CN117191724A - Development method of LMWH and UFH 2-1 calibration curve of anti-Xa assay kit (chromogenic substrate method) - Google Patents
Development method of LMWH and UFH 2-1 calibration curve of anti-Xa assay kit (chromogenic substrate method) Download PDFInfo
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- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 title claims abstract description 240
- 238000011088 calibration curve Methods 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 45
- 230000001858 anti-Xa Effects 0.000 title claims abstract description 14
- 239000003593 chromogenic compound Substances 0.000 title claims abstract description 12
- 238000003149 assay kit Methods 0.000 title claims abstract description 11
- 238000011161 development Methods 0.000 title abstract description 8
- 229960002897 heparin Drugs 0.000 claims abstract description 86
- 229920000669 heparin Polymers 0.000 claims abstract description 86
- 239000003055 low molecular weight heparin Substances 0.000 claims abstract description 52
- 238000004364 calculation method Methods 0.000 claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 230000000694 effects Effects 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 238000002835 absorbance Methods 0.000 claims description 54
- 230000008859 change Effects 0.000 claims description 51
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 8
- 238000005259 measurement Methods 0.000 claims description 4
- 241001146702 Candidatus Entotheonella factor Species 0.000 claims description 3
- CLOMYZFHNHFSIQ-UHFFFAOYSA-N clonixin Chemical compound CC1=C(Cl)C=CC=C1NC1=NC=CC=C1C(O)=O CLOMYZFHNHFSIQ-UHFFFAOYSA-N 0.000 claims description 3
- 238000003908 quality control method Methods 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 239000002994 raw material Substances 0.000 abstract description 4
- 239000003146 anticoagulant agent Substances 0.000 abstract description 3
- QNZCBYKSOIHPEH-UHFFFAOYSA-N Apixaban Chemical compound C1=CC(OC)=CC=C1N1C(C(=O)N(CC2)C=3C=CC(=CC=3)N3C(CCCC3)=O)=C2C(C(N)=O)=N1 QNZCBYKSOIHPEH-UHFFFAOYSA-N 0.000 abstract description 2
- 101001129803 Homo sapiens Paired mesoderm homeobox protein 2A Proteins 0.000 abstract description 2
- 102100031686 Paired mesoderm homeobox protein 2A Human genes 0.000 abstract description 2
- 229960003886 apixaban Drugs 0.000 abstract description 2
- KANJSNBRCNMZMV-ABRZTLGGSA-N fondaparinux Chemical compound O[C@@H]1[C@@H](NS(O)(=O)=O)[C@@H](OC)O[C@H](COS(O)(=O)=O)[C@H]1O[C@H]1[C@H](OS(O)(=O)=O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O[C@@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O4)NS(O)(=O)=O)[C@H](O3)C(O)=O)O)[C@@H](COS(O)(=O)=O)O2)NS(O)(=O)=O)[C@H](C(O)=O)O1 KANJSNBRCNMZMV-ABRZTLGGSA-N 0.000 abstract description 2
- 229960001318 fondaparinux Drugs 0.000 abstract description 2
- 239000003112 inhibitor Substances 0.000 abstract description 2
- 229960001148 rivaroxaban Drugs 0.000 abstract 1
- KGFYHTZWPPHNLQ-AWEZNQCLSA-N rivaroxaban Chemical compound S1C(Cl)=CC=C1C(=O)NC[C@@H]1OC(=O)N(C=2C=CC(=CC=2)N2C(COCC2)=O)C1 KGFYHTZWPPHNLQ-AWEZNQCLSA-N 0.000 abstract 1
- 239000000463 material Substances 0.000 description 3
- 229940127219 anticoagulant drug Drugs 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000004019 antithrombin Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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Abstract
The invention discloses a development method of an anti-Xa assay kit (chromogenic substrate method) LMWH and UFH2 in 1 calibration curve, which improves parameter setting of a detection instrument through a calibration calculation formula, achieves the aim of taking a calibration product of low molecular heparin LMWH and common heparin UFH in 2 as 1, saves production raw materials, stock and transportation of the calibration product, increases cost performance of the heparin calibration product, and has a certain promotion effect on research, registration and production of calibration products for measuring anti-Xa activity of Xa factor inhibitor anticoagulant drugs such as fondaparinux ARIX, rivaroxaban R IVA, apixaban APIX, edeox, nelimostat and the like.
Description
Technical Field
The invention relates to the technical field of calibration curve methods, in particular to a development method of an anti-Xa determination kit (chromogenic substrate method) LMWH and UFH 2-1 calibration curve.
Background
The inlet reagent of the Xa-resistant assay kit usually adopts a 3-point linear calibration curve of low molecular heparin LMWH and common heparin UFH calibrator 2 and 1.
International standard NIBSC, once low molecular heparin LMWH: international standard NIBSC for 11/176 and plain heparin UFH: 07/328 respectively compares the Shanghai Zhenyuan reagent on a SYSMEX-CS5100 coagulometer and the imported reagent on an original coagulometer, and the result shows that the assigned result of Shanghai Zhenyuan completely accords with the range of relative deviation CV minus or plus 15% |, the test value of common heparin UFH on a target value point with the theoretical value of 0.40IU/ml in the assigned result of imported reagent is 0.31IU/ml, the relative deviation CV=22.5% and the absolute deviation B minus 0.09IU/ml, and the negative deviation is obviously overrun.
When the interference factors such as the matrix effect are excluded, it is considered that the following factors may exist:
the low molecular heparin LMWH and the common heparin UFH have different reaction rates in the measurement of Xa-resisting activity due to the obvious difference of molecular weight and purity, so that the calibration curve and the linear range of the low molecular heparin LMWH and the common heparin UFH have different reaction rates;
whether the imported reagent producer really solves the key skills of the reagent kit calibrator and the calibration curve 2 and 1 in technology;
because heparin anticoagulants depend on antithrombin AT bridging Xa factor to play a thousand-fold anticoagulation effect, the internal connection of 3 reactants determines that the detection reaction and the calibration curve should not be a straight line but should be a curve with more than 2 times;
is a 3-point 2-in-1 linear calibration curve capable of reflecting the true results of Xa more accurately than Logistic Log4P, log P?
The above problems are properly resolved by first-order reference substance assignment with traceability metric interchangeability conditions.
For this reason we propose a development method of an anti-Xa assay kit (chromogenic substrate method) LMWH and UFH2 in 1 calibration curve.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a development method of an anti-Xa assay kit (chromogenic substrate method) LMWH and UFH2 in-1 calibration curve, which achieves the aim of combining a low-molecular heparin LMWH and common heparin UFH calibrator 2 into 1, saves the production raw materials, the stock and the transportation of the calibrator, and increases the cost performance of the heparin calibrator.
In order to solve the technical problems, the invention provides the following technical scheme:
a developing method of an anti-Xa assay kit (chromogenic substrate method) LMWH and UFH 2-1 calibration curve comprises the following steps:
as production reagents with the same batch number and calibration materials with the same batch number which meet the performance verification requirement, on the same detection instrument, the same item is detected under the relatively stable quality control environment condition, and the test of the calibration curve and the sample (under the condition of removing the interference factors of the sample) is true, definite and reliable;
under the above conditions, the international standard NIBSC: LMWH11/176 and UFH07/328 respectively de-assign calibrator labeled low molecular heparin LMWH and plain heparin UFH, the results of which are stable, determined and reliable, including that the differential performance between the low molecular heparin LMWH and plain heparin UFH on the international standard reference curve and calibrator calibration curve is relatively stable and can be estimated;
according to the calibration formula of LogisticLog 5P: absorbance change rate r=r 0 +K/{1+EXP[-(a+bLnC+c*C)](wherein R is) 0 For the absorbance change rate of 0 value, K is the reciprocal of the factor number (i.e. factor number=1/K), a, b and c are the intercept, slope and concentration index slope of the target tangent line respectively, the absorbance change rate delta R difference between the LMWH of the low molecular heparin and the UFH international standard reference curve of the common heparin under the given condition of 5 concentrations is calculated, and the absorbance change rate R of 5 targets of the LMWH international standard reference curve of the low molecular heparin is calculated L +Δr=absorbance change rate RU of standard UFH international standard reference curve for plain heparin, i.e.: the absorbance change rate RU of 5 concentration targets of the common heparin UFH calibration curve after the absorbance change rate delta R is reduced can be estimated through the absorbance change rate RL of 5 concentration targets of the calibration curve of the low molecular heparin LMWH, and the corresponding concentration is kept unchanged, and only 5 parameters of R, K, a, b, c are changed;
when a new sample concentration is tested, a low-molecular heparin LMWH concentration result can be obtained only through a low-molecular heparin LMWH reference or calibrator curve, and a common heparin UFH concentration result can be obtained at the same time;
the conversion factor of converting low molecular heparin LMWH into common heparin UFH is added into the parameter setting of common heparin UFH test item, and the Xa-UFH activity concentration of the same set of calibrator with the low molecular heparin LMWH can be detected through the hidden common heparin UFH calibration curve.
The absorbance change rate difference method comprises the following steps:
under the condition that 5 target point concentrations of international standard reference or calibrator configuration between low molecular heparin LMWH and common heparin UFH are corresponding to the same, setting reference or referenceThe serial numbers of the 5 target mark values of the calibration standard are i=1, 2,3,4 and 5 respectively, and the absorbance change rate R of 5 concentration target spots i ＝R 0i +K i /{1+EXP[-(a i +b i LnC+c i *C)]}；
Setting the absorbance change rate R of 5 concentration targets of international standard reference or calibrator of low molecular heparin LMWH Li ＝R 0Li +K Li /{1+EXP[-(a Li +b Li LnC+c Li *C)]International standard reference or calibrator for UFH of plain heparin, 5 concentration target absorbance rate R Ui ＝R 0Ui +K Ui /{1+EXP[-(a Ui +b Ui LnC+c Ui *C)]Then DeltaR 0i ＝ΔR 0Ui -ΔR 0Li ，ΔK i ＝K Ui -ΔK Li ，Δa i ＝a Ui -a Li ，Δb i ＝b Ui -b Li ，Δc i ＝c Ui -c Li ；
The 5 concentration target point parameters of the international standard reference or calibrator of the common heparin UFH are respectively as follows: r is R 0Ui ＝ΔR 0i +R 0Li ，K Ui ＝ΔK i +K Li ，a Ui ＝Δa i +a Li ，b Ui ＝Δb i +b Li ，c Ui ＝Δc i +c Li ；
According to the converted common heparin UFH absorbance change rate R Ui ＝R 0Ui +K Ui /{1+EXP[-(a Ui +b Ui LnC+c Ui *C)]A calibration formula is used for calculating the absorbance change rate R of the common heparin UFH with 5 concentration targets Ui The theoretical value concentration of the international standard UFH assignment or the marked value concentration of the calibrator is taken as the abscissa, and the absorbance change rate R of the common heparin UFH is obtained through conversion Ui Preparing a common heparin UFH calibration curve for the ordinate;
the conversion parameter information of the common heparin UFH calibration curve is set in a common heparin UFH project on a detection instrument, and as long as the parameter of the low molecular heparin LMWH calibration curve is unchanged, the common heparin UFH calibration curve is kept constant.
The concentration difference method comprises the following steps:
most detectors are not capable of modifying the absorbance change rate R, in which case the formula for the scaling parameter calculation process is calculated in accordance with polynominal 5P: lnC =a+b [ (R-R) 0 )/100]+c*[(R-R 0 )/100] 2 +d[(R-R 0 )/100] 3 Calculating R under an international standard reference curve or a calibrator calibration curve of the low molecular heparin LMWH 0Li 、K Li 、a Li 、b Li 、c Li 、d Li R is then 0Ui ＝ΔR 0i +R 0Li ，K Ui ＝ΔK i +K Li ，a Ui ＝Δa i +a Li ，b Ui ＝Δb i +b Li ，c Ui ＝Δc i +c Li ，d Ui ＝Δd i +d Li ；
R is as described above 0Ui 、K Ui 、a Ui 、b Ui 、c Ui 、d Ui Parameters are respectively substituted into formulas of a calculation process of the polynominal 5P calculation scaling parameters: lnC =a+b [ (R-R) 0 )/100]+c*[(R-R 0 )/100] 2 +d[(R-R 0 )/100] 3 5 target concentration log values LnC are calculated i Converting into corresponding concentration values;
setting the conversion parameter information of the common heparin UFH calibration curve into a common heparin UFH project on a detection instrument, wherein the common heparin UFH calibration curve is kept constant as long as the parameters of the low molecular heparin LMWH calibration curve are unchanged;
when detecting a common heparin UFH sample, the absorbance change rate R (dOD/min) under a low molecular heparin LMWH reference curve or a calibration curve obtained in the anti-Xa-UFH measurement project is directly converted into the absorbance change rate R (dOD/min) of the common heparin UFH through a set program, and then the concentration or content display is calculated.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the parameter setting of the detecting instrument is improved through the calibration calculation formula, so that the purpose of combining 2 of the calibrator of the low molecular heparin LMWH and the common heparin UFH is achieved, the production raw materials, the inventory and the transportation of the calibrator are saved, and the cost performance of the heparin calibrator is increased.
Detailed Description
In order that the manner in which the above recited features, objects and advantages of the present invention are obtained will become readily apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Based on the examples in the embodiments, those skilled in the art can obtain other examples without making any inventive effort, which fall within the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Examples
The invention provides a developing method of an anti-Xa determination kit (chromogenic substrate method) LMWH and UFH 2-1 calibration curve, which comprises the following steps:
as production reagents with the same batch number and calibration materials with the same batch number which meet the performance verification requirement, on the same detection instrument, the same item is detected under the relatively stable quality control environment condition, and the test of the calibration curve and the sample (under the condition of removing the interference factors of the sample) is true, definite and reliable;
under the above conditions, the international standard NIBSC: LMWH11/176 and UFH07/328 respectively de-assign calibrator labeled low molecular heparin LMWH and plain heparin UFH, the results of which are stable, determined and reliable, including that the differential performance between the low molecular heparin LMWH and plain heparin UFH on the international standard reference curve and calibrator calibration curve is relatively stable and can be estimated;
according to the calibration formula of LogisticLog 5P: absorbance change rate r=r 0 +K/{1+EXP[-(a+bLnC+c*C)](wherein R is) 0 The absorbance change rate is 0, K is the reciprocal of the factor number (i.e. factor number=1/K), a, b and c are the intercept, slope and concentration index slope of the target tangent, and the low molecular liver is calculated under the given condition of 5 concentrationsThe difference value of absorbance change rate delta R between the plain LMWH and the common heparin UFH international standard reference curve is calculated by the absorbance change rate R of 5 targets of the low molecular heparin LMWH international standard reference curve L +Δr=absorbance change rate RU of standard UFH international standard reference curve for plain heparin, i.e.: the absorbance change rate RU of 5 concentration targets of the common heparin UFH calibration curve after the absorbance change rate delta R is reduced can be estimated through the absorbance change rate RL of 5 concentration targets of the calibration curve of the low molecular heparin LMWH, and the corresponding concentration is kept unchanged, and only 5 parameters of R, K, a, b, c are changed;
when a new sample concentration is tested, a low-molecular heparin LMWH concentration result can be obtained only through a low-molecular heparin LMWH reference or calibrator curve, and a common heparin UFH concentration result can be obtained at the same time;
the conversion factor of converting low molecular heparin LMWH into common heparin UFH is added into the parameter setting of common heparin UFH test item, and the Xa-UFH activity concentration of the same set of calibrator with the low molecular heparin LMWH can be detected through the hidden common heparin UFH calibration curve.
Operating program
The absorbance change rate difference method comprises the following steps:
under the condition that 5 target point concentrations of the international standard reference or calibrator configuration between the low molecular heparin LMWH and the common heparin UFH are identical, setting the serial numbers of 5 target point marking values of the reference or calibrator to be i=1, 2,3,4,5 respectively, and then setting the absorbance change rate R of 5 concentration target points i ＝R 0i +K i /{1+EXP[-(a i +b i LnC+c i *C)]}；
Setting the absorbance change rate R of 5 concentration targets of international standard reference or calibrator of low molecular heparin LMWH Li ＝R 0Li +K Li /{1+EXP[-(a Li +b Li LnC+c Li *C)]International standard reference or calibrator for UFH of plain heparin, 5 concentration target absorbance rate R Ui ＝R 0Ui +K Ui /{1+EXP[-(a Ui +b Ui LnC+c Ui *C)]Then DeltaR 0i ＝ΔR 0Ui -ΔR 0Li ，ΔK i ＝K Ui -ΔK Li ，Δa i ＝a Ui -a Li ，Δb i ＝b Ui -b Li ，Δc i ＝c Ui -c Li ；
The 5 concentration target point parameters of the international standard reference or calibrator of the common heparin UFH are respectively as follows: r is R 0Ui ＝ΔR 0i +R 0Li ，K Ui ＝ΔK i +K Li ，a Ui ＝Δa i +a Li ，b Ui ＝Δb i +b Li ，c Ui ＝Δc i +c Li ；
According to the converted common heparin UFH absorbance change rate R Ui ＝R 0Ui +K Ui /{1+EXP[-(a Ui +b Ui LnC+c Ui *C)]A calibration formula is used for calculating the absorbance change rate R of the common heparin UFH with 5 concentration targets Ui The theoretical value concentration of the international standard UFH assignment or the marked value concentration of the calibrator is taken as the abscissa, and the absorbance change rate R of the common heparin UFH is obtained through conversion Ui Preparing a common heparin UFH calibration curve for the ordinate;
the conversion parameter information of the common heparin UFH calibration curve is set in a common heparin UFH project on a detection instrument, and as long as the parameter of the low molecular heparin LMWH calibration curve is unchanged, the common heparin UFH calibration curve is kept constant.
The concentration difference method comprises the following steps:
most detectors are not capable of modifying the absorbance change rate R, in which case the formula for the scaling parameter calculation process is calculated in accordance with polynominal 5P: lnC =a+b [ (R-R) 0 )/100]+c*[(R-R 0 )/100] 2 +d[(R-R 0 )/100] 3 Calculating R under an international standard reference curve or a calibrator calibration curve of the low molecular heparin LMWH 0Li 、K Li 、a Li 、b Li 、c Li 、d Li R is then 0Ui ＝ΔR 0i +R 0Li ，K Ui ＝ΔK i +K Li ，a Ui ＝Δa i +a Li ，b Ui ＝Δb i +b Li ，c Ui ＝Δc i +c Li ，d Ui ＝Δd i +d Li ；
R is as described above 0Ui 、K Ui 、a Ui 、b Ui 、c Ui 、d Ui Parameters are respectively substituted into formulas of a calculation process of the polynominal 5P calculation scaling parameters: lnC =a+b [ (R-R) 0 )/100]+c*[(R-R 0 )/100] 2 +d[(R-R 0 )/100] 3 5 target concentration log values LnC are calculated i Converting into corresponding concentration values;
setting the conversion parameter information of the common heparin UFH calibration curve into a common heparin UFH project on a detection instrument, wherein the common heparin UFH calibration curve is kept constant as long as the parameters of the low molecular heparin LMWH calibration curve are unchanged;
when detecting a common heparin UFH sample, the absorbance change rate R (dOD/min) under a low molecular heparin LMWH reference curve or a calibration curve obtained in the anti-Xa-UFH measurement project is directly converted into the absorbance change rate R (dOD/min) of the common heparin UFH through a set program, and then the concentration or content display is calculated.
According to the development method of the anti-Xa assay kit (chromogenic substrate method) LMWH and UFH2 in-1 calibration curve, the parameter setting of a detection instrument is improved through a calibration calculation formula, so that the aim of taking the calibration product 2 of low-molecular heparin LMWH and common heparin UFH as 1 is achieved, the production raw materials, the stock and the transportation of the calibration product are saved, the cost performance of the heparin calibration product is increased, and the development, registration and production of the calibration product for the anti-Xa activity assay of Xa inhibitor anti-coagulant medicaments such as fondaparinux ARIX, rivaroban RIVA, apixaban APIX, edeox and Nelimostat are promoted to a certain extent.
It should be noted that in this document, terms such as "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A method for developing an anti-Xa assay kit (chromogenic substrate method) LMWH and UFH2 in 1 calibration curve, comprising the steps of:
s1, detecting the same item on the same detection instrument under relatively stable quality control environment conditions as production reagents with the same batch number and calibration products with the same batch number which meet the performance verification requirements, wherein the test of a calibration curve and a sample (under the condition of removing interference factors of the sample) of the calibration curve and the sample is real, definite and reliable;
s2, under the condition, using an international standard NIBSC: LMWH11/176 and UFH07/328 respectively de-assign calibrator labeled low molecular heparin LMWH and plain heparin UFH, the results of which are stable, determined and reliable, including that the differential performance between the low molecular heparin LMWH and plain heparin UFH on the international standard reference curve and calibrator calibration curve is relatively stable and can be estimated;
s3, according to a calibration formula of the Logistic Log 5P: absorbance change rate r=r 0 +K/{1+EXP[-(a+bLnC+c*C)](wherein R is) 0 For the absorbance change rate of 0 value, K is the reciprocal of the factor number (i.e. factor number=1/K), a, b and c are the intercept, slope and concentration index slope of the target tangent line respectively, the absorbance change rate delta R difference between the LMWH of the low molecular heparin and the UFH international standard reference curve of the common heparin under the given condition of 5 concentrations is calculated, and the absorbance of 5 targets of the LMWH international standard reference curve of the low molecular heparin is calculatedRate of change R L +Δr=absorbance change rate RU of standard UFH international standard reference curve for plain heparin, i.e.: the absorbance change rate RU of 5 concentration targets of the common heparin UFH calibration curve after the absorbance change rate delta R is reduced can be estimated through the absorbance change rate RL of 5 concentration targets of the calibration curve of the low molecular heparin LMWH, and the corresponding concentration is kept unchanged, and only 5 parameters of R, K, a, b, c are changed;
s4, when testing new sample concentration, obtaining a low-molecular heparin LMWH concentration result only through a low-molecular heparin LMWH reference or calibrator curve, and simultaneously obtaining a common heparin UFH concentration result;
s5, adding a conversion factor of converting the low-molecular heparin LMWH into the common heparin UFH in the parameter setting of the common heparin UFH test item, and detecting the Xa-UFH activity concentration of the same set of calibrator with the low-molecular heparin LMWH through a hidden common heparin UFH calibration curve.
2. The method for developing an anti-Xa assay kit (chromogenic substrate method) LMWH and UFH2 in 1 calibration curve according to claim 1, wherein: the operation procedure comprises an absorbance change rate difference method, which comprises the following steps:
s1, under the condition that 5 target point concentrations of international standard reference or calibrator configuration between low molecular heparin LMWH and common heparin UFH are identical correspondingly, setting the serial numbers of 5 target point reference values of the reference or calibrator to be i=1, 2,3,4 and 5 respectively, and then setting the absorbance change rate R of 5 concentration target points i ＝R 0i +K i /{1+EXP[-(a i +b i LnC+c i *C)]}；
S2, setting the absorbance change rate R of 5 concentration target points of an international standard reference or calibrator of low molecular heparin LMWH Li ＝R 0Li +K Li /{1+EXP[-(a Li +b Li LnC+c Li *C)]International standard reference or calibrator for UFH of plain heparin, 5 concentration target absorbance rate R Ui ＝R 0Ui +K Ui /{1+EXP[-(a Ui +b Ui LnC+c Ui *C)]Then DeltaR 0i ＝ΔR 0Ui -ΔR 0Li ，ΔK i ＝K Ui -ΔK Li ，Δa i ＝a Ui -a Li ，Δb i ＝b Ui -b Li ，Δc i ＝c Ui -c Li ；
S3, 5 concentration target point parameters of an international standard reference or a calibrator of the common heparin UFH are respectively: r is R 0Ui ＝ΔR 0i +R 0Li ，K Ui ＝ΔK i +K Li ，a Ui ＝Δa i +a Li ，b Ui ＝Δb i +b Li ，c Ui ＝Δc i +c Li ；
S4, obtaining the UFH absorbance change rate R of the common heparin according to conversion Ui ＝R 0Ui +K Ui /{1+EXP[-(a Ui +b Ui LnC+c Ui *C)]A calibration formula is used for calculating the absorbance change rate R of the common heparin UFH with 5 concentration targets Ui The theoretical value concentration of the international standard UFH assignment or the marked value concentration of the calibrator is taken as the abscissa, and the absorbance change rate R of the common heparin UFH is obtained through conversion Ui Preparing a common heparin UFH calibration curve for the ordinate;
s5, setting the conversion parameter information of the common heparin UFH calibration curve into a common heparin UFH project on a detection instrument, and keeping the common heparin UFH calibration curve constant as long as the parameters of the low-molecular heparin LMWH calibration curve are unchanged.
3. The method for developing an anti-Xa assay kit (chromogenic substrate method) LMWH and UFH2 in 1 calibration curve according to claim 1, wherein: the operation procedure comprises a concentration difference method, which comprises the following steps:
s1, most detectors cannot modify the absorbance change rate R, and in this case, the formula of the calibration parameter calculation process is calculated according to polynominal 5P: lnC =a+b [ (R-R) 0 )/100]+c*[(R-R 0 )/100] 2 +d[(R-R 0 )/100] 3 Calculating R under an international standard reference curve or a calibrator calibration curve of the low molecular heparin LMWH 0Li 、K Li 、a Li 、b Li 、c Li 、d Li R is then 0Ui ＝ΔR 0i +R 0Li ，K Ui ＝ΔK i +K Li ，a Ui ＝Δa i +a Li ，b Ui ＝Δb i +b Li ，c Ui ＝Δc i +c Li ，d Ui ＝Δd i +d Li ；
S2, R is as described above 0Ui 、K Ui 、a Ui 、b Ui 、c Ui 、d Ui Parameters are respectively substituted into formulas of a calculation process of the polynominal 5P calculation scaling parameters: lnC =a+b [ (R-R) 0 )/100]+c*[(R-R 0 )/100] 2 +d[(R-R 0 )/100] 3 5 target concentration log values LnC are calculated i Converting into corresponding concentration values;
s3, setting the conversion parameter information of the common heparin UFH calibration curve into a common heparin UFH project on a detection instrument, wherein the common heparin UFH calibration curve is kept constant as long as the parameters of the low-molecular heparin LMWH calibration curve are unchanged;
s4, when the common heparin UFH sample is detected, the absorbance change rate R (dOD/min) under a low molecular heparin LMWH reference curve or a calibration curve obtained in the anti-Xa-UFH measurement project is directly converted into the absorbance change rate R (dOD/min) of the common heparin UFH through a set program, and then the concentration or content display is calculated.
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