CN103743837A - Human anti-thrombin III heparin combination ratio detection method - Google Patents
Human anti-thrombin III heparin combination ratio detection method Download PDFInfo
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Abstract
The invention discloses a human anti-thrombin III heparin combination ratio detection method. The detection method comprises the steps of using an efficient size exclusion chromatography column and a sodium salt buffer solution with mobile phases comprising isopropanol and phosphoric acid at pH of 6.0-7.5 to calculate a combination ratio between the human anti-thrombin III and the heparin through a ratio of an area of characteristic peak and a corresponding standard curve; adding inactivated human anti-thrombin III into a standard product of the human anti-thrombin III according to a ratio, and manufacturing a standard curve according to change of the ratio of the area of characteristic peak. According to the method, the human factors are excluded; the accuracy and the precision of results are high, the experiment operation steps are simple, the detection time can be shortened, the experiment cost is reduced and the working efficiency of detectors is greatly improved.
Description
Technical field
The present invention relates to biological medicine and detect analysis technical field, particularly relate to a kind of Human Antithrombin Ⅲ's Heparin-binding ratio detection method.
Background technology
Human Antithrombin Ⅲ's (Human Antithrombin III, AT-III) by liver cell and vascular endothelial cell, secreted, a kind of sugary 10% strand α 2 glycoprotein, relative molecular weight is 58-64 kD, isoelectric point 4.8 ~ 5.3,432 amino acid residues, consist of, in human normal plasma, antithrombin Ⅲ content is 0.12 ~ 0.3g/L.It regulates blood coagulation activity by the activity of the activated clotting factors such as Trombin inhibiting, FIXa, FXa, FXIa, FX II a, maintains blood normal physiological balance.Human Antithrombin Ⅲ's goods are a kind of plasma proteins medicines that obtained by human normal plasma purifying, and purity is high and keep its natural physiologically active, is mainly used in treating congenital or acquired antithrombase deficiency disease.Its clinical indication is extensive, is the anticoagulant that has potentiality.A little less than the anticoagulation of antithrombin Ⅲ, wait a moment, but with Heparin-binding after, anticoagulating active and anticoagulation significantly strengthen.Therefore the binding ability of antithrombin Ⅲ and heparin directly affects its biologically active.
Human Antithrombin Ⅲ's separation and purification and virus inactivation technology (as thermal treatment) can cause a minority's antithrombin Ⅲ molecular structure to change, and lose and heparin-bounding ability, thereby lose activity.Therefore European Pharmacopoeia requires ratio that can be shared with heparin-bounding molecule in the quantitative Human Antithrombin Ⅲ of detection goods, can not be lower than 60%.The detection method that European Pharmacopoeia provides is crossed immunoelectrophoresis detection method, and the method has following shortcoming: 1. need preparation or buy the anti-human antithrombin Ⅲ antiserum of rabbit, price, length consuming time, complex operation difficulty; 2. electrophoresis time is long, approximately 20 hours; 3. omnidistance requirement of temperature controlled 4 ℃ during electrophoresis; 4. electrophoresis completes poststaining decolouring difficulty; 5. Normal Agarose Gel is frangible, not easy to operate; 6. need manual measurement baseline, peak height, calculating peak area, subjective factor is obvious, and computational solution precision is lower; 7. there is no relevant reagent kit product, standard is difficult to realize unified.
Summary of the invention
The object of the invention is to provide a kind of method of Human Antithrombin Ⅲ of mensuration and Heparin-binding ratio, overcomes the shortcoming of Human Antithrombin Ⅲ and Heparin-binding ratio detection method and reagent in prior art.
Purified Human Antithrombin Ⅲ's preparation is the pharmaceutical grade protein that a kind of purity is higher, by efficient size exclusion chromatograph, single chromatographic peak can be detected; While adding excessive heparin in Human Antithrombin Ⅲ's preparation, keep Human Antithrombin Ⅲ's molecule of biologically active (with heparin-bounding ability) reversible non-covalent combination to occur with heparin molecule, molecule quantitative change is large, can form a chromatographic peak (peak 1) that retention time is shorter, and cannot because molecular weight does not change, form in situ a chromatographic peak (peak 2) with heparin-bounding Human Antithrombin Ⅲ's molecule (losing bioactive molecule).Adopt the chromatographic condition through optimizing, comprise the selection of mobile phase, the adjustment of damping fluid, can realize the baseline separation of two chromatographic peaks.Calculate respectively the area of two chromatographic peaks, the corresponding typical curve of ratio substitution of peak 1 area and two peak area sums can calculate Heparin-binding ratio value (being expressed as a percentage).
Human Antithrombin Ⅲ's Heparin-binding ratio detection method technical scheme of the present invention, comprises the following steps:
(1) solution preparation, prepares respectively Human Antithrombin Ⅲ and the standard solution of damping fluid, mobile phase, heparin solution, Human Antithrombin Ⅲ's solution, inactivation;
(2), to the Human Antithrombin Ⅲ's preparation standard solution that adds in proportion inactivation in Human Antithrombin Ⅲ's standard items, adopt high performance liquid chromatography, according to the variation production standard curve of characteristic peak area ratio;
(3) need testing solution is adopted to high performance liquid chromatography, by the ratio of characteristic peak area and the combination ratio of corresponding typical curve calculating Human Antithrombin Ⅲ and heparin.
Concrete scheme of the present invention is:
1. solution preparation
Phosphate buffer: phosphate solution 0.1 ~ 0.3mol/L, with NaOH or hydrochloric acid solution, regulate pH to 6.0 ~ 7.5, stand-by.
Mobile phase: isopropyl alcohol: phosphate buffer=1:99.
Heparin solution: with the dilution of above-mentioned mobile phase or dissolve liquaemin to certain concentration, every milliliter of contained heparin units (IU) should be 30 ~ 150 times of Human Antithrombin Ⅲ's units (IU) in every milliliter of testing sample.
Human Antithrombin Ⅲ's solution: by Human Antithrombin Ⅲ's standard items or test sample with above-mentioned mobile phase dilution or be dissolved to 1 ~ 50 mg/ml.The Heparin-binding ratio of Human Antithrombin Ⅲ's standard items is known, and is not less than 90%.Need testing solution and the above-mentioned heparin solution equal-volume preparing got after dilution mix.
The Human Antithrombin Ⅲ of inactivation: will add Bicine solution (Bicine(N-bis-(hydroxyethyl) glycocoll of 50mmol/L), the NaHCO of 100mmol/L in Human Antithrombin Ⅲ's standard items
3, pH 8.3); The trinitro-benzene-sulfonic acid that adds again 10 ~ 30mmol/L, 25 ℃ of constant temperature are processed 30min; Add the Tris damping fluid (50mmol/L Tris – HCl, 100mmol/L NaCl, pH7.4) of 20 times of volumes to mix.After ultrafiltration concentration, through HiPrep Sephacryl S-200 HR molecular sieve chromatography purifying, obtain deactivated monomeric protein.Through HPLC, detect content of monomer > 95%, not with Heparin-binding, retention time is consistent with Human Antithrombin Ⅲ's standard items.
Standard solution: Human Antithrombin Ⅲ's standard items are prepared to (after preparation, total protein content and testing sample are consistent) according to different proportion respectively with the inactivation Human Antithrombin Ⅲ of known protein concentration.Human Antithrombin Ⅲ's standard items proportion, between 0 ~ 100%, at least should have the standard solution of 3 kinds of different proportions; Within the proportional range that the Heparin-binding ratio of test sample should cover at standard solution.The above-mentioned solution preparing is mixed with isopyknic above-mentioned heparin solution respectively.
2. high-efficient liquid phase chromatogram condition
Chromatographic column: efficient size exclusion chromatograph post, as TOSOH BIOSCIENCE TSK-GEL G3000SW, TSKgel G3000SWXL, TSK gel SuperSW 3000, or the product of similar aperture, granularity and post bed height.
Flow velocity: 0.2 ~ 0.8ml/min.
Detecting device: UV280nm.
Sample size: 5 ~ 20 μ L
3. high performance liquid chromatography experiment
Standard solution and need testing solution are carried out to chromatography experiment according to above-mentioned chromatographic condition successively.
4. collection of illustrative plates is processed with result and is calculated
Peak area integration: adopt conventional peak area integration method, as the method for HPLC system software automatic integration, calculate peak area.Calculate peak area ratio A=peak 1 area/(1 area+peak, peak, 2 areas).
Typical curve: the peak area ratio A value of accurate solution chromatogram is carried out to linear regression as X-axis and Y-axis respectively with corresponding Heparin-binding ratio (being designated as B), obtain the regression equation of B=aA+b, R
2>=0.95.
By the A value substitution typical curve regression equation of test sample, can calculate the Heparin-binding ratio of test sample.
The present invention with respect to the advantage of prior art is: adopt HPLC method to replace crossed immunoelectrophoresis method, method is more simple, easily row, standardization.
Detection method beneficial effect of the present invention is specific as follows:
1, prepare inactivation Human Antithrombin Ⅲ monomer simple compared with the anti-human antithrombin Ⅲ serum of rabbit.Remove long immune detection work from.
2, solution preparation and sample preparation are simple.
3, HPLC detection is convenient, in the short time, can obtain testing result.
4, by HPLC instrument, carry out automatic integration, result degree of accuracy and accuracy are higher, have got rid of artificial interference.
5, meet the requirement of GMP, result is easy to preserve, and is difficult for revising, and has realized the trackability of testing result.
Accompanying drawing explanation
Figure 1 shows that the efficient size exclusion chromatograph figure of the present invention.
Wherein, chromatogram a is Human Antithrombin Ⅲ's standard items;
Chromatogram b is the Human Antithrombin Ⅲ of inactivation;
Chromatogram c is heparin;
Chromatogram d is Human Antithrombin Ⅲ's standard items: Human Antithrombin Ⅲ's protein concentration of inactivation is than being 50:50;
Peak 1 is heparin Human Antithrombin Ⅲ combination peak;
Peak 2 is Human Antithrombin Ⅲ peak;
Peak 3 is heparin peak.
embodiment:
In order to understand better the present invention, with instantiation, describe technical scheme of the present invention in detail below, but the present invention is not limited thereto.
Embodiment 1
1. reagent and sample please be shown in Table 1:
Table 1
2. solution preparation:
Phosphate buffer: weigh sodium dihydrogen phosphate 12.0g, be dissolved in 950ml purified water, regulate pH to 7.4 with the sodium hydroxide solution of 0.2mol/L,, mix to 1L with purified water constant volume.
Mobile phase: get above-mentioned phosphate buffer 495ml, add 5ml isopropyl alcohol, mix ultrasound wave processing 30min and carry out degassed.
Heparin solution: dilute heparin sodium injection to 5000IU/ml with above-mentioned mobile phase.
The Human Antithrombin Ⅲ of inactivation: will in Human Antithrombin Ⅲ's standard items, add Bicine solution (Bicine(N-bis-(hydroxyethyl) glycocoll of 50mmol/L), the NaHCO3 of 100mmol/L, pH 8.3); The trinitro-benzene-sulfonic acid that adds again 10 ~ 30mmol/L, 25 ℃ of constant temperature are processed 30min; Add the Tris damping fluid (50mmol/LTris – HCl, 100mmol/L NaCl, pH7.4) of 20 times of volumes to mix.After ultrafiltration concentration, through HiPrep Sephacryl S-200 HR molecular sieve chromatography purifying, obtain deactivated monomeric protein.Through HPLC, detect content of monomer > 95%, not with Heparin-binding, retention time is consistent with Human Antithrombin Ⅲ's standard items.
Standard solution: Human Antithrombin Ⅲ's standard items are prepared to (total protein content and testing sample are consistent) according to the ratio of protein content as shown in table 2 respectively with the inactivation Human Antithrombin Ⅲ of known protein concentration:
Table 2
3. sample preparation
Test sample is diluted to 9mg/ml with mobile phase.Test sample after dilution and above-mentioned 5 kinds of standard solution mix respectively (Human Antithrombin Ⅲ: heparin activity is than being 1:100) with above-mentioned heparin solution equal-volume, hatch 10 minutes for 37 ℃.
4. high-efficient liquid phase chromatogram condition
Use efficient size exclusion chromatograph post, TOSOH BIOSCIENCE TSK-GEL G3000SW, mobile phase pre-equilibration 1h for flow velocity: 0.6ml/min.Detecting device wavelength set is UV280nm.The test sample of handling well and standard solution be sample introduction successively, each sample size 10 μ L.After each sample introduction, set elution time 1h.Test sample sample introduction 5 times, as parallel experiment.
5. result is calculated:
Peak area integration: adopt the Breeze2.0 software of Waters HPLC system, automatic integration calculates peak area.Calculate peak area ratio A=peak 1 area/(1 area+peak, peak, 2 areas).
Typical curve: the peak area ratio A value of 5 kinds of standard solution chromatograms is carried out to linear regression as X-axis and Y-axis respectively with corresponding theoretical Heparin-binding ratio (being designated as B), obtain the regression equation of B=aA+b.By the A value substitution typical curve regression equation of test sample, can calculate the Heparin-binding ratio of test sample.
Concrete outcome is as shown in table 3:
Table 3
6. crossed immunoelectrophoresis detection method detects test sample:
Crossed immunoelectrophoresis method concrete steps according to < < European Pharmacopoeia > > (EP7.5) regulation are measured test sample, replicate determination 5 times:
Crossed immunoelectrophoresis: with the Ago-Gel of barbital electrophoretic buffer (pH8.4, the heparin that contains 15IU/ml) preparation 10g/L, topple over the gel that 5ml melts on the glass plate of 5cm * 5cm, 4 ℃ of cooling 30min.A hole, diameter 2mm are made a call to respectively in place at adjacent two each 1cm of angular distance edge of gel.A hole adds the Human Antithrombin Ⅲ's test sample to be measured 5 μ L that are diluted to 1IU/ml, and another hole adds 5 μ L bromophenol blues as indicator, and under 7v/cm voltage, electrophoresis is until indicator migrates to end, and in electrophoresis process, temperature keeps below 4 ℃.The electrophoresis region that retains test sample 1.5cm * 5cm, excise remaining gel, 5% the sero-fast gel of the anti-human antithrombin Ⅲ of rabbit (the 10g/L agarose of containing of the new preparation of 3.5ml is toppled in remaining space, the barbital electrophoretic buffer of pH8.4), after cooled and solidified, engage completely with original remaining gel, gel is turned to 90 °, and the voltage of setting 2v/cm carries out two dimensional electrophoresis, about 16h.
Dyeing: by the filter paper parcel that soaks into physiological sodium chloride solution for gel, compress 2h, constantly more renew filter paper, unreacted antiserum in gel is diffused out completely, dye and decolour with acid blue 92, occur two precipitation peaks on gel.
Result is calculated: calculate the ratio (manually integration) that accounts for all precipitation peak area sums near the peak area of anode, be the Heparin-binding ratio of test sample.Result statistics is as shown in table 4:
Table 4
7. the comparison of two kinds of assay methods:
The measurement result of two kinds of assay methods (method that method provided by the invention and European Pharmacopoeia provide) is through one-way analysis of variance, result shows that two kinds of detection methods do not have significant difference (89.16% ± 1.37% VS 90.26% ± 9.21%) to the testing result of same test sample, and this crossed immunoelectrophoresis method result that illustrates that method measurement result provided by the invention provides with European Pharmacopoeia is consistent.But the crossed immunoelectrophoresis method (1.54% VS10.20%) that the coefficient of variation (CV) of method repeated detection result provided by the invention provides much smaller than European Pharmacopoeia, has better degree of accuracy.
Embodiment 2:
1. reagent and sample are as shown in table 5:
Table 5
2. solution preparation:
Phosphate buffer: weigh sodium dihydrogen phosphate 20.0g, be dissolved in 950ml purified water, regulate pH to 6.4 with the sodium hydroxide solution of 0.2mol/L,, mix to 1L with purified water constant volume.
Mobile phase: get above-mentioned phosphate buffer 495ml, add 5ml isopropyl alcohol, mix ultrasound wave processing 30min and carry out degassed.
Heparin solution: dilute heparin sodium injection to 2000IU/ml with above-mentioned mobile phase.
The Human Antithrombin Ⅲ of inactivation: will add Bicine solution (Bicine(N-bis-(hydroxyethyl) glycocoll of 50mmol/L), the NaHCO of 100mmol/L in Human Antithrombin Ⅲ's standard items
3, pH 8.3); The trinitro-benzene-sulfonic acid that adds again 10 ~ 30mmol/L, 25 ℃ of constant temperature are processed 30min; Add the Tris damping fluid (50mmol/LTris – HCl, 100mmol/L NaCl, pH7.4) of 20 times of volumes to mix.After ultrafiltration concentration, through HiPrep Sephacryl S-200 HR molecular sieve chromatography purifying, obtain deactivated monomeric protein.Through HPLC, detect content of monomer > 95%, not with Heparin-binding, retention time is consistent with Human Antithrombin Ⅲ's standard items.
Standard solution: Human Antithrombin Ⅲ's standard items are prepared to (total protein content and testing sample are consistent) according to the ratio of protein content shown in table 6 respectively with the inactivation Human Antithrombin Ⅲ of known protein concentration:
Table 6
。
3. sample preparation
Test sample is diluted to 9mg/ml with mobile phase.Test sample after dilution and above-mentioned 5 kinds of standard solution mix respectively (Human Antithrombin Ⅲ: heparin activity is than being 1:40) with above-mentioned heparin solution equal-volume, hatch 10 minutes for 37 ℃.
4. high-efficient liquid phase chromatogram condition
Use efficient size exclusion chromatograph post, TOSOH BIOSCIENCE TSK-GEL G3000SW, mobile phase pre-equilibration 1h for flow velocity: 0.6ml/min.Detecting device wavelength set is UV280nm.The test sample of handling well and standard solution be sample introduction successively, each sample size 10 μ L.After each sample introduction, set elution time 1h.Test sample sample introduction 5 times, as parallel experiment.
5. result is calculated:
Peak area integration: adopt the Breeze2.0 software of Waters HPLC system, automatic integration calculates peak area.Calculate peak area ratio A=peak 1 area/(1 area+peak, peak, 2 areas).
Typical curve: the peak area ratio A value of 5 kinds of standard solution chromatograms is carried out to linear regression as X-axis and Y-axis respectively with corresponding theoretical Heparin-binding ratio (being designated as B), obtain the regression equation of B=aA+b.By the A value substitution typical curve regression equation of test sample, can calculate the Heparin-binding ratio of test sample.
Concrete outcome is as shown in table 7:
Table 7
6. crossed immunoelectrophoresis detection method detects test sample:
Crossed immunoelectrophoresis method concrete steps according to < < European Pharmacopoeia > > (EP7.5) regulation are measured test sample, replicate determination 5 times:
Crossed immunoelectrophoresis: with the Ago-Gel of barbital electrophoretic buffer (pH8.4, the heparin that contains 15IU/ml) preparation 10g/L, topple over the gel that 5ml melts on the glass plate of 5cm * 5cm, 4 ℃ of cooling 30min.A hole, diameter 2mm are made a call to respectively in place at adjacent two each 1cm of angular distance edge of gel.A hole adds the Human Antithrombin Ⅲ's test sample to be measured 5 μ L that are diluted to 1IU/ml, and another hole adds 5 μ L bromophenol blues as indicator, and under 7v/cm voltage, electrophoresis is until indicator migrates to end, and in electrophoresis process, temperature keeps below 4 ℃.The electrophoresis region that retains test sample 1.5cm * 5cm, excise remaining gel, 5% the sero-fast gel of the anti-human antithrombin Ⅲ of rabbit (the 10g/L agarose of containing of the new preparation of 3.5ml is toppled in remaining space, the barbital electrophoretic buffer of pH8.4), after cooled and solidified, engage completely with original remaining gel, gel is turned to 90 °, and the voltage of setting 2v/cm carries out two dimensional electrophoresis, about 16h.
Dyeing: by the filter paper parcel that soaks into physiological sodium chloride solution for gel, compress 2h, constantly more renew filter paper, unreacted antiserum in gel is diffused out completely, dye and decolour with acid blue 92, occur two precipitation peaks on gel.
Result is calculated: calculate the ratio (manually integration) that accounts for all precipitation peak area sums near the peak area of anode, be the Heparin-binding ratio of test sample.Result statistics is as shown in table 8:
Table 8
。
7. the comparison of two kinds of assay methods:
The measurement result of two kinds of assay methods (method that method provided by the invention and European Pharmacopoeia provide) is through one-way analysis of variance, result shows that two kinds of detection methods do not have significant difference (91.49% ± 0.61% VS 90.26% ± 9.21%) to agreeing to the testing result of test sample, and this crossed immunoelectrophoresis method result that illustrates that the measurement result of method provided by the invention provides with European Pharmacopoeia is consistent.But the crossed immunoelectrophoresis method (0.67% VS10.20%) that the coefficient of variation (CV) of method repeated detection result provided by the invention provides much smaller than European Pharmacopoeia, has better degree of accuracy.
Claims (6)
1. Human Antithrombin Ⅲ's Heparin-binding ratio detection method, is characterized in that, comprises the following steps:
(1) solution preparation, prepares respectively Human Antithrombin Ⅲ and the standard solution of damping fluid, mobile phase, heparin solution, Human Antithrombin Ⅲ's solution, inactivation;
(2), to the Human Antithrombin Ⅲ's preparation standard solution that adds in proportion inactivation in Human Antithrombin Ⅲ's standard items, adopt high performance liquid chromatography, according to the variation production standard curve of characteristic peak area ratio;
(3) need testing solution is adopted to high performance liquid chromatography, by the ratio of characteristic peak area and the combination ratio of corresponding typical curve calculating Human Antithrombin Ⅲ and heparin.
2. Human Antithrombin Ⅲ's Heparin-binding ratio detection method according to claim 1, is characterized in that, solution preparation is specially:
Phosphate buffer: phosphate solution 0.1 ~ 0.3mol/L, regulates pH to 6.0 ~ 7.5 with NaOH or hydrochloric acid solution;
Mobile phase: isopropyl alcohol: phosphate buffer=1:99;
Heparin solution: with the dilution of above-mentioned mobile phase or dissolve liquaemin to certain concentration, every milliliter of contained heparin units is 30 ~ 150 times of Human Antithrombin Ⅲ's units in every milliliter of testing sample;
Human Antithrombin Ⅲ's solution: Human Antithrombin Ⅲ's standard items or test sample are diluted or are dissolved to 1 ~ 50mg/ml with above-mentioned mobile phase, and the Heparin-binding ratio of Human Antithrombin Ⅲ's standard items is known, and is not less than 90%; Need testing solution and the above-mentioned heparin solution equal-volume preparing got after dilution mix;
The Human Antithrombin Ⅲ of inactivation: will add Bicine solution in Human Antithrombin Ⅲ's standard items; The trinitro-benzene-sulfonic acid that adds again 10 ~ 30mmol/L, 25 ℃ of constant temperature are processed 30min; Add the Tris damping fluid of 20 times of volumes to mix, after ultrafiltration concentration, through HiPrep Sephacryl S-200 HR molecular sieve chromatography purifying, obtain deactivated monomeric protein, through HPLC, detect content of monomer > 95%, not with Heparin-binding, retention time is consistent with Human Antithrombin Ⅲ's standard items;
Standard solution: respectively according to different proportion preparation, Human Antithrombin Ⅲ's standard items proportion, between 0 ~ 100%, at least should have the standard solution of 3 kinds of different proportions by the inactivation Human Antithrombin Ⅲ of Human Antithrombin Ⅲ's standard items and known protein concentration; Within the proportional range that the Heparin-binding ratio of test sample should cover at standard solution; The above-mentioned solution preparing is mixed with isopyknic above-mentioned heparin solution respectively.
3. Human Antithrombin Ⅲ's Heparin-binding ratio detection method according to claim 1, is characterized in that, high performance liquid chromatography chromatographic column is efficient size exclusion chromatograph post.
4. Human Antithrombin Ⅲ's Heparin-binding ratio detection method according to claim 1, is characterized in that, high performance liquid chromatography chromatographic condition is flow velocity: 0.2 ~ 0.8ml/min; Detecting device wavelength: UV280nm; Sample size: 5 ~ 20 μ L.
5. Human Antithrombin Ⅲ's Heparin-binding ratio detection method according to claim 1, it is characterized in that, while adding excessive heparin in Human Antithrombin Ⅲ's preparation, keep bioactive Human Antithrombin Ⅲ's molecule reversible non-covalent combination to occur with heparin molecule, molecule quantitative change is large, can form a chromatogram peak-to-peak 1 that retention time is shorter, and cannot because molecular weight does not change, form in situ a chromatogram peak-to-peak 2 with heparin-bounding Human Antithrombin Ⅲ's molecule.
6. Human Antithrombin Ⅲ's Heparin-binding ratio detection method according to claim 5, is characterized in that, adopts peak area integration method, calculates peak area; Calculate peak area ratio A=peak 1 area/(1 area+peak, peak, 2 areas); The peak area ratio A value of accurate solution chromatogram is carried out to linear regression as X-axis and Y-axis respectively with corresponding Heparin-binding ratio B, obtain the regression equation of B=aA+b, R
2>=0.95.
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|---|---|---|---|---|
| CN110672567A (en) * | 2019-09-26 | 2020-01-10 | 南通大学 | Low molecular weight heparin gold nano material and application thereof in heparanase detection |
| CN110672567B (en) * | 2019-09-26 | 2022-01-11 | 南通大学 | Low molecular weight heparin gold nano material and application thereof in heparanase detection |
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