CN109239061A - A kind of liquid-type Antiprothrombin antibodies assay kit - Google Patents
A kind of liquid-type Antiprothrombin antibodies assay kit Download PDFInfo
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- CN109239061A CN109239061A CN201811071001.XA CN201811071001A CN109239061A CN 109239061 A CN109239061 A CN 109239061A CN 201811071001 A CN201811071001 A CN 201811071001A CN 109239061 A CN109239061 A CN 109239061A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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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
- G01N33/86—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
The present invention provides a kind of liquid-type Antiprothrombin antibodies assay kit, belongs to medicine in-vitro diagnosis field.The kit, including reagent R1, reagent R2, quality-control product and calibration object；The reagent R1 includes fibrin ferment, heparin or its salt, the first buffer and protective agent；The reagent R2 includes the chromophoric substrate of fibrin ferment, the second buffer, stabilizer and the second preservative.R1 and reagent R2 is liquid-type in antithrombin Ⅲ detection kit of the invention, and corkage is to use, easy to use without redissolving, and storage is valid up to 1 year, and not by the interference of HCII in plasma sample, the detection range of linearity is wide, and highest detection limit can reach 155%.
Description
Technical field
The invention belongs to medicine in-vitro diagnosis fields, and in particular to a kind of liquid-type Antiprothrombin antibodies measurement reagent
Box.
Background technique
Antithrombin Ⅲ is a kind of serine protease inhibitor (serine protease inhibitor) in blood plasma.
Factorⅱa, VII, Ⅸ a, Ⅹ a, Ⅻ a activated centre contain serine residue, belong to serine protease (serine
protease).Arginine residues on antithrombin Ⅲ molecule, can in conjunction with the serine residue of these enzyme active centers and
It is allowed to inactivate.In blood, each molecule antithrombin Ⅲ can form compound in conjunction with a molecule fibrin ferment, to make
Thrombin inactivation.Chromogenic assay is now widely used in laboratory research and in-vitro diagnosis.Its principle be it is artificial synthesized first can
With by the compound of thromboplastin catalytic pyrolysis to be measured, and compound connects upper chromogenic substance, in the detection process chromogenic substance
It can be dissociated, make occur color change in test sample, the activity of tested thromboplastin can be extrapolated according to color change.
The activity of antithrombin Ⅲ (AT- III) is grasped as disseminated intravascular coagulation (DIC), liver illness, nephrosis
Finger when monitoring, ill-condition analysis, prognosis judgement and the heparin therapy or use III concentrate formulation of AT- of the diseases such as syndrome are offerd medicine
Mark is of great significance.AT- III is as the most important obstruction factor of factor Xa in blood, it controls blood
Solidification and fibrinous dissolution.The level of AT- III changes according to various diseases, symptom in its blood.AT- III in blood
Level reduction, which may result in heparin therapy effect, to be presented.III pathologic of AT- increases: show blood anticoagulant increased activity,
It is mainly seen in oral anticoagulation, Acute hemorrhage etc..III pathologic of AT- reduces: common are congenital III deficiency disease of AT-；Blood
State and when thrombotic diseases before bolt, blood anticoagulant declines, as DIC high coagulates phase, myocardial infarction, angina pectoris, cerebrovascular disease
Change, pregnant disease, Deep vain thrombosis, nephrotic syndrome etc.；Synthesis is reduced, such as severe liver disease；
The presently disclosed detection to antithrombase mainly has three classes method, is freezing method, immunization and color development bottom respectively
Object method.Freezing method is that III activity of AT- in sample is directly or indirectly measured by plasma coagulation time, and freezing method can be direct
Using test plasma can also by test plasma heat defibrination then use, but directly use test plasma when have it is more
Kind protease participates in reaction, measurement result susceptible.Test plasma heats defibrination processing, trivial operations, Er Qiehui
The reduction for leading to the level of AT- III is unfavorable for accurately calculating.Immunological analysis method includes single radial immunodiffusion method, is immunized
The concentration of AT- III in turbidimetry and enzyme-linked immunosorbent assay sample.This method specificity is good, high sensitivity, still
Cumbersome, time-consuming, inactive AT- III has also assisted in immunological response, and III concentration of AT- of detection can not reflect completely
III activity level of AT-.For the patient that this AT- III caused by reduced due to the activity of AT- III is lacked, testing result is inaccurate
Really.Substrate color development method be fibrin ferment in reagent formed in the presence of heparin with the antithrombase in test plasma fibrin ferment and
The compound of antithrombase is suppressed the activity of fibrin ferment.Remaining no and antithrombin Ⅲ forms the solidifying of compound
Hemase acts on the special chromophoric substrate of fibrin ferment, and substrate cleavage is generated 4- nitroaniline (4-nitroaniline, pNA) colour developing
Group, this reaction can cause the variation in 405 nanometers absorbances, and the amount of the 4- nitroaniline generated is resisted in sample
What the activity of fibrin ferment was inversely proportional.Therefore, using the variation of absorbance at measurement 405nm, antithrombase in blood plasma can be calculated
III activity level due to this method high sensitivity, accuracy is good, detection time is short, a variety of automatic analyzers can be suitable for
Device, and under the development of production domesticization detection reagent, clinical detection expense is significantly reduced, using also all the more extensive in clinic.
Although still having testing result by blood mostly in its continuous mode in fact, this method is widely used in clinical application
The case where a variety of interfering substances influence in slurry.Such as heparin cofactor II, it is inhibited to fibrin ferment.About
70% thrombin-inhibiting activity is caused by antithrombin Ⅲ.Therefore the substrate color development method of fibrin ferment cannot accurately reflect blood
The true horizon of antithrombin Ⅲ in slurry.
In order to reduce the influence of heparin cofactor II, in United States Patent (USP) US5985582 and Chinese patent CN102690862A,
A kind of Antithrombin III assay kit (Chromogenic assay) is disclosed, kit includes heparin derivatives, fibrin ferment and color development
Substrate reagent；The reagent is digested to obtain heparin derivatives before sample is added in heparin.This adds increased reagents
The cost of production, technique become comparatively laborious.Due to technical costs height, and the prior art is enterprising in full automatic blood-coagulation instrument
Need to change many parameters when row III Activity determination of AT-, it is inconvenient for operation, as a result also it is easy to appear error.
Demers et al. (Thrombosis and Hemostasia, 69 (3), pp.231-235 (1993)) report uses
FXa substitutes fibrin ferment, not will receive the inhibition of HCII, Chinese patent CN107153043A and CN106153612A disclose one kind
Type Antiprothrombin antibodies measure reagent, and substitute fibrin ferment with FXa, but higher with FX analysis cost, and FXa is unstable
Fixed, trivial operations are not suitable for automatic analyzer.
The detection reagent of antithrombin Ⅲ currently on the market uses freeze-dried powder preparation more, however freeze-dried powder reagent needs ice
Drying process is lyophilized, technics comparing is complicated, and needs to redissolve when in use, and during redissolution, the difference of sample-adding amount can all cause
Poor between freeze-dried powder preparation bottle, difference between batch is very big.
Summary of the invention
That the purpose of the present invention is to solve existing Antiprothrombin antibodies assay kit stability is poor, sensitivity is low
The problem of, and a kind of liquid-type Antiprothrombin antibodies assay kit is provided.
The present invention provides a kind of liquid-type Antiprothrombin antibodies assay kit, including reagent R1, reagent R2, quality-control product
And calibration object；
The reagent R1 includes fibrin ferment, heparin or its salt, the first buffer and protective agent；
The reagent R2 includes the chromophoric substrate of fibrin ferment, the second buffer, stabilizer and the second preservative.
Preferably, any one of the fibrin ferment in people, ox, pig thrombiase, the concentration of the fibrin ferment
For 8-20U/mL.
Preferably, the heparin or its salt are preferably heparin or heparin sodium, and concentration is preferably 12U/ml.
Preferably, the protective agent includes amino acid, polyalcohol, surfactant, metal ion chelation agent, inorganic salts
With the first preservative；
The amino acid is selected from one or more of glycine, lysine, arginine or histidine, quality volume basis
Than for 1%-20%；
The polyalcohol is selected from one or more of xylitol, mannitol, sorbierite, and quality percent by volume is
0.1%-10%；
The surfactant is selected from one or more of Tween-20, Tween-80, TritonX-100, quality volume
Percentage is 0.01%-0.5%；
The metal ion chelation agent is selected from disodium ethylene diamine tetraacetate, sodium potassium tartrate tetrahydrate, ammonium citrate, quality volume hundred
Divide than being 5%-20%；
The inorganic salts are selected from sodium chloride, ammonium sulfate, calcium chloride, potassium sulfate, and quality percent by volume is 0.5%-10%.
Preferably, the chromophoric substrate is selected from Tos-Gly-Pro-Arg-pNA (ChromozymTH), Boc-D-
One or more of Arg-Gly-Arg-pNA2HCl or H-D-Phe-Pip-Arg-pNA2HCl.
Preferably, the stabilizer is casein, quality percent by volume 1-10%.
Preferably, it is slow to be respectively selected from trishydroxymethylaminomethane-hydrochloric acid for first buffer and the second buffer
One or more of fliud flushing, HEPES buffer solution, disodium hydrogen phosphate-citrate buffer solution, barbital sodium-hydrochloride buffer.
Preferably, it is big mould to be respectively selected from Sodium azide, Proclin300, celebrating for first preservative and the second preservative
Element, thimerosal.
Preferably, the quality-control product is added buffer, freeze drying protectant and preservative freeze-drying and is made by animal blood plasma.
Preferably, the calibration object is will to mix after normal healthy people plasma collection, then carries out III activity inspection of AT-
It surveys, detected value should add buffer, freeze drying protectant and preservative, be lyophilized after packing in 80%-120%, then anti-with WHO
Fibrin ferment international standard substance blood plasma carries out traceability definite value to the calibration object.
Beneficial effects of the present invention
The present invention provides a kind of liquid-type Antiprothrombin antibodies assay kit, including reagent R1, reagent R2, quality-control product
And calibration object；Stabilizer casein is used to prepare fibrin ferment chromophoric substrate liquid by the kit, enables to fibrin ferment color development bottom
Object is stably dissolved in aqueous solution.R1 and reagent R2 is liquid-type in antithrombin Ⅲ detection kit of the invention, is opened
Bottle is used, easy to use without redissolving, and storage is valid up to 1 year, can effectively solve the problem that the stability problem of reagent, is stablized
Property good, high sensitivity, specificity it is good, not by the interference of HCII in plasma sample, detect that the range of linearity is wide, and highest detection limit can reach
To 155%.
Detailed description of the invention
Fig. 1 is the calibration curve of antithrombin Ⅲ.
Fig. 2 is the linearity curve of antithrombin Ⅲ.
Specific embodiment
Term as used in the present invention generally there are those of ordinary skill in the art usually to manage unless otherwise indicated
The meaning of solution.The present invention is described in further detail combined with specific embodiments below and referring to data.It should be understood that the embodiment is
In order to demonstrate the invention, it rather than limits the scope of the invention in any way.
The present invention provides a kind of liquid-type Antiprothrombin antibodies assay kit, including reagent R1, reagent R2, quality-control product
And calibration object；
The reagent R1 includes fibrin ferment, heparin or its salt, the first buffer and protective agent；
The reagent R2 includes the chromophoric substrate of fibrin ferment, the second buffer, stabilizer and the second preservative.
Any one of fibrin ferment of the present invention in people, ox, pig thrombiase, preferably thrombin of beef, described is solidifying
The concentration of hemase is 8-20U/mL.
Heparin of the present invention or its salt are preferably heparin or heparin sodium, and concentration is preferably 12U/ml；
First buffer of the present invention be preferably tris-HCI buffer, HEPES buffer solution,
One or more of disodium hydrogen phosphate-citrate buffer solution, barbital sodium-hydrochloride buffer, more preferable disodium hydrogen phosphate-lemon
Lemon acid buffer, the working concentration of buffer are preferably 0.01-0.5mol/L.
Protective agent of the present invention preferably includes amino acid, polyalcohol, surfactant, metal ion chelation agent, inorganic
Salt and the first preservative；
The amino acid is preferably selected from one or more of glycine, lysine or histidine, more preferable lysine, work
Make concentration quality percent by volume 1%-20%；
The polyalcohol is preferably selected from one or more of xylitol, mannitol, sorbierite, more preferable xylitol, work
Make concentration quality percent by volume 0.1%-10%；
The surfactant is preferably selected from one or more of Tween-20, Tween-80, Triton X-100, more excellent
Select Triton X-100, working concentration quality percent by volume 0.01%-0.5%；
The metal ion chelation agent is preferably selected from disodium ethylene diamine tetraacetate, sodium potassium tartrate tetrahydrate, ammonium citrate, more preferably
Disodium ethylene diamine tetraacetate, working concentration quality percent by volume 5%-20%；
The inorganic salts are preferably selected from sodium chloride, ammonium sulfate, calcium chloride, potassium sulfate, preferably calcium chloride, working concentration quality
Percent by volume 0.5%-10%, the calcium chloride of this concentration range can guarantee can also be improved while the ionic strength of buffer it is solidifying
The stability of hemase；
First preservative of the present invention is preferably Sodium azide, Proclin300, gentamicin, thimerosal, more preferably
Proclin300, working concentration quality percent by volume are 0.01-1%.
Chromophoric substrate of the present invention is preferably selected from Tos-Gly-Pro-Arg-pNA (ChromozymTH), Boc-D-Arg-
One or more of Gly-Arg-pNA2HCl or H-D-Phe-Pip-Arg-pNA2HCl.More preferable Boc-D-Arg-
Gly-Arg-pNA·2HCl.It is preferred that working concentration is 0.1mmol/L-4mmol/L, the city fibrin ferment chromophoric substrate Jun Shi
It sells available.Thus, it is possible to effectively improve the efficiency of Antiprothrombin antibodies measurement.
Stabilizer of the present invention is preferably casein, and preferably working concentration is quality percent by volume 1-10%.It should
Casein in concentration range can be such that fibrin ferment chromophoric substrate is stably dissolved in aqueous solution, due to not needing to be lyophilized
Journey, therefore production technology is simplified, and reduces production cost.
Second buffer of the present invention can be identical with the first buffer, can also be different, is preferably selected from trihydroxy methyl
Aminomethane-hydrochloride buffer, HEPES buffer solution, disodium hydrogen phosphate-citrate buffer solution, in barbital sodium-hydrochloride buffer
One or more, more preferable disodium hydrogen phosphate-citrate buffer solution, the working concentration of buffer is 0.01-0.5mol/L.
Second preservative of the present invention can be identical with the first preservative, can also be different, be preferably selected from Sodium azide,
Proclin300, gentamicin, thimerosal, more preferable Proclin300, working concentration quality percent by volume are 0.01-1%.
Quality-control product of the present invention is added buffer, freeze drying protectant, preservative and is lyophilized and is made by commercially available animal blood plasma.
The animal blood plasma comes from one or more of pig, ox, horse, sheep, dog.The buffer is preferably 5-40mM
Tris-HCl buffer；Freeze drying protectant is preferably selected from glycine, lysine, histidine, sorbierite, xylitol, sweet dew
Alcohol, one or more of combination, the glycine of more preferable 1%-10% and the sorbierite of 0.1%-5%；Preservative is preferably
Working concentration quality percent by volume is the Proclin300 of 0.01-1%；The freeze temperature is preferably -60 DEG C, when freeze-drying
Between preferably 12 hours.
The preparation of calibration object of the present invention is will to mix after normal healthy people plasma collection, and it is living then to carry out AT- III
Property detection, detected value should add buffer, freeze drying protectant and preservative in 80%-120%, be lyophilized after packing, then use
WHO antithrombase international standard substance blood plasma carries out traceability definite value to the calibration object.
The buffer for preparing calibration object is preferably the Tris-HCl buffer of 5-40mM, and freeze drying protectant is preferably selected from
Glycine, lysine, histidine, sorbierite, xylitol, mannitol, one or more of combination, more preferable 1%-10%'s
The sorbierite of glycine and 0.1%-5%；The preservative is preferably that working concentration quality percent by volume is 0.01-1%
Proclin300.The freeze temperature is preferably -60 DEG C, and freeze-drying time is preferably 12 hours.
In the examples below, the various processes and method being not described in detail are conventional methods as known in the art.Under
Material used in example, reagent, device, instrument, equipment etc. are stated, unless otherwise specified, is commercially obtained.
The present invention is further described combined with specific embodiments below:
The preparation of embodiment 1 reagent R1, reagent R2, quality-control product and calibration object
The protectant preparation of reagent R1: 2% is added using disodium hydrogen phosphate-citrate buffer solution of the 10mM of pH 8.0 and is relied
Propylhomoserin, 0.5% xylitol, 0.02%Triton X-100,5%EDTA, 1% calcium chloride, 0.1% Proclin300.
The preparation of reagent R1: thrombin of beef need to be made into high concentration in advance, and 10mg/ml is spare；Heparin sodium need to be configured in advance
1mg/ml=180U/ml is spare, prepares disodium hydrogen phosphate-citrate buffer solution of 10mM, prepared protective agent, addition is added
900ml pure water sufficiently dissolves, and adjusts pH to 8.0, and height is added than cattle on the hoof fibrin ferment (visiing English purchased from Shenyang), until concentration is 12U/
Ml, it is 12U/ML that heparin sodium to concentration, which is added,.With pure water constant volume final volume to 1000ml.
Reagent R2 is prepared, by 2.25g chromophoric substrate Boc-D-Arg-Gly-Arg-pNA2HCl, 20g casein, 1g
Proclin300 is added in disodium hydrogen phosphate-citrate buffer solution of 10mM, with hydrochloric acid tune pH to 5.0, with pure water constant volume
Final volume is to 1000ml.
The preparation of III calibration object of AT-: it is added in buffer after healthy human normal plasma is mixed, buffer is by 20mM
The glycine of Tris-HCl and 4%, 0.1% sorbierite, 0.1% Proclin300 dissolution, and adjusting pH value is 7.5, with
1ml/ bottles of packing freeze-dryings.Definite value is carried out to III calibration object of AT- with WHO antithrombase international standard substance blood plasma 08/258, definite value is
90%.
The preparation of III quality-control product of AT-: after the mixing of commercially available animal blood plasma, addition 20mM Tris-HCl buffer, 4% it is sweet
Propylhomoserin, 0.1% sorbierite, 0.1% Proclin300 dissolution, and adjusting pH value is 7.0, is lyophilized, is used with 1ml/ bottles of packing
The value of AT-III reagent test quality-control product in the normal range, definite value 101%.
The preparation of embodiment 2 reagent R1, reagent R2, quality-control product and calibration object
The protectant preparation of reagent R1: using pH 7.5 10mM Tris-HCl buffer be added 1% cysteine,
1% trehalose, 0.01% Tween-20,0.5% potassium chloride, 0.1% sucrose, 0.1% Sodium azide.
The preparation of reagent R1: thrombin of beef need to be made into high concentration in advance, and 10mg/ml is spare；Heparin sodium need to be configured in advance
1mg/ml=180U/ml is spare, and the Tris-HCl buffer, the prepared protective agent of addition, addition 900ml for preparing 10mM are pure
Water sufficiently dissolves, and adjusts pH to 7.5, height is added than cattle on the hoof fibrin ferment (visiing English purchased from Shenyang), heparin is added until concentration is 9U/ml
Sodium to concentration is 9U/ML.With pure water constant volume final volume to 1000ml.
The preparation of reagent R2: by 2.5g chromophoric substrate Tos-Gly-Pro-Arg-pNA, 10g bovine serum albumin(BSA), 1g
Proclin300 is added in the glycine-HCI buffer of 20mM, adjusts pH to 5.0, extremely with pure water constant volume final volume
1000ml。
The preparation of III calibration object of AT-: it is added in buffer after healthy human normal plasma is mixed, buffer is by 20mM PBS
With 2% glycine, 0.1% sorbierite, 0.2% Proclin300 dissolution, and adjust pH value be 7.5, with 1ml/ bottle divide
Dress freeze-drying.Definite value, definite value 92% are carried out to III calibration object of AT- with WHO antithrombase international standard substance blood plasma 08/258.
The preparation of III quality-control product of AT-: after the mixing of commercially available animal blood plasma, addition 20mM PBS buffer solution, 2% glycine,
0.1% sorbierite, 0.2% Proclin300 dissolution, and adjusting pH value is 7.5, with 1ml/ bottle packing freeze-dryings, uses AT-III
The value of reagent test quality-control product in the normal range, definite value 104%.
The preparation of embodiment 3 reagent R1, reagent R2, quality-control product and calibration object
The protectant preparation of reagent R1: 2% arginine, 1% sweet is added using the HEPES buffer solution of the 10mM of pH 7.5
Oil, 0.01% Tween-80,1% sodium chloride, 0.1% sucrose, 0.2% gentamicin.
The preparation of reagent R1: thrombin of beef need to be made into high concentration in advance, and 10mg/ml is spare；Heparin sodium need to be configured in advance
1mg/ml=180U/ml is spare, prepares the PBS buffer solution of 10mM, pH 7.5, prepared protective agent is added, high more solidifying than cattle on the hoof
Hemase (visits English purchased from Shenyang), until concentration is 10U/ml, heparin sodium to concentration is 5U/ml.
The preparation of reagent R2: by 2.75g chromophoric substrate H-D-Phe-Pip-Arg-pNA2HCl, 10g bovine serum albumin
White, 1.5g Proclin300 is added in the PBS buffer solution of 10mM, pH 7.5.
The preparation of III calibration object of AT-: it is added in buffer after healthy human normal plasma is mixed, buffer is by 10mM
The glycine of HEPES and 1%, 0.5% mannitol, the dissolution of 0.2% gentamicin, and adjusting pH value is 7.4, with 1ml/ bottles
Packing freeze-drying.Definite value, definite value 95% are carried out to III calibration object of AT- with WHO antithrombase international standard substance blood plasma 08/258.
The preparation of III quality-control product of AT-: after commercially available animal blood plasma mixing, the sweet ammonia of 10mM HEPES buffer solution, 1% is added
Acid, 0.5% mannitol, the dissolution of 0.2% gentamicin, and adjusting pH value is 7.2, is lyophilized with 1ml/ bottles of packing, uses AT-
The value of III reagent test quality-control product in the normal range, definite value 99%.
The liquid-type Antiprothrombin antibodies assay kit that embodiment 1 is prepared is done into calibration curve, line of going forward side by side
Property range test, repeatability detection, the detection of quality-control product homogeneity, Detection of Stability and the anti-interference of heparin cofactor II is ground
Study carefully.
1, calibration curve makes
Reagent preparation R1 and reagent R2 is prepared as described in Example 1.Taking concentration is 90% antithrombin Ⅲ calibration object,
Sample is prepared according to following ratio with antithrombin Ⅲ calibration object and buffer, 7:4,1:1,1:2,1:4,1:8 use Sysmex
CA7000 detects sample, and OD405 absorbance change rate and corresponding antithrombin Ⅲ concentration are taken logarithmic linear side
Journey makes calibration curve, as shown in Figure 1.
The detection of 2 ranges of linearity
Sample is 155% from human plasma,freeze-dried's value, and active concentration is proportionally diluted to buffer is respectively
96.92%, 67.88%, 38.84%, 9.8%, detection is repeated three times with Sysmex coagulo meter CA7000, is averaged, is done
Linear regression curves, the range of linearity 10%~155% have preferable linear relationship, and it is anti-to meet detection for correlation coefficient r > 0.99
The active requirement of fibrin ferment III.Linear result is as shown in Figure 2 and Table 1.
Table 1
4 repeatability of embodiment is investigated
Under the conditions of repeatability, with normal the retest of Quality Control blood plasma 10 times, CV value is calculated.The results are shown in Table 2, display
Repeated CV=1.2%.
Table 2
5 quality-control product homogeneity of embodiment is investigated
5 bottles of quality-control products are taken, every bottle is tested 1 time, and the average value of each 5 measurement results of parameter is calculatedAnd standard deviation
SD1；Separately with 1 bottle of follow-on test 5 times in above-mentioned 5 bottles of quality-control products, the average value of each 5 measurement results of parameter is calculatedWith
Standard deviation SD2；Homogeneity between calculating bottle outlet.It is as shown in table 3:
Table 3
| Between bottle | In bottle |
| 100.6 | 101.8 |
| 99.1 | 100.1 |
| 98.8 | 99.8 |
| 100.5 | 100.8 |
| 102.1 | 99.6 |
| SD1 | 1.325519 |
| SD2 | 0.895545 |
| Between SD bottles | 0.977241 |
| mean | 100.22 |
| CV | 0.98% |
Test result shows homogeneity CV=0.98% between quality-control product bottle, illustrates that the quality-control product homogeneity prepared is very good.
6 study on the stability of embodiment
Of the invention and competing product reagent R1 and reagent R2 are respectively placed in 2-8 DEG C and 37 DEG C, sample every 7 days, right respectively
Quality-control product (101%) is measured.Test result is as shown in table 4.
Table 4
Test result shows, liquid instant Antiprothrombin antibodies measurement reagent of the present invention is compared with prior art
Liquid reagent has higher stability at 4 DEG C and 37 DEG C.Reagent of the present invention can be stablized 14 days at 37 DEG C.Illustrate stability very
It is good.
Anti-interference research of the embodiment 7 to heparin cofactor II
Plasma sample is purchased certainly with interfering substance 80U/ml heparin cofactor II (HC II) respectively
(HYPHENBioMed) (specific concentration and mixed method see the table below 5) is mixed in ratio as shown in the table:
Plasma sample of the table 5 containing HCII interfering substance
| HCII concentration (U/ml) | Interfering substance stoste dosage (ul) | Blood plasma dosage (ul) | Physiological saline dosage (ul) |
| 0 | 0 | 1140 | 60 |
| 1 | 15 | 1140 | 45 |
| 2 | 30 | 1140 | 30 |
| 3 | 45 | 1140 | 15 |
| 4 | 60 | 1140 | 0 |
Contrast agent uses freeze-dried powder type antithrombin Ⅲ (AT- III) assay kit (color development of SIEMENS company production
Substrate method), using Sysmex CA7000 instrument, respectively to the plasma sample containing HCII interfering substance listed in table 5
Antithrombin activity detection is carried out, finds out the mean value of measurement result respectively as measured value.
Table 6
| HCII concentration (U/ml) | 0 | 1 | 2 | 3 | 4 |
| Contrast agent measured value | 102.2 | 103.9 | 104.6 | 109.1 | 118.3 |
| Relative deviation | 0.0% | 1.7% | 2.3% | 6.8% | 15.8% |
| Measured value of the present invention | 102.6 | 101.1 | 103.8 | 104.2 | 108.5 |
| Relative deviation | 0.0% | - 1.50% | 1.20% | 1.60% | 5.80% |
Summarize: anti-interference test result shows combination when HCII concentration is in 4U/ml or less, this reagent has good
Anti-jamming effectiveness, hence it is evident that better than liquid-type antithrombin Ⅲ (AT- III) assay kit (chromophoric substrate of Jing Pin company production
Method).
Claims (10)
1. a kind of liquid-type Antiprothrombin antibodies assay kit, which is characterized in that including reagent R1, reagent R2, quality-control product
And calibration object；
The reagent R1 includes fibrin ferment, heparin or its salt, the first buffer and protective agent；
The reagent R2 includes the chromophoric substrate of fibrin ferment, the second buffer, stabilizer and the second preservative.
2. a kind of liquid-type Antiprothrombin antibodies assay kit according to claim 1, which is characterized in that described solidifying
Any one of hemase in people, ox, pig thrombiase, the concentration of the fibrin ferment are 8-20U/mL.
3. a kind of liquid-type Antiprothrombin antibodies assay kit according to claim 1, which is characterized in that described
Heparin or its salt are heparin or heparin sodium, concentration 12U/ml.
4. a kind of liquid-type Antiprothrombin antibodies assay kit according to claim 1, which is characterized in that the guarantor
Protecting agent includes amino acid, polyalcohol, surfactant, metal ion chelation agent, inorganic salts and the first preservative；
The amino acid is selected from one or more of glycine, lysine, arginine or histidine, and quality percent by volume is
1%-20%；
The polyalcohol is selected from one or more of xylitol, mannitol, sorbierite, and quality percent by volume is 0.1%-
10%；
The surfactant is selected from one or more of Tween-20, Tween-80, TritonX-100, quality volume basis
Than for 0.01%-0.5%；
The metal ion chelation agent is selected from disodium ethylene diamine tetraacetate, sodium potassium tartrate tetrahydrate, ammonium citrate, quality percent by volume
For 5%-20%；
The inorganic salts are selected from sodium chloride, ammonium sulfate, calcium chloride, potassium sulfate, and quality percent by volume is 0.5%-10%.
5. a kind of liquid-type Antiprothrombin antibodies assay kit according to claim 1, which is characterized in that described
Chromophoric substrate is selected from Tos-Gly-Pro-Arg-pNA (ChromozymTH), Boc-D-Arg-Gly-Arg-pNA2HCl or H-
One or more of D-Phe-Pip-Arg-pNA2HCl.
6. a kind of liquid-type Antiprothrombin antibodies assay kit according to claim 1, which is characterized in that described
Stabilizer is casein, quality percent by volume 1-10%.
7. a kind of liquid-type Antiprothrombin antibodies assay kit according to claim 1, which is characterized in that described
First buffer and the second buffer are respectively selected from tris-HCI buffer, HEPES buffer solution, phosphoric acid hydrogen
One or more of disodium-citrate buffer solution, barbital sodium-hydrochloride buffer.
8. a kind of liquid-type Antiprothrombin antibodies assay kit according to claim 1, which is characterized in that described
First preservative and the second preservative are respectively selected from Sodium azide, Proclin300, gentamicin, thimerosal.
9. a kind of liquid-type Antiprothrombin antibodies assay kit according to claim 1, which is characterized in that the matter
Control product are added buffer, freeze drying protectant and preservative freeze-drying and are made by animal blood plasma.
10. a kind of liquid-type Antiprothrombin antibodies assay kit according to claim 1, which is characterized in that described
Calibration object is then to carry out III Activity determination of AT-, detected value should be in 80%- for mixing after normal healthy people plasma collection
120%, addition buffer, freeze drying protectant and preservative are lyophilized after packing, then use WHO antithrombase international standard substance blood
Slurry carries out traceability definite value to the calibration object.
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| CN201811071001.XA CN109239061A (en) | 2018-09-14 | 2018-09-14 | A kind of liquid-type Antiprothrombin antibodies assay kit |
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| CN201811071001.XA CN109239061A (en) | 2018-09-14 | 2018-09-14 | A kind of liquid-type Antiprothrombin antibodies assay kit |
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| CN109239061A true CN109239061A (en) | 2019-01-18 |
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| CN201811071001.XA Pending CN109239061A (en) | 2018-09-14 | 2018-09-14 | A kind of liquid-type Antiprothrombin antibodies assay kit |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110346357A (en) * | 2019-07-16 | 2019-10-18 | 山东艾科达生物科技有限公司 | One kind being used for Antithrombin III assay reagent |
| CN111826418A (en) * | 2020-08-28 | 2020-10-27 | 保定天岳生物工程有限公司 | Antithrombin III determination kit |
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| US11891588B2 (en) | 2019-07-31 | 2024-02-06 | Ecolab Usa Inc. | Personal protective equipment free delimer compositions o |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040043428A1 (en) * | 2002-08-29 | 2004-03-04 | Roche Diagnostics Corporation | Specificity in the determination of antithrombin |
| CN102690862A (en) * | 2012-06-08 | 2012-09-26 | 上海太阳生物技术有限公司 | Kit (Developing substrate method) for testing antithrombase III (AT-III) |
| CN103238072A (en) * | 2010-12-09 | 2013-08-07 | 奥姆里克斯生物药品有限公司 | Immunoassay method for thrombin detection |
| CN105223348A (en) * | 2014-05-26 | 2016-01-06 | 北京九强生物技术股份有限公司 | The latex enhancing immune turbidimetry detection kit of people's Antithrombin III |
| CN105466920A (en) * | 2015-11-20 | 2016-04-06 | 鲁翌 | A rapid antithrombin III detecting kit based on interaction between thrombin and a chromogenic substrate and a detecting method thereof |
| CN107727587A (en) * | 2017-09-22 | 2018-02-23 | 宁波瑞源生物科技有限公司 | A kind of Antithrombin III assay kit and its detection method |
-
2018
- 2018-09-14 CN CN201811071001.XA patent/CN109239061A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040043428A1 (en) * | 2002-08-29 | 2004-03-04 | Roche Diagnostics Corporation | Specificity in the determination of antithrombin |
| CN103238072A (en) * | 2010-12-09 | 2013-08-07 | 奥姆里克斯生物药品有限公司 | Immunoassay method for thrombin detection |
| CN102690862A (en) * | 2012-06-08 | 2012-09-26 | 上海太阳生物技术有限公司 | Kit (Developing substrate method) for testing antithrombase III (AT-III) |
| CN105223348A (en) * | 2014-05-26 | 2016-01-06 | 北京九强生物技术股份有限公司 | The latex enhancing immune turbidimetry detection kit of people's Antithrombin III |
| CN105466920A (en) * | 2015-11-20 | 2016-04-06 | 鲁翌 | A rapid antithrombin III detecting kit based on interaction between thrombin and a chromogenic substrate and a detecting method thereof |
| CN107727587A (en) * | 2017-09-22 | 2018-02-23 | 宁波瑞源生物科技有限公司 | A kind of Antithrombin III assay kit and its detection method |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110346357B (en) * | 2019-07-16 | 2021-10-26 | 山东艾科达生物科技有限公司 | Reagent for determining antithrombin III |
| CN110346357A (en) * | 2019-07-16 | 2019-10-18 | 山东艾科达生物科技有限公司 | One kind being used for Antithrombin III assay reagent |
| US11891588B2 (en) | 2019-07-31 | 2024-02-06 | Ecolab Usa Inc. | Personal protective equipment free delimer compositions o |
| CN111826418A (en) * | 2020-08-28 | 2020-10-27 | 保定天岳生物工程有限公司 | Antithrombin III determination kit |
| CN112051259B (en) * | 2020-08-31 | 2022-08-19 | 武汉生之源生物科技股份有限公司 | Nitric oxide detection kit and detection method |
| CN112051259A (en) * | 2020-08-31 | 2020-12-08 | 武汉生之源生物科技股份有限公司 | Nitric oxide detection kit and detection method |
| CN113234792A (en) * | 2021-04-06 | 2021-08-10 | 北京九强生物技术股份有限公司 | Quality control composition for blood coagulation detection |
| CN113092786A (en) * | 2021-04-09 | 2021-07-09 | 北京美联泰科生物技术有限公司 | Buffer solution and application thereof in central nervous system specific protein detection kit |
| CN114250265A (en) * | 2021-12-15 | 2022-03-29 | 深圳传世生物医疗有限公司 | Liquid type factor X determination kit |
| CN114617903A (en) * | 2022-03-15 | 2022-06-14 | 中国人民解放军总医院第一医学中心 | Composition for plasma freeze-drying and application thereof |
| CN114755427A (en) * | 2022-06-13 | 2022-07-15 | 深圳市帝迈生物技术有限公司 | Anti Xa activity assay kit of external source addition antithrombin |
| CN114755427B (en) * | 2022-06-13 | 2022-11-08 | 深圳市帝迈生物技术有限公司 | Anti Xa activity assay kit of external source addition antithrombin |
| CN114813287A (en) * | 2022-06-29 | 2022-07-29 | 深圳市帝迈生物技术有限公司 | Calibrator for heparin anticoagulation detection and preparation method thereof |
| CN115814099A (en) * | 2022-12-19 | 2023-03-21 | 成都蓉生药业有限责任公司 | Dry heat treatment stabilizer for human blood coagulation factor IX, preparation and preparation method thereof |
| CN115814099B (en) * | 2022-12-19 | 2024-04-26 | 成都蓉生药业有限责任公司 | Dry heat treatment stabilizer for human coagulation factor IX, preparation and preparation method thereof |
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Application publication date: 20190118 |