CN107727872A - A kind of kit of heparin determination - Google Patents
A kind of kit of heparin determination Download PDFInfo
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- CN107727872A CN107727872A CN201710779609.7A CN201710779609A CN107727872A CN 107727872 A CN107727872 A CN 107727872A CN 201710779609 A CN201710779609 A CN 201710779609A CN 107727872 A CN107727872 A CN 107727872A
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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
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
- G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
Abstract
The invention belongs to medical science in-vitro diagnosis field, and in particular to a kind of kit of heparin determination, include pig F Ⅹ a, acharan sulfate lyases and chromophoric substrate.The a of F Ⅹ are prepared by Swine plasma described in kit of the present invention, solve blood plasma source, are adapted to China's actual conditions, and it is convenient to prepare, and reduces cost；Kit of the present invention can be used for the detection of a variety of heparin class medicines using a standard curve, easy to operate without formulating the standard curve of a plurality of different heparin class medicines；Kit detection plasma sample of the present invention is without dilution, and testing result high sensitivity, stability is good, and accuracy is high.
Description
Technical field
The invention belongs to medical science in-vitro diagnosis field, and in particular to a kind of kit of heparin determination, more particularly, to one
The heparin determination kit of kind Chromogenic assay (one-step method) detection heparin activity.
Background technology
Heparin is a kind of acidic mucopolysaccharide, is synthesized by the mast cell for being distributed in intestinal mucosa.Content pole in normal human blood
It is few, its anticoagulation very little under physiological conditions.It can be by accelerating antithrombase, HC II to play anticoagulation, to TFPI and PROTEIN C
System also has an impact.Natural heparin is inhomogenous, its molecular weight (Wang Zhen justice thrombus and hemostasis basis reason between 3-57kD
By with clinic [M] third edition Shanghai：Shanghai scientific basic publishing house, 2004:119-119).Clinically using heparin as anti-freezing
Agent medicine and widely use, be mainly used in the treatment and operation on vessels of heart of thrombotic diseases, haemodialysis, extracorporal circulatory system waited
The anti-freezing processing of journey.
Heparin is that a kind of the most frequently used anticoagulant therapy agent, heparin class medicine occupy pole in antithrombotic reagent in clinical medicine
The big market share, wherein accounted for LMWHs class medicine leading again.As medicine, the accurate detection nothing of heparin activity function
Important meaning is respectively provided with by the dynamic monitoring of patient in the quality control for production process or clinical treatment.Heparin at present
Assay method be mainly freezing method and Chromogenic assay (US4234682, US 4948724, the A of US 5308755,
CN104048931A、CN 103063592 A、CN103323416A；Ten C H,Lamping R J,Henny C P,et
al.Automated amidolytic method for determining heparin,a heparinoid,and a
low-Mr heparin fragment,based on their anti-Xa activity[J].Clinical
Chemistry,1984,30(6):860-864).Freezing method is more traditional heparin activity assay method, its is cumbersome,
Specificity is poor, time and effort consuming, accuracy are low, and Chromogenic assay utilize contain in heparin sugar chain it is high by affine five with antithrombase
Sugared domain, internal antithrombase (AT) can be specifically bound, form heparin-antithrombin compounds, heparin-antithrombase
Compound can suppress the general principle of the chromogenic reaction of factor X (or factor II) the hydrolysis chromophoric substrate of activation,
The characteristics of with high sensitivity, easy to operate, high specificity.Research is shown in Anti-Ⅹa interaction process, need to only be contained
Heparin core pentasaccharides domain sugar chain activates AT and then anticoagulant purpose；During anti-II a factor interactions not
Only need heparin to include core pentasaccharides domain, also to include at least 18 monose compositions sugar chain (Lane D A,
Denton J,Flynn A M,et al.Anticoagulant activities of heparin oligosaccharides
and their neutralization by platelet factor 4.[J].Biochemical Journal,1984,
218(3):725-732).Useful Chromogenic assay substitutes the trend of blood clotting method in the world at present.In the 6th heparin state
In the standard items cooperation demarcation of border, WHO determines Anti-Ⅹa and anti-II a factor actives as the scaling method recommended,
American Pharmacopeia the 37th edition and European Pharmacopoeia the 8.3rd edition are using this method measure heparin potency.Version in 2015《Middle traditional Chinese medicines
Allusion quotation》Freezing method is also changed to Chromogenic assay measure heparin and resists II a factor actives by the assay method of heparin potency.
Clinically there are many business on the market at present using kit measurement effectively to react concentration of the heparin in blood plasma
The heparin determination kits of product is sold, and most products are Chromogenic assay and mostly imported product.On the market using hair
The heparin determination kit of color substrate method has two kinds, and one kind is two-step method, and the method detects sample independent of the AT contents in blood plasma
Total heparin content in product blood plasma.The heparin determination kit of the method has two classes, and antithrombase is included in a kind of commercial reagent box
(AT), a of the ox F Ⅹ or a of people F Ⅹ, buffer solution and chromophoric substrate, such as Sekisui companies
Heparin (a of Anti-F Ⅹ), Heparin-0020009400, Siemens company of IL companies
Heparin, Stago companyThe a of Biophen Heparin anti-Ⅹ of Heparin and Aniara companies
(2stages) etc., antithrombase (AT), a of ox F II, buffer solution and chromophoric substrate is included in another kind of commercial reagent box, such as
Sekisui companiesHeparin (a of Anti-F II) and Aniara companies Biophen Heparin
A of anti-II (2stages) etc., such reagent are not used to detect low molecular weight heparin (LMWH).Another is one-step method,
The a of ox F Ⅹ and chromophoric substrate are included in the method commercial reagent box, such as the Liquid Heparin-0020300100 of IL companies,
Aniara Biophen Heparin LRT (liquid) and Biophen Heparin (lyophilized) etc., the method are detected in sample blood plasma
Play the heparin of anticoagulation.
Management of the China to blood is stricter, and people's blood source is limited, and a of ox F Ⅹ preparation method simplicity and origin is
Long, a components of F Ⅹ are exhausted in the preferable a sources of F Ⅹ and commercially available heparin determination kit (Chromogenic assay) thus in patent report
It is most of to come from ox blood.However, compare ox blood resource, China's pig blood resource more horn of plenty, utilization rate is but high.It is in addition, clinical
The heparin class medicament categories of upper application are more, need to formulate according to different types of heparin during Chromogenic assay measure heparin at present
Corresponding new standard curve.This reduces the simplicity of heparin determination kit (Chromogenic assay) to a certain extent.
The content of the invention
It is an object of the invention to for deficiency existing for current existing heparin determination kit, there is provided a kind of chromophoric substrate
Method (one-step method) detects the kit of heparin activity, and the kit is lower to a certain degree to solve the source of blood plasma, more meets Chinese state
Feelings, while prepare conveniently, it is easy to operate, there is higher sensitivity and stability, testing result accuracy is high, and market competition is excellent
Gesture is strong.
To realize the purpose of the present invention, the present invention provides following technical scheme.
A kind of kit of heparin determination, include pig F Ⅹ a, acharan sulfate lyases and chromophoric substrate.
Wherein, a of pig F Ⅹ are activation Swine plasma factor X, can pass through known point by those skilled in the art
From purification process, extraction purification is prepared from Swine plasma, such as with reference to Church W R et al. document (Church W R,
Mann K G.A simple purification of human Factor X using a high affinity
monoclonal antibody immunoadsorbant[J].Thrombosis Research,1985,38(4):417-
424.)。
The acharan sulfate lyases in kit of the present invention is similar to heparinaseⅡ property, can
Modification and shearing glycosaminoglycan chains, have certain splitting action to heparin and heparin sulfate, can be led to by those skilled in the art
Cross known isolation and purification method to be prepared from Bacteroides stercoris, such as the document with reference to Kim B T et al.
(Kim B T,Hong S W,Kim W S,et al.Purification and characterization of acharan
sulfate lyases,two novel heparinases,from Bacteroides stercoris,HJ-15[J]
.European Journal of Biochemistry,2001,268(9):2635-2641.)。
During the activity of Chromogenic assay detection heparin, chromophoric substrate is as a substrates of F Ⅹ, it is desirable to has higher
Sensitiveness and preferably water-soluble, can just effectively improve the sensitivity of detection, ensure the accuracy of testing result.As excellent
Select, the chromophoric substrate in kit of the present invention is Suc-Ile-Glu- (γ-Piperidyl)-Gly-Arg-pNA
HCl, Z-D-Arg-Gly-Arg-pNAHCl or Bz-Ile-Glu (- OR)-Gly-Arg-pNAHCl.More preferably Suc-
Ile-Glu-(γ-Piperidyl)-Gly-Arg-pNA·HCl。
In some embodiments, the kit includes R1 reagents and R2 reagents, and the R1 reagents include a of pig F Ⅹ, institute
State R2 reagents and include acharan sulfate lyases and chromophoric substrate.That is a of pig F Ⅹ separately as kit one of them
Component, and another component using acharan sulfate lyases and chromophoric substrate as kit.
R1 reagents described in kit of the present invention and R2 reagents are freeze dried.
The R1 lyophilized preparations freeze-dried are made up of the reagent of a of pig F Ⅹ, buffer, stabilizer and excipient.
Buffer in the R1 reagents is selected from any one of phosphate buffer and Tris buffer solutions, is preferably
Tris-HCl buffer solutions, concentration 10-50mM, pH scope are 6.5-9.0.
The stabilizer and excipient in the R1 reagents select stabilizer and figuration well known by persons skilled in the art
Agent.The stabilizer is at least one of BSA, PEG6000, EDTA and Aprotinin, preferably BSA and/or PEG6000, and concentration is
1-10mg/ml.The excipient is at least one of Dextran 40, sucrose and mannitol, preferably mannitol, concentration 50-
200mM。
The R2 lyophilized preparations are by acharan sulfate lyases, chromophoric substrate, buffer, dextran sulfate, steady
The reagent for determining agent and excipient freeze-dried is made.
Buffer in the R2 reagents is selected from any one of phosphate buffer and Tris buffer solutions, is preferably
Tris-HCl buffer solutions, concentration 10-50mM, pH scope are 6.5-9.0.
The dextran sulfate concentration in the R2 reagents is 0.01-0.025g/L, and dextran sulfate can reduce sample
The influence to heparin determination such as PF4 in product blood plasma.
Stabilizer and excipient in the R2 reagents select stabilizer and excipient well known by persons skilled in the art.It is excellent
Choosing, the stabilizer is at least one of BSA, PEG6000 and EDTA, and preferably PEG6000, its concentration is 1-10mg/ml.Institute
Excipient is stated as at least one of Dextran 40, sucrose and mannitol, preferably mannitol, concentration 50-200mM.
During the kit detection sample heparin activity shown in the present invention, detection method can be end-point method or move
State method.
Present invention also offers a kind of method of heparin determination, the lyases of sulfate containing acharan and chromophoric substrate
Reagent directly mixed with testing sample blood plasma after being incubated 0.5-2 minutes at 37 DEG C, add a of F containing pig Ⅹ reagent mixing it is equal
It is even, after being acted on 3 minutes at 37 DEG C, absorbance of the above-mentioned sample under 405nm wavelength is read, according to known standard curve
The heparin content in testing sample blood plasma is calculated.
Acharan sulfate lyases can make the heparin fraction in testing sample degrade, but retain it and combined with AT
Ability.Acharan sulfate lyases enzyme activity is defined as under given conditions, and 1 μm of ol heparin of conversion is unsaturated in 1min
Enzyme amount needed for oligonucleotide chain, referred to as an international unit (IU or U).
It is worth noting that, during any of the above-described reagent detects sample heparin activity, the freeze-dried type reagent is used
The concentration of each component is defined as its working concentration after distilled water redissolves.
In some embodiments, the kit includes R1 reagents and R2 reagents, and the R1 reagents include a of pig F Ⅹ, institute
State R2 reagents and include acharan sulfate lyases and chromophoric substrate.Wherein, the working concentration of a of pig F Ⅹ is preferably
0.1-0.45U/ml, more preferably 0.25U/ml.The working concentration of the acharan sulfate lyases is preferably 0.05U/
Ml-0.7U/ml, more preferably 0.5U/ml；The working concentration of the chromophoric substrate is preferably 1.2-3.0mg/ml, more preferably
1.5mg/ml.The R2 reagents are 1 with sample blood plasma mixed volume ratio:1.
Compared with prior art, kit of the present invention at least has one of following features：
(1) a of F Ⅹ are prepared by Swine plasma, solve blood plasma source, are adapted to China's actual conditions, and it is convenient to prepare, and reduces into
This；
(2) standard curves can be used for the detection of a variety of heparin class medicines, without formulating a plurality of different heparin class medicines
Standard curve, it is easy to operate；
(3) plasma sample is detected without dilution, and testing result high sensitivity, stability is good, and accuracy is high.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 is the calibration curve of kit of the present invention；
Fig. 2 is kit of the present invention and the correlation analysis of existing commercial reagent box testing result.
Embodiment
The invention discloses a kind of heparin determination kit.Those skilled in the art can use for reference present disclosure, suitably change
Enter technological parameter realization.In particular, all similar replacements and change are aobvious for a person skilled in the art
And be clear to, they are considered as being included in the present invention.The method and product of the present invention is carried out by preferred embodiment
Description, related personnel substantially can not depart from present invention, method described herein is modified in spirit and scope or
Suitably change with combining, to realize and using the technology of the present invention.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention
Case is clearly and completely described, it is clear that and described embodiment is only part of the embodiment of the present invention, rather than all
Embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art institute under the premise of creative work is not made
The every other embodiment obtained, belongs to the scope of protection of the invention.
Unless otherwise specified, reagent involved in the embodiment of the present invention is commercially available prod, can pass through business canal
Road purchase obtains.Wherein, document (Church W R, Mann K G.As of a of pig F Ⅹ with reference to Church W R et al.
simple purification of human Factor X using a high affinity monoclonal
Antibody immunoadsorbant [J] .Thrombosis Research, 1985,38 (4):417-424.) it is prepared.
The chromophoric substrate is purchased from gill biochemistry (Shanghai) Co., Ltd., such as Suc-Ile-Glu- (γ-Piperidyl)-Gly-
Arg-pNA·HCl。
The composition and preparation method of the heparin determination kit of embodiment 1
R1 reagents are mainly formulated by following raw material：The a concentration of pig F Ⅹ is 1.25U/ml, and Tris 50mM, BSA are
6.64mg/ml, PEG-4000 are 10mg/ml and mannitol is 100mM, and pH to 8.4 is adjusted with 6M HCl, with 1ml/ bottles point
Dress is lyophilized.
R2 reagents are formed by following preparation of reagents：Chromophoric substrate Suc-Ile-Glu- (γ-Piperidyl)-Gly-Arg-
PNAHCl is 3.0mg/ml, and acharan sulfate lyases is 1.0U/ml, Tris 50mM, dextran sulfate 0.02g/
L, PEG-4000 is 10mg/ml and mannitol is 100mM, and pH to 7.5 is adjusted with 6M HCl, lyophilized with the packing of 1ml/ bottles.
The assay method of the detection kit of embodiment 2
(1) the gained R1 reagents of embodiment 1, R2 reagents are subjected to redissolution reconstruction：
Every bottle of R1 reagent 5ml distilled water redissolves, and every bottle of R2 reagent 2ml distilled water redissolves.
(2) so that BioTek ELx800 ELIASAs operate (end-point method) as an example, illustrated according to instrument, set analysis program：Survey
The a length of 405nm of standing wave；
The heparin of known potency is diluted to 100U/ml heparin with physiological saline first.Take 10 μ l100U/ml liver
Element, the dilution of 990 μ l human plasmas standard items is added, obtain 1.0U/ml Heparin Standard product.Continue the dilution of employment plasma standard
1.0U/ml Heparin Standard product, prepare 0.0,0.2,0.5,0.8, the sample of 1.0U/ml 5 standard points of heparin, incubated at 37 DEG C
Educate 30 seconds.Take 50 μ l dilute after sample, add 50 μ l R2 reagents 37 DEG C be incubated 60 seconds, add 50 μ l R1 reagents at 37 DEG C
It is incubated 180 seconds, adds the acetic acid solution terminating reactions of 50 μ l 20%, absorbance (OD is determined under 405nm405nm).Per Guan Chong
Repetition measurement is fixed 3 times, with OD405nmAverage be ordinate, corresponding heparin activity is abscissa, make " activity-absorbance " mark
Directrix curve.Similarly formulate low molecular weight heparin standard curve.The measurement result of heparin and each standard point of low molecular weight heparin is such as
Shown in table 1, according to test of linearity result, the standard curve of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) is very close, and
There is preferable coefficient correlation, with the significant difference between SPSS 17.0 software test, two standard curves, analysis of covariance knot
Fruit is shown under the level of confidence level 95%, and the assay Sig.P values of two standard curve intercepts and slope are respectively 0.693
With 0.524,0.05 is all higher than, i.e., there was no significant difference for the intercept and slope of two standard curves, and both can be merged into one
Shared standard curve.Therefore as shown in figure 1, taking the overall average of each two groups of determination datas of standard point to formulate standard curve, mark
Directrix curve equation is：Y=-0.5168x+1.0273, correlation coefficient r=0.9952.
Each standard point measurement result analysis of table 1
Sample to be tested is taken, ibid the absorbance of method measure sample, substitutes into standard curve, you can treat test sample to calculate
The activity of heparin in this.
This kit is applied to (Biochemical Analyzer or blood on various brands and the semi-automatic and full automatic detector of model
Solidifying analyzer).As France is thought up to high STA Compact coagulo meters, U.S.'s Beckman Kurt ACL elite coagulo meters, Germany
BE Compact X coagulo meters etc., design parameter can suitably be adjusted according to instrument is different.
The analytical performance of 3 kit of the present invention of embodiment is assessed
(1) sensitivity
Using 0.0U/ml Heparin Standard product as dummy, the gained of embodiment 1 kit of the present invention, the institute of embodiment 2 are used
Detection method is stated, replication 20 times, calculates this sample OD405nmAverageWith standard deviation (SD), subtract twice with blank average
Standard deviation substitutes into calibrating curve equation, calculates minimum detection limit, and its result is as shown in table 2.
The minimum detection limit of table 2 is analyzed
The result of table 2 is shown：The minimum detection limit of kit detection heparin of the present invention is up to 0.02U/ml, less than commercially available heparin
Detection kit (Chromogenic assay, French Aniara companies BIOPHEN Heparin kits, article No.：221006) specification
Described in minimum detection limit (0.05U/ml), i.e. kit of the present invention detection heparin high sensitivity.
(2) stability
R1, R2 freeze dried are both placed in normal temperature after distilled water redissolves in the gained of embodiment 1 kit of the present invention
Under (18-25 DEG C).The reagent of certain volume is drawn daily, with theoretical value 0.2U/ml LMWH, 0.25U/ml UFH, 0.5U/
Ml UFH and 0.75U/ml LMWH Heparin Standard product are detection sample, are then examined according to detection method described in embodiment 2
Survey, the daily replication of single sample 10 times, commercially available heparin determination kit (Chromogenic assay) is synchronously measured.Single sample
The daily absorbance measurement average result of product and the softwares of SPSS 17.0 carry out the significance test result of linear regression analysis slope
As shown in table 3 and table 4.
The kit reagent stability result analysis of the present invention of table 3
The French Aniara companies BIOPHEN Heparin kit reagents stability result analysis of table 4
Table 3 and the result of table 4 are shown：Reagent is placed 10 days under (18-25 DEG C) equally at normal temperatures after redissolving, in confidence level
Under 95% level, statistically there are no significant (P >=0.05) for the slope of regression line of kit measurement result of the present invention, and
French Aniara companies BIOPHEN Heparin kit (article No.s：221006) slope of regression line has conspicuousness (P ＜ 0.05),
Kit i.e. of the present invention can be stablized at room temperature after redissolving to be preserved 10 days, hence it is evident that higher than commercially available kit.This result is said
Bright kit of the invention can reduce waste, so as to reduce cost.
Embodiment 4 and the comparison of commercialized product
From Ruijin Hospital, Shanghai Jiao Tong University School of Medicine and Longhua Hospital affiliated Shanghai University Of Chinese Traditional Medicine be in hospital and
Totally 200 parts of the patient blood sample through heparin therapy is randomly selected in outpatient service.Blood presses 9:1 ratio and 0.109mol/L citric acids
After sodium anti-freezing liquid mixes, 3000r/min is centrifuged 15 minutes, isolated blood plasma.With the gained of embodiment 1 kit of the present invention and method
Aniara companies of state BIOPHEN Heparin kit (article No.s：221006) both phase relations are calculated to sample measures respectively
Number, and carry out linear regression.As a result correlation coefficient r=0.9935 of two kinds of kits, equation of linear regression y=are shown
1.0104x-0.0087 see Fig. 2.
According to the requirement (r ＞ 0.975) of U.S. clinical Laboratory Standard tissue (CLSI) file, kit of the present invention and
The detection data of French Aniara companies import reagent box have good uniformity.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (7)
1. a kind of kit of heparin determination, it is characterised in that include pig F Ⅹ a, acharan sulfate lyases and color development
Substrate.
2. kit according to claim 1, it is characterised in that the chromophoric substrate be Suc-Ile-Glu- (γ-
Piperidyl)-Gly-Arg-pNAHCl, Z-D-Arg-Gly-Arg-pNAHCl or Bz-Ile-Glu (- OR)-Gly-
Arg-pNA·HCl。
3. kit according to claim 1 or 2, it is characterised in that comprising R1 reagents and R2 reagents, the R1 reagents bag
The a of F containing pig Ⅹ, the R2 reagents include acharan sulfate lyases and chromophoric substrate.
4. kit according to claim 3, it is characterised in that the R1 reagents and R2 reagents are freeze-dried reagent, wherein
The R1 reagents are made up of a of pig F Ⅹ, buffer, stabilizer and excipient, and the R2 reagents are cracked by acharan sulfate
Enzyme, chromophoric substrate, buffer, dextran sulfate, stabilizer and excipient are made.
5. kit according to claim 4, it is characterised in that the buffer is that phosphate buffer or Tris are buffered
Liquid, pH 6.5-9.0；The stabilizer is at least one of BSA, PEG6000, EDTA and Aprotinin；The excipient is the right side
Revolve at least one of sugared acid anhydride 40, lactose and mannitol.
A kind of 6. method of heparin determination, it is characterised in that the reagent of the lyases of sulfate containing acharan and chromophoric substrate is straight
Connect and mixed with testing sample blood plasma after being incubated 0.5-2 minutes at 37 DEG C, the reagent for adding a of F containing pig Ⅹ is well mixed, in 37
After being acted on 3 minutes at DEG C, absorbance of the above-mentioned sample under 405nm wavelength is read, is calculated according to known standard curve
To the heparin content in testing sample blood plasma.
7. according to the method for claim 6, it is characterised in that the working concentration of a of pig F Ⅹ is 0.1-0.45U/ml；
The working concentration of the acharan sulfate lyases is 0.05U/ml-0.7U/ml；The working concentration of the chromophoric substrate
For 1.2-3.0mg/ml.
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| CN113174425A (en) * | 2021-04-20 | 2021-07-27 | 普迈德（北京）科技有限公司 | Heparin quantitative detection kit and application thereof |
| CN114184562A (en) * | 2021-11-19 | 2022-03-15 | 北京赛升药业股份有限公司 | Method for determining urokinase activity by chromogenic substrate method |
| 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 |
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| US6140062A (en) * | 1997-06-27 | 2000-10-31 | Universiteit Maastricht | Method of determining the heparin content |
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