, which is difficult to realize, so the patent has misleading process; the patent application No. CN200810083554.7 discloses a liquid kaolin APTT reagent which is stable for at least 30 days at 37 ℃, but does not disclose a specific process, and the sensitivity of the reagent is different depending on the activator, and the performance of the APTT reagent is unknown.
In recent years, a plurality of synthetic phospholipids are used in combination for the purpose of clinical sensitivity, disease identification, or quality improvement (see, for example, US 2011/159597). In addition, as the types of synthetic phospholipids, 3 kinds of Phosphatidylethanolamine (PE), Phosphatidylcholine (PC) and Phosphatidylserine (PS) are mixed and contained in the reagent, which is the main stream, but the synthetic phospholipids are many in types, difficult to obtain and expensive, and the ratio of each component is difficult to control, and the dissolution problem is also existed, and the precipitation is partially caused, which affects the quality of the reagent. The existing reagent has the problems of ellagic acid and phospholipid precipitation, ellagic acid buffer solution, raw material treatment such as ellagic acid loss of activity under alkaline conditions, uniform phospholipid emulsification, and the problem of the proportion of the ellagic acid buffer solution to the phospholipid, which directly influence the quality of the domestic reagent, so that the market is still occupied by siemens, Sitah, Waifen and the like.
Therefore, a kit which has simple manufacturing process and high stability and can directly replace imported high-stability Activated Partial Thromboplastin Time (APTT) is expected by the market.
Disclosure of Invention
The invention aims to provide an ellagic acid reagent and a preparation method thereof, which comprise the production processes of phospholipid emulsification, ellagic acid dissolution, addition sequence of all raw materials, APTT time adjustment and the like, and the processes are simple and rapid and are all described in detail.
Another object of the present invention is to provide an activated partial thromboplastin time measuring reagent which has good stability, is stable for at least 30 days at 37 ℃ and is stable for at least 30 days at 5 + -3 ℃ after uncovering.
Another objective of the invention is to provide an APTT kit, which can directly replace an imported high-stability Activated Partial Thromboplastin Time (APTT) kit.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a preparation method of an ellagic acid reagent, which comprises the following steps:
adding ellagic acid into calcium-free purified water, and rapidly stirring to uniformly disperse the ellagic acid; firstly, adding an alkaline buffer solution or inorganic base; then quickly adding an acidic buffer solution or organic acid and inorganic acid within 10 minutes after adding the ellagic acid, and adjusting the pH to 7.1 +/-0.1; then adding metal ions; then adding amino acid and derivatives thereof to obtain a buffer solution;
in addition, adding 2-8 ℃ calcium-free purified water into the rabbit brain phospholipid, and uniformly mixing and emulsifying at 2-8 ℃ to obtain emulsified phospholipid;
and adding the emulsified phospholipid into the buffer solution, and quickly stirring and uniformly mixing to obtain the ellagic acid reagent.
Further, in a preferred embodiment of the present invention, the blending emulsification is performed by using a hollow blending tube and a hollow grinding tube, and the specific method is as follows: inserting a grinding tube into a mixing tube, adding ice water at 2-8 ℃ into the grinding tube, adding rabbit brain phospholipid into the mixing tube, adding calcium-free purified water at 2-8 ℃ into the mixing tube, ensuring that the whole emulsification process is in an environment of 2-8 ℃, and grinding substances in the mixing tube by using the grinding tube.
Further, in the preferred embodiment of the invention, the alkaline buffer is at least one of sodium acetate, Tris-base and Hepes-Na; the inorganic base is at least one of sodium hydroxide and potassium hydroxide.
Further, in the preferred embodiment of the present invention, the acidic buffer is at least one of Tris-hcl, Hepes and dihydrogen phosphate; the organic acid is at least one of formic acid, acetic acid and oxalic acid; the inorganic acid is hydrochloric acid.
Further, in a preferred embodiment of the present invention, the metal ion is at least one of magnesium ion, iron ion, and manganese ion.
Further, in a preferred embodiment of the present invention, the amino acid and the derivative thereof are at least one of alanine, glycine, tetronic acid, arginine, histidine, glutamic acid, and salts thereof.
Further, in the preferred embodiment of the present invention, a preservative is added at any time after the addition of the acidic buffer or the organic acid or the inorganic acid, and the preservative is at least one of sodium azide, proclin300 and gentamicin.
The invention provides an ellagic acid reagent, which is prepared by adopting the preparation method of the ellagic acid reagent.
The invention provides a reagent for measuring the time of activated partial thromboplastin, which comprises the ellagic acid reagent and a calcium chloride reagent, wherein the calcium chloride reagent is a phosphate buffer solution prepared from calcium chloride.
The invention provides an APTT kit which comprises the activated partial thromboplastin time measuring reagent.
The ellagic acid reagent and the preparation method thereof, the activated partial thromboplastin time determination reagent and the APTT kit have the beneficial effects that: the preparation method of the ellagic acid reagent provided by the embodiment of the invention comprises the steps of adding ellagic acid into calcium-free purified water, and quickly stirring to uniformly disperse the ellagic acid; firstly, adding an alkaline buffer solution or inorganic base; then quickly adding an acidic buffer solution or organic acid and inorganic acid within 10 minutes after adding the ellagic acid, and adjusting the pH to 7.1 +/-0.1; then adding metal ions; then adding amino acid and derivatives thereof to obtain a buffer solution; in addition, adding 2-8 ℃ calcium-free purified water into the rabbit brain phospholipid, and uniformly mixing and emulsifying at 2-8 ℃ to obtain emulsified phospholipid; adding the emulsified phospholipid into the buffer solution, and quickly stirring and uniformly mixing to obtain the ellagic acid reagent. The reagent for measuring the activated partial thromboplastin time comprises the ellagic acid reagent and the calcium chloride reagent, wherein the calcium chloride reagent is a phosphate buffer solution prepared from calcium chloride, the reagent has good stability, can be stable for at least 30 days at 37 ℃, and is stable for at least 30 days at 5 +/-3 ℃ after the cover of the reagent is opened. The APTT kit provided by the embodiment of the invention comprises the activated partial thromboplastin time measuring reagent, and can directly replace an imported high-stability APTT kit.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a flow chart of a method for formulating an ellagic acid reagent according to an embodiment of the present invention;
FIG. 2 is a plot of correlation scattergrams for different samples in an example of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The ellagic acid reagent and the method for preparing the same, the activated partial thromboplastin time measurement reagent, and the APTT kit according to the embodiment of the present invention will be specifically described below.
The embodiment of the invention provides a preparation method of an ellagic acid reagent, which is shown in a figure 1 and comprises the following steps:
(1) preparing a buffer solution:
adding ellagic acid into calcium-free purified water, rapidly stirring to uniformly disperse, wherein the rotation number is about 500-1000 r/min, the specific rotation speed is determined according to the size of a stirring rotor, and the standard that the solution does not overflow is adopted, such as: 1000ml of ellagic acid reagent was prepared, and the stirring rotor was 15 × 100mm (diameter-length) and rotated at 500r/min, and the stirring rotor was 8 × 40mm (diameter-length) and rotated at 1000 r/min. Ellagic acid is less soluble and the pH of purified water is about 5.6, so even if stirring is accelerated for 24 hours, complete dissolution cannot be achieved. The used ellagic acid has no special requirement and can be analyzed and purified, and in the embodiment, 0.2-0.5 mmol of ellagic acid is preferably adopted.
The alkali buffer or inorganic base is added first, and the ellagic acid is rapidly dissolved under the alkali condition, so that the ellagic acid can be completely dissolved about 2 minutes after the alkali buffer or inorganic base is added. The alkaline buffer solution is at least one of sodium acetate, Tris-base and Hepes-Na; the inorganic base is at least one of sodium hydroxide and potassium hydroxide. In this embodiment, preferably, 0.3 to 0.8mmol of sodium acetate is added.
Then quickly adding an acidic buffer solution or organic acid and inorganic acid within 10 minutes after adding the ellagic acid, adjusting the pH to 7.1 +/-0.1, wherein the ellagic acid is easily soluble in alkali, but has poor stability in an alkaline solution and is easily decomposed, so that the ellagic acid is not suitable for being prepared into a solution for long-time analysis and scientific research, and the acidic buffer solution needs to be quickly added to ensure the stability of the ellagic acid, thereby forming the optimal pH of the APTT reagent. The acidic buffer solution is at least one of Tris-hcl, Hepes and dihydrogen phosphate; the organic acid is at least one of formic acid, acetic acid and oxalic acid; the inorganic acid is hydrochloric acid. In this embodiment, 0.8 to 1.3mmol of Tris-hcl is preferably added.
And then, adding metal ions, wherein the metal ions and the ellagic acid form a stable chelate to accelerate the activation effect of the ellagic acid, and the adding amount of the metal ions directly influences the measured value of the formed reagent and the correlation with a comparative Siemens reagent. The metal ions are at least one of magnesium ions, iron ions and manganese ions, and 0.02-0.03 mmol of iron ions are preferably added in the embodiment.
And then adding amino acid and derivatives thereof to prevent the ellagic acid from precipitating and ensure the quality of the reagent to obtain a buffer solution. The amino acid and its derivative are at least one of alanine, glycine, tetronic acid, arginine, histidine, glutamic acid and its salt, and arginine hydrochloride 1.5% and glycine 1.5% are preferably added in this embodiment.
When the buffer solution is prepared, the preservative can be added at any time after the acidic buffer solution or the organic acid and the inorganic acid are added, and the preservative is at least one of sodium azide, proclin300 and gentamicin.
(2) Preparing emulsified phospholipid: adding calcium-free purified water at the temperature of 2-8 ℃ into rabbit cephalin, and uniformly mixing and emulsifying in an ice bath at the temperature of 2-8 ℃ to obtain emulsified phospholipid. The animal phospholipid adopted in the embodiment is taken from rabbit brain, preferably 0.3-0.35 g/L of rabbit brain phospholipid, and the raw material is sold in a large amount in the market.
The blending emulsification of this embodiment is performed by using a hollow blending tube and a hollow grinding tube, and the specific method is as follows: inserting a grinding tube into a mixing tube, adding ice water at 2-8 ℃ into the grinding tube, adding rabbit brain phospholipid into the mixing tube, adding calcium-free purified water at 2-8 ℃ into the mixing tube, ensuring that the whole emulsification process is in an environment of 2-8 ℃, and grinding substances in the mixing tube by using the grinding tube. The mixing tube and the grinding tube form a device similar to the grinding device, the phospholipid is quickly emulsified in a liquid low-temperature environment, thorough, uniform and quick emulsification is guaranteed, the unique, convenient and quick emulsification process is adopted, the emulsification is complete, the phospholipid particles are uniform, the activity of the phospholipid particles is not damaged, and the uniform liquid emulsion is prepared.
(3) Adding the emulsified phospholipid into the buffer solution according to a specific proportion, and quickly stirring and uniformly mixing to obtain a yellow uniform dispersion liquid, namely the ellagic acid reagent.
The embodiment of the invention provides an ellagic acid reagent, which is prepared by adopting the preparation method of the ellagic acid reagent.
The embodiment of the invention provides a reagent for measuring activated partial thromboplastin time, which mainly comprises two parts, namely an ellagic acid reagent and a calcium chloride reagent, wherein the calcium chloride reagent is a phosphate buffer solution with a proper concentration prepared by calcium chloride, the calcium chloride reagent used in the embodiment is prepared by 0.03mmol of calcium chloride, 0.5mmol of disodium hydrogen phosphate and 0.10 mmol of sodium dihydrogen phosphate, and the pH value is 7.10. The application method of the activated partial thromboplastin time measuring reagent comprises the following steps: 50ul of the sample was added to the hemagglutination cup and incubated at 37 ℃ for 60S; subsequently, 50ul of the above-mentioned APTT assay was added thereto, and incubated at 37 ℃ for 60S; and finally, adding 50ul of the calcium chloride solution into a hemagglutination cup, uniformly mixing the sample, the APTT reagent and the calcium chloride reagent, and measuring the coagulation time after 240 seconds.
The embodiment of the invention provides an APTT kit which comprises an activated partial thromboplastin time determination reagent.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example provides an ellagic acid reagent prepared by the following method:
(1) adding 0.4mmol ellagic acid into calcium-free purified water, and rapidly stirring to uniformly disperse.
0.5mmol of sodium acetate was added.
1mmol Tris-hcl was added rapidly within 10 minutes after the addition of ellagic acid, adjusting the pH to 7.1. + -. 0.1.
0.025mmol of iron ions were added.
Arginine hydrochloride 1.5% and glycine 1.5% were added.
Adding preservative sodium azide.
(2) Preparing emulsified phospholipid: adding calcium-free purified water with the temperature of 5 ℃ into 0.32g/L rabbit cephalin, and uniformly mixing and emulsifying in an ice bath with the temperature of 5 ℃ to obtain the emulsified phospholipid.
(3) Adding the emulsified phospholipid into the buffer solution according to a specific proportion, and quickly stirring and uniformly mixing to obtain a yellow uniform dispersion liquid, namely the ellagic acid reagent.
The present embodiment also provides an activated partial thromboplastin time measuring reagent comprising the above-mentioned ellagic acid reagent and calcium chloride reagent.
Example 2
This example provides an ellagic acid reagent prepared by the following method:
(1) adding 0.2mmol ellagic acid into calcium-free purified water, and rapidly stirring to uniformly disperse.
0.4mmol Tris-base was added.
0.8mmol Hepes was added rapidly within 10 minutes after the addition of ellagic acid, and the pH was adjusted to 7.1. + -. 0.1.
0.02mmol of magnesium ions was added.
Alanine was added.
The preservative proclin300 was added.
(2) Preparing emulsified phospholipid: adding calcium-free purified water with the temperature of 3 ℃ into 0.3 g/L rabbit cephalin, and uniformly mixing and emulsifying in an ice bath with the temperature of 3 ℃ to obtain the emulsified phospholipid.
(3) Adding the emulsified phospholipid into the buffer solution according to a specific proportion, and quickly stirring and uniformly mixing to obtain a yellow uniform dispersion liquid, namely the ellagic acid reagent.
The present embodiment also provides an activated partial thromboplastin time measuring reagent comprising the above-mentioned ellagic acid reagent and calcium chloride reagent.
Example 3
This example provides an ellagic acid reagent prepared by the following method:
(1) adding 0.5mmol ellagic acid into calcium-free purified water, and rapidly stirring to uniformly disperse.
0.8mmol of sodium hydroxide was added.
About 0.5ml of acetic acid was rapidly added within 10 minutes after the addition of ellagic acid, and the pH was adjusted to 7.1. + -. 0.1.
0.03mmol of manganese ions were added.
Histidine and glutamic acid were added.
The preservative gentamicin is added.
(2) Preparing emulsified phospholipid: adding calcium-free purified water with the temperature of 8 ℃ into 0.35 g/L rabbit cephalin, and uniformly mixing and emulsifying in an ice bath with the temperature of 8 ℃ to obtain the emulsified phospholipid.
(3) Adding the emulsified phospholipid into the buffer solution according to a specific proportion, and quickly stirring and uniformly mixing to obtain a yellow uniform dispersion liquid, namely the ellagic acid reagent.
The present embodiment also provides an activated partial thromboplastin time measuring reagent comprising the above-mentioned ellagic acid reagent and calcium chloride reagent.
Example 4
This example provides an ellagic acid reagent prepared by the following method:
(1) adding 1.5mmol ellagic acid into calcium-free purified water, and rapidly stirring to uniformly disperse.
2.5mol/L sodium acetate was added.
1mol/L Tris-hcl was added rapidly within 10 minutes after the addition of ellagic acid, and the pH was adjusted to 7.1. + -. 0.1.
0.02mmol of ferric chloride was added.
Arginine hydrochloride 1.5% and glycine 1.5% were added.
Adding 0.5g/L of preservative gentamicin.
(2) Preparing emulsified phospholipid: adding calcium-free purified water of 5 deg.C into rabbit brain phospholipid of 0.32g/L, and emulsifying in ice bath of 5 deg.C to obtain emulsified phospholipid.
(3) Adding the emulsified phospholipid into the buffer solution according to a specific proportion, and quickly stirring and uniformly mixing to obtain a yellow uniform dispersion liquid, namely the ellagic acid reagent.
Example 5
This example provides an ellagic acid reagent that is prepared in substantially the same manner as example 4, except that: in this example, 3mmol of ellagic acid and 0.04mmol of ferric chloride were used.
Example 6
This example provides an ellagic acid reagent that is prepared in substantially the same manner as example 4, except that: in this example, 6mmol of ellagic acid and 0.06mmol of ferric chloride were used.
Example 7
This example provides an ellagic acid reagent prepared by the following method:
(1) adding 3mmol ellagic acid into calcium-free purified water, and rapidly stirring to uniformly disperse.
2.5mol/L sodium acetate was added.
1mol/L Tris-hcl was added rapidly within 10 minutes after the addition of ellagic acid, and the pH was adjusted to 7.1. + -. 0.1.
0.04mmol of ferric chloride was added.
Arginine hydrochloride 0.5% and glycine 0.5% were added.
Adding 0.5g/L of preservative gentamicin.
(2) Preparing emulsified phospholipid: adding calcium-free purified water of 5 deg.C into rabbit cephalin 0.16g/L, and emulsifying in ice bath of 5 deg.C to obtain emulsified phospholipid.
(3) Adding the emulsified phospholipid into the buffer solution according to a specific proportion, and quickly stirring and uniformly mixing to obtain a yellow uniform dispersion liquid, namely the ellagic acid reagent.
Example 8
This example provides an ellagic acid reagent that is prepared in substantially the same manner as example 7, except that: this example uses 1.5% arginine hydrochloride and 1.5% glycine.
Example 9
This example provides an ellagic acid reagent that is prepared in substantially the same manner as example 7, except that: this example uses arginine hydrochloride 3% and glycine 3%.
The following tests were carried out by assay on different ellagic acid reagents and activated partial thromboplastin time measuring reagents.
1. Respectively adopting ice bath emulsification, normal temperature emulsification and other modes to dissolve and process phospholipid, and the specific method comprises the following steps:
a. and (3) emulsification in ice bath: adding ice water of 2-8 ℃ into a grinding tube, adding rabbit brain phospholipid into a mixing tube, adding calcium-free purified water of 2-8 ℃ into the mixing tube, inserting the grinding tube into the mixing tube, and grinding the substances in the mixing tube by using the grinding tube.
b. And (3) emulsification at normal temperature: and (3) at normal temperature, calcium-free purified water is filled into the grinding pipe, the grinding pipe is inserted into the mixing pipe, and the grinding pipe is used for grinding the substances in the mixing pipe.
c. Dissolving in other modes: rabbit brain phospholipid is added into a 10ml centrifugal tube, then calcium-free purified water (without temperature limitation) is added, the rabbit brain phospholipid is smashed into small particles as much as possible by a glass rod, and then the rabbit brain phospholipid is evenly stirred by hand or is evenly mixed by means of other modes of vibration.
And comparing data of the phospholipids subjected to ice bath emulsification, normal-temperature emulsification and other dissolving treatment with a Siemens reagent: quality control measurement 1/2, quality control repeatability 10 sets of data, dispersion state of the solution (uniform dispersion, whether particles are visible), and comparison results are shown in table 1:
TABLE 1 comparison of data for phospholipid treatment with different emulsification modes
As can be seen from Table 1, compared with the phospholipid dissolving treatment mode of normal temperature emulsification or other modes, the phospholipid treated by the ice bath emulsification mode has good properties and the lowest coefficient of variation, and meets the product requirements.
2. Three groups of data of the addition amount of the ellagic acid-metal ions, namely quality control 1/2, are compared with those of a Siemens reagent, and the comparison result is shown in Table 2:
TABLE 2 comparison of data on different addition amounts of ellagic acid-metal ion
As can be seen from Table 2, the optimum ratio of ellagic acid to metal ion in example 5 was obtained at different addition levels of ellagic acid-metal ion, and the measured value of quality control was consistent with that of imported Siemens APTT ellagic acid reagent.
3. Three groups of data of the addition amount of phospholipid-arginine hydrochloride-glycine are shown in the table 3, wherein the quality control is 1/2, and compared with the quality control of a Siemens reagent, the stability of the phospholipid-arginine hydrochloride-glycine addition amount data is 37-30 days, and the stability of the phospholipid-arginine hydrochloride-glycine addition amount data is 30 days after uncovering at the temperature of 5 +/-3 ℃, and the comparison results are shown in the table 3:
TABLE 3 comparison of data for different amounts of phospholipid arginine hydrochloride glycine
As can be seen from table 3, the different addition amounts of phospholipid-arginine hydrochloride-glycine: in examples 7-9, the quality control values of example 8 were consistent with those of Siemens APTT ellagic acid reagent, and example 8 had good stability and was consistent with that of imported reagents by performing 37 ℃ heat stability test and 2-8 ℃ decap test with Siemens APTT ellagic acid reagent.
4, the reagent test patient samples are compared with Siemens reagent test patient samples (50 samples in total, No. 1-30 normal samples, No. 31-40 liver disease family samples and No. 41-50 cardiovascular family samples) for methodology correlation, and the correlation coefficient R is 0.998(R is 0.998)20.996), the results are shown in table 4:
TABLE 4 correlation comparison of different samples
The data in table 4 are subjected to correlation analysis, fig. 2 is a correlation scatter diagram, and as can be seen from table 4 and fig. 2, compared with 50 clinical blood coagulation samples, the correlation between the ellagic acid reagent of the present invention and the imported siemens APTT ellagic acid reagent is high through measurement and correlation analysis.
In conclusion, the ellagic acid reagent and the preparation method thereof provided by the embodiment of the invention comprise production processes of phospholipid emulsification, ellagic acid dissolution, addition sequence of raw materials, APTT time adjustment and the like, and the processes are simple and rapid and are all described in detail; the reagent for measuring the activated partial thromboplastin time has good stability, can be stable for at least 30 days at 37 ℃, and is stable for at least more than 30 days at 5 +/-3 ℃ after the cover of the reagent is opened; the APTT kit provided by the embodiment of the invention can directly replace an imported high-stability Activated Partial Thromboplastin Time (APTT) kit.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (4)
1. The preparation method of the ellagic acid reagent is characterized by comprising the following steps:
adding ellagic acid into calcium-free purified water, and rapidly stirring to uniformly disperse the ellagic acid; firstly, adding an alkaline buffer solution or inorganic base; then quickly adding an acidic buffer solution or organic acid and inorganic acid within 10 minutes after adding ellagic acid, adjusting the pH to 7.1 +/-0.1, and then adding a preservative within any time, wherein the preservative is at least one of sodium azide, proclin300 and gentamicin; then adding metal ions; then adding amino acid and derivatives thereof to obtain a buffer solution;
the alkaline buffer solution is at least one of sodium acetate, Tris-base and Hepes-Na; the inorganic base is at least one of sodium hydroxide and potassium hydroxide; the acidic buffer solution is at least one of Tris-hcl, Hepes and dihydric phosphate; the organic acid is at least one of formic acid, acetic acid and oxalic acid; the inorganic acid is hydrochloric acid; the metal ions are at least one of magnesium ions, iron ions and manganese ions; the amino acid and its derivatives are at least one of alanine, glycine, tetronic acid, arginine, histidine, glutamic acid and its salts;
in addition, adding 2-8 ℃ calcium-free purified water into the rabbit brain phospholipid, and uniformly mixing and emulsifying at 2-8 ℃ to obtain emulsified phospholipid; the mixing emulsification is carried out by adopting a hollow mixing tube and a hollow grinding tube, and the specific method comprises the following steps: inserting a grinding tube into a mixing tube, adding ice water at 2-8 ℃ into the grinding tube, adding rabbit brain phospholipid into the mixing tube, adding calcium-free purified water at 2-8 ℃ into the mixing tube, ensuring that the whole emulsification process is in an environment of 2-8 ℃, and grinding substances in the mixing tube by using the grinding tube;
and adding the emulsified phospholipid into the buffer solution, and quickly stirring and uniformly mixing to obtain the ellagic acid reagent.
2. An ellagic acid reagent produced by the method of formulating an ellagic acid reagent of claim 1.
3. An activated partial thromboplastin time measuring reagent comprising the ellagic acid reagent of claim 2 and a calcium chloride reagent, wherein the calcium chloride reagent is a phosphate buffer solution formulated with calcium chloride.
4. An APTT kit comprising the activated partial thromboplastin time measuring reagent of claim 3.
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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 |
| CN110887970B (en) * | 2019-11-29 | 2023-10-31 | 北京赛科希德科技股份有限公司 | Extraction buffer solution, rabbit brain extraction solution, 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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| DE3625090A1 (en) * | 1986-07-24 | 1988-01-28 | Behringwerke Ag | AGENT FOR THE THERAPY FACTOR VIII-RESISTANT HAEMOPHILY A AND METHOD FOR THE PRODUCTION THEREOF |
| US5055412A (en) * | 1989-03-21 | 1991-10-08 | Proksch Gary J | Factor sensitive reagent for testing of blood coagulation containing ellagic acid and divalent metal ions and method of making the same |
| ATE177536T1 (en) * | 1993-06-30 | 1999-03-15 | Diagnostische Forsch Stiftung | MEASUREMENT OF ACTIVATED PARTIAL THROMBOPLASTIN TIME (APTT) IN A ONE-STEP REACTION |
| CN101221189B (en) * | 2007-01-12 | 2012-08-15 | 上海太阳生物技术有限公司 | External diagnostic reagent kit used for measuring activated partial thromboplastin time |
| US9921232B2 (en) * | 2014-09-26 | 2018-03-20 | Abbott Point Of Care Inc. | Ellagic acid formulations for use in coagulation assays |
| JP2017049040A (en) * | 2015-08-31 | 2017-03-09 | シスメックス株式会社 | Reagent, reagent kit and method for measuring activated partial thromboplastin time |
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