Heparin assay kit and heparin content assay method for non-diagnostic purposes

Abstract

The present invention relates to the field of medical in vitro diagnosis, and in particular to a heparin assay kit and a method for determining heparin content for non-diagnostic purposes. The kit includes R1 reagent and R2 reagent, wherein R1 reagent contains a chromogenic substrate, heparin hydrolase, lysine, Triton X-100, and a first buffer; and R2 reagent contains FⅩa, arginine, NP-40, magnesium chloride, and a second buffer. The kit of the present invention solves the source of plasma to a certain extent, is more in line with China's national conditions, is easy to prepare, simple to operate, has high sensitivity, stability, and anti-interference ability, and has high accuracy in test results and strong market competitive advantages.

Description

Technical Field

[0001] The present invention relates to the field of medical in vitro diagnosis, in particular to a heparin determination kit and a heparin content determination method for non-diagnostic purposes. Background Art

[0002] Heparin is an acidic mucopolysaccharide synthesized by mast cells located in the intestinal mucosa. Its content in normal human blood is extremely low, and its anticoagulant effect is minimal under physiological conditions. It exerts its anticoagulant effect by accelerating antithrombin and HCII, and also affects the TFPI and protein C systems. Natural heparin is heterogeneous, with a molecular weight ranging from 3 to 57 kD (Wang Zhenyi. Basic Theory and Clinical Practice of Thrombosis and Hemostasis [M]. 3rd ed. Shanghai: Shanghai Science Foundation Press, 2004: 119-119). It is widely used clinically as an anticoagulant, primarily for the treatment of thrombotic diseases and anticoagulation in cardiovascular surgery, hemodialysis, and extracorporeal circulation.

[0003] Heparin is one of the most commonly used anticoagulant therapeutic agents in clinical medicine. Heparin-based drugs occupy a large market share among antithrombotic drugs, among which low-molecular-weight heparins are the dominant ones. As a drug, accurate detection of heparin activity is of great significance both for quality control of the production process and for dynamic monitoring of patients during clinical treatment. Heparin determination methods mainly include coagulation method and chromogenic substrate method (US4234682, US4948724, US5308755A, CN104048931A, CN103063592A, CN103323416A; Ten CH, Lamping RJ, Henny CP, et al. Automated amidolytic method for determining heparin, a heparinoid, and a low-Mr heparinfragment, based on their anti-Xa activity [J]. Clinical Chemistry, 1984, 30(6): 860-864). The chromogenic substrate method utilizes the basic principle that the heparin sugar chain contains a pentasaccharide domain with high affinity for antithrombin, which can specifically bind to AT in the body to form a heparin-antithrombin complex. The heparin-antithrombin complex can inhibit the color development reaction of the activated coagulation factor X (or coagulation factor II) by hydrolyzing the chromogenic substrate. It has the characteristics of high sensitivity, simple operation and strong specificity. The antithrombotic and anticoagulant activities of heparin are related to the characteristic composition and relative mass of the heparin molecule. Studies have shown that in the process of anti-factor Xa interaction, only the sugar chain containing the core pentasaccharide domain of heparin is required to activate AT and thus achieve the purpose of anticoagulation; in the process of anti-factor IIa interaction, heparin must not only contain the core pentasaccharide domain, but also include sugar chains composed of at least eighteen monosaccharides (Lane DA, Denton J, Flynn AM, et al. Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4. [J]. Biochemical Journal, 1984, 218 (3): 725-732).

[0004] There are two types of heparin assay kits on the market that use the chromogenic substrate method. One is a two-step method that includes the addition of antithrombin (AT) and is independent of the AT content in the sample plasma to detect the total heparin content in the sample plasma. The other is a one-step method that relies on the AT content in the sample plasma (the AT content in plasma is in the range of 35% to 130%). The assay is based on the principle of competitive inhibition and detects heparin that acts as an anticoagulant in the sample plasma, such as IL's LiquidHeparin-0020300100, Aniara Biophen HeparinLRT, and STAGO's. -LiquidAnti-Xa. Obviously, the one-step kits commonly used for heparin detection on the market are basically liquid reagents, which makes their stability limited to some extent.

[0005] Analysis of the application of heparin detection kits currently available on the market revealed:

[0006] First, there are many types of heparin drugs used clinically. Chromogenic substrate assays typically require the development of new standard curves based on the specific type of heparin. Furthermore, studies have shown significant differences in the sensitivity of commercially available assays to direct factor Xa inhibitors (Dogne, Jean-Michel, Sabor, et al. Heparin-calibrated chromogenic anti-Xa assays are not suitable for assessing the presence of significant direct factor Xa inhibitor levels [J]. Thrombosis Research An International Journal on Vascular Obstruction Hemorrhage & Hemostasis, 2017). This, to some extent, reduces the simplicity of heparin assay kits (chromogenic substrate assays).

[0007] Secondly, most commercially available test kits contain dextran sulfate to reduce the interference of PF4 and other substances in sample plasma on heparin detection. However, some studies have shown that the presence of dextran sulfate can lead to excessively high heparin test results under high-concentration unfractionated heparin treatment (Ignjatovic V, Summerhayes R, Gan A, et al. Monitoring Unfractionated Heparin (UFH) Therapy: Which Anti Factor Xa Assay is Appropriate? [J]. Thrombosis Research, 2007, 120(3): 347-351.). Therefore, in such cases, it is also important to reduce the interference of PF4 while ensuring the accuracy of the test results.

[0008] Furthermore, my country has strict blood regulations, limiting access to human blood. Furthermore, bovine FXa production methods are simple and well-established. Consequently, the preferred FXa source in patent reports and the FXa component in commercially available heparin assay kits (chromogenic substrate method) are largely derived from bovine blood. However, compared to bovine blood, my country has a more abundant pig blood resource, yet its utilization rate is low. Summary of the Invention

[0009] In light of this, the present invention provides a heparin assay kit and a method for determining heparin content for non-diagnostic purposes. This kit addresses the plasma source issue to a certain extent, is more suitable for China's national conditions, and is easy to prepare and operate. It also offers high sensitivity, stability, and anti-interference capabilities, resulting in highly accurate test results and a strong market competitive advantage.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The invention provides a heparin determination kit, which comprises an R1 reagent and an R2 reagent. The R1 reagent comprises a chromogenic substrate, heparin hydrolase, lysine, Triton X-100, and a first buffer; and the R2 reagent comprises FⅩa, arginine, NP-40, magnesium chloride, and a second buffer.

[0012] Preferably, in the R1 reagent, the chromogenic substrate is any one of S-2732, S-2765 and S-2222; preferably, the chromogenic substrate is S-2732.

[0013] Preferably, the heparin hydrolase is heparinase II or acharan sulfate lyase;

[0014] Preferably, the first buffer is selected from one or more of HEPES, Tris-HCl or phosphate buffer, with a pH of 6.5 to 9.0, preferably 7.0 to 8.0.

[0015] Preferably, the first buffer is HEPES and Tris-HCl buffer.

[0016] Preferably, in the R2 reagent, FXa is porcine FXa or bovine FXa, preferably porcine FXa.

[0017] Preferably, the second buffer is selected from one or more of HEPES, Tris-HCl or phosphate buffer, with a pH of 6.5 to 9.0, preferably 7.5 to 8.4.

[0018] Preferably, the second buffer is Tris-HCl buffer.

[0019] Preferably, the R1 reagent further comprises a first lyoprotectant, which is selected from any one of mannitol, BSA, PEG6000 or aprotinin, or a mixture of several thereof;

[0020] Preferably, the first lyoprotectant is mannitol.

[0021] Preferably, the R2 reagent further comprises a second lyoprotectant, which is selected from any one of mannitol, BSA, PEG6000 or aprotinin, or a mixture of several of them.

[0022] Preferably, the second lyoprotectant is a mixture of mannitol and BSA.

[0023] Preferably, the working concentration of each component in the R1 reagent is:

[0024]

[0025]

[0026] The working concentrations of the components in the R2 reagent are:

[0027]

[0028] Preferably, in the R2 reagent, magnesium chloride is 0.05 to 0.25 mol / L. Preferably, the working concentrations of the components in the R1 reagent are:

[0029]

[0030] The working concentrations of the components in the R2 reagent are:

[0031]

[0032] In a specific embodiment provided by the present invention, the pH value of the first buffer is 7.5, and the pH value of the second buffer is 8.4.

[0033] In the present invention, the R1 reagent or the R2 reagent is a liquid reagent or a freeze-dried reagent.

[0034] The present invention also provides a method for determining heparin content for non-diagnostic purposes, wherein the heparin determination kit according to any one of claims 1 to 8 is used to determine the content of a sample, comprising the following steps:

[0035] ① Mix R1 reagent with the sample and incubate at 37°C for 0.5-2 minutes;

[0036] ② Add R2 reagent to the mixture obtained in step ①, incubate at 37°C for 3-4 minutes, and read the absorbance of the reaction product at a wavelength of 405 nm;

[0037] ③According to the known standard curve, the heparin content in the sample is obtained.

[0038] Preferably, the sample is a liquid sample, R1 reagent and R2 reagent are liquid reagents, or R1 reagent and R2 reagent are reconstituted freeze-dried reagents, and the volume ratio of R1 reagent, sample and R2 reagent is (1-2):(1-2):(1-2).

[0039] Preferably, the sample is a liquid sample, and the volume ratio of the R1 reagent, the sample, and the R2 reagent is 1:1:1.

[0040] The present invention provides a heparin assay kit and a method for determining heparin content for non-diagnostic purposes. The kit comprises reagent R1 and reagent R2. Reagent R1 contains a chromogenic substrate, heparin hydrolase, lysine, Triton X-100, and a first buffer; reagent R2 contains FXa, arginine, NP-40, magnesium chloride, and a second buffer. Compared with existing technologies, the kit of the present invention has the following advantages:

[0041] (1) The FXa used in the present invention is prepared from porcine plasma, which solves the plasma source problem, is suitable for China's national conditions, is easy to prepare, and reduces costs;

[0042] (2) A single standard curve obtained by the kit of the present invention can be used for the detection of multiple heparin drugs, eliminating the need to develop multiple standard curves for different heparin drugs, and thus the operation is simple;

[0043] (3) The lysine added to the R1 reagent of the present invention works synergistically with Triton X-100 to effectively improve the stability of the chromogenic substrate and heparin hydrolase, and ensure the stability of the enzyme activity during the freeze-drying process;

[0044] (4) The present invention can improve the stability of FXa factor activity compared to conventional kits by synergistically combining arginine and NP-40 added to the R2 reagent, and ensure the stability of enzyme activity during the freeze-drying process;

[0045] (5) The present invention found that the synergistic effect of adding TritonX-100, NP-40 and magnesium chloride to the reagent can improve the anti-interference effect during the detection process of the kit and improve its detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a correlation analysis between the test results of the kit of the present invention and existing commercially available kits. DETAILED DESCRIPTION

[0047] The present invention discloses a heparin assay kit and a method for determining heparin content for non-diagnostic purposes. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0048] The invention provides a heparin determination kit, which comprises an R1 reagent and an R2 reagent. The R1 reagent comprises a chromogenic substrate, heparin hydrolase, lysine, Triton X-100, and a buffer; and the R2 reagent comprises a porcine FⅩa reagent, arginine, NP-40, magnesium chloride, and a buffer.

[0049] The R1 and R2 reagents in the above kit can be liquid or freeze-dried. In the process of detecting the heparin activity of the sample using the above kit, the detection method can be an endpoint method or a kinetic method. The specific steps are as follows:

[0050] ① Mix R1 reagent and sample plasma in a volume ratio of 1:1, mix and incubate at 37°C for 0.5-2 minutes;

[0051] ② Add R2 reagent and mix it with sample plasma in a volume ratio of 1:1. After incubation at 37°C for 3 minutes, read the absorbance of the sample at a wavelength of 405 nm.

[0052] ③ Calculate the heparin content in the sample plasma based on the known standard curve.

[0053] Definition of working concentration: During the detection of heparin activity in a sample using any of the above reagents, the concentration of each component in the analytical mixture formed by mixing the R1 and R2 reagents (liquid reagents or freeze-dried reagents reconstituted with distilled water) with the sample plasma is defined as its working concentration.

[0054] In the process of detecting heparin activity using a chromogenic substrate method, as an FXa substrate, it is required to have high sensitivity and good water solubility to effectively improve the sensitivity of the detection and ensure the accuracy of the detection results. In the kit of the present invention, the chromogenic substrate in the R1 reagent is any one of Suc-Ile-Glu-(γ-Piperidyl)-Gly-Arg-pNA·HCl (S-2732), ZD-Arg-Gly-Arg-pNA·HCl (S-2765), and Bz-Ile-Glu(-OR)-Gly-Arg-pNA·HCl (S-2222); the preferred chromogenic substrate is Suc-Ile-Glu-(γ-Piperidyl)-Gly-Arg-pNA·HCl (S-2732), and its working concentration is 1.2-3.0 mg / mL, with an optimal working concentration of 1.5 mg / mL.

[0055] In the kit of the present invention, the heparin hydrolase contained in the R1 reagent is heparinase II or acharan sulfate lyase, wherein the properties of acharan sulfate lyase are similar to those of heparinase II; both can modify and cleave glycosaminoglycan chains and have a certain cleavage effect on heparin and heparan sulfate. Heparinase II can be purchased or, like acharan sulfate lyase, can be prepared from the corresponding strain by a person skilled in the art using known isolation and purification methods, such as those described in Kim BT et al. (Kim BT, Hong SW, Kim WS, 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). In a preferred embodiment, the working concentration of heparin hydrolase is 0.1 U / mL to 0.7 U / mL, with an optimal working concentration of 0.5 U / mL. Within this working concentration range, it is able to partially degrade heparin in the sample while retaining its ability to bind to AT. The enzymatic activity of heparinase II or acharan sulfate lyase is defined as the amount of enzyme required to convert 1 μmol of heparin unsaturated oligosaccharide chains in 1 minute under specific conditions, and is referred to as one international unit (IU or U).

[0056] The buffer in the R1 reagent of the present invention is selected from one or more of HEPES, Tris and phosphate buffer, with a pH range of 6.5-9.0. In some embodiments, a Tris-HEPES buffer is preferred, wherein the concentration of Tris in the buffer is 10 to 50 mM and the concentration of HEPES is 2 to 10 mM. To ensure that the R1 reagent has a sufficient level of solid content, the buffer further includes any one or a mixture of mannitol, BSA, PEG6000 and aprotinin, preferably mannitol.

[0057] In some embodiments of the present invention, lysine and TritonX-100 in the composition of the R1 reagent are used together as a stabilizer and a freeze-dried enzyme activity protectant, the concentration of lysine is 1-10%, and the content of TritonX-100 is 0.2-0.5 mL / L.

[0058] In the kit of the present invention, porcine FXa in the R2 reagent is extracted and purified from porcine plasma according to patent (CN201710780196.4), and its working concentration is 0.1-0.45 U / mL, with an optimal working concentration of 0.25 U / mL.

[0059] The buffer in the R2 reagent of the present invention is selected from one or more of HEPES, Tris and phosphate buffer, with a pH range of 6.5-9.0. In some embodiments, Tris-HCl buffer is preferred, the concentration of Tris in the buffer is 10 to 50 mM, and the preferred pH range is pH 7.5-8.4. To ensure that the R2 reagent has a sufficient level of solid content, the buffer further includes any one or a mixture of mannitol, BSA, PEG6000 and aprotinin, preferably mannitol mixed with BSA.

[0060] In some embodiments of the present invention, arginine and NP-40 in the composition of the R2 reagent are used together as a stabilizer and a freeze-dried enzyme activity protectant, the concentration of arginine is 1-10%, and the content of NP-40 is 0.05-2 mL / L.

[0061] In an embodiment of the present invention, the R2 reagent further includes a magnesium salt, preferably magnesium chloride, with a concentration of 0.01 to 0.25 mol / L. The presence of the magnesium salt not only ensures the ionic strength of the R2 reagent, but also effectively improves the sensitivity of the kit detection.

[0062] Unless otherwise specified, the materials, reagents, and instruments used in the following examples can be obtained from commercial sources.

[0063] The present invention will be further described below in conjunction with the embodiments:

[0064] Example 1 Preparation of the kit of the present invention and the control experimental group

[0065] (1) Kit of the present invention

[0066] Table 1: Formula of the kit of the present invention (liquid reagent) (experimental group 1)

[0067]

[0068] Table 2: Formula of the kit of the present invention (lyophilized reagent) (experimental group 2 and experimental group 3)

[0069]

[0070] Note: ① All lyophilized reagents were freeze-dried in 1 mL / vial aliquots. Reconstitute the lyophilized product with 2 mL of distilled water before use. ② The R1 reagent in Experimental Group 2 was prepared using heparinase II; the R1 reagent in Experimental Group 3 was prepared using acharan sulfate lyase.

[0071] (2) Preparation of control experimental group

[0072] Table 3 Control experimental group formula

[0073]

[0074]

[0075]

[0076] Example 2 Establishment of the detection method of the kit of the present invention

[0077] (1) Screening of heparin hydrolase concentration

[0078] Taking the BioTek ELx800 microplate reader (endpoint method) as an example, set the analysis program according to the instrument instructions: the measurement wavelength is 405 nm;

[0079] First, dilute heparin of known titer with normal saline to 100U / mL heparin. Take 10μL of 100U / mL heparin and add 990μL of human plasma standard to dilute it to obtain 1.0U / mL heparin standard. Continue to dilute the 1.0U / mL heparin standard with human plasma standard to prepare samples of 5 standard points of heparin at 0.0, 0.2, 0.5, 0.8, and 1.0U / mL, and incubate at 37°C for 30 seconds. Take 50μL of the diluted sample, add 50μL of R1 reagent and incubate at 37°C for 60 seconds, then add 50μL of R2 reagent and incubate at 37°C for 180 seconds, then add 50μL of 20% acetic acid solution to terminate the reaction, and measure the absorbance value (OD) at 405nm. 405nm Each tube was measured 3 times, and the OD 405nmThe mean of the heparin activity was plotted on the ordinate, and the corresponding heparin activity on the abscissa. A standard curve for activity versus absorbance was constructed. Similarly, a standard curve for low molecular weight heparin was constructed. The results of the heparin and low molecular weight heparin assays at each standard point for experimental group 1 and control groups 1 to 4, along with the curve analysis results, are shown in Table 4. Linearity tests revealed that the standard curves for UFH and LMWH from different reagent groups exhibited good correlation coefficients. SPSS 17.0 software was used to test for significant differences between the two standard curves (calibration curves for UFH and LMWH from the same reagent group). Analysis of covariance results showed that at a 95% confidence level, when the heparinase II content in reagent R1 was between 0.1 and 0.7 U / mL, the intercept and slope of the two standard curves exhibited no significant difference. Therefore, the two standard curves could be combined into a single standard curve, or either curve could be used for testing. Due to the degradation of heparin structure by heparinase II, the degraded heparin component retains its activity with AT, but its chain length becomes shorter. Therefore, the standard curve ultimately adopted the standard curve of low molecular weight heparin. The LMWH standard curve equation for experimental group 1 is: y = -0.5064x + 1.0221.

[0080] Table 4 Summary and analysis of the determination results of standard curves of different reagent groups (liquid reagents)

[0081]

[0082] (2) Screening of heparin hydrolase species and establishment of the detection method of the kit of the present invention

[0083] The same experimental method as in Example 1(1) was used to determine the standard points of heparin and low molecular weight heparin using experimental groups 2 to 3 and control groups 5 to 7, respectively. In control group 7, the order of adding the R1 reagent and the R2 reagent was reversed. The results are shown in Table 5. According to the linear test results, the standard curves of UFH and LMWH in different reagent groups all had good correlation coefficients. SPSS 17.0 software was used to test the significant difference between the two standard curves (calibration curves of UFH and LMWH in the same reagent group). The covariance analysis results showed that at a confidence level of 95%, when the type of heparin hydrolase in the R1 reagent was heparinase II or acharan sulfate lyase, the intercept and slope test results of the two standard curves had Sig.P values ​​greater than 0.05. When the formulation consisted of heparinase I and heparinase III, the intercept and slope test results of the two standard curves had Sig.P values ​​less than 0.05. That is, in the formulation combination of the kit of the present invention, only when heparinase II and acharan sulfate lyase are used can the standard curves used for UFH and LMWH determination use the same calibration curve, thereby ensuring the accuracy of the sample test results. Due to the degradation of heparin structure by heparinase II, the degraded heparin component retains its activity with AT, but its chain length becomes shorter. Therefore, the standard curve ultimately adopted the standard curve of low molecular weight heparin. The LMWH standard curve equation for experimental group 2 is: y = -0.5008x + 1.0307.

[0084] In addition, the results in Table 5 also show that when the kit of the present invention is used for detection, the order of adding the R1 reagent and the R2 reagent cannot be interchanged. The detection steps are: ① R1 reagent is mixed with the sample plasma in a volume ratio of 1:1, mixed and incubated at 37°C for 0.5-2 minutes; ② R2 reagent is added, and the volume ratio of the mixture with the sample plasma is 1:1. After incubation at 37°C for 3 minutes, the absorbance value of the above sample at a wavelength of 405 nm is read; ③ The heparin content in the sample plasma is calculated based on the known standard curve.

[0085] Table 5 Summary and analysis of the determination results of the standard curves of different reagent groups (lyophilized reagents)

[0086]

[0087] This kit is suitable for use on various brands and models of semi-automatic and fully-automatic analyzers (biochemical analyzers or coagulation analyzers) that utilize the chromogenic substrate method, such as the French STA-REvolution coagulometer, the American Beckman Coulter ACL TOP series coagulometer, and the Siemens CA / CS series coagulometer. Specific parameters can be adjusted appropriately based on the instrument.

[0088] Example 3 Analytical Performance Evaluation of the Kit of the Present Invention

[0089] (1) Sensitivity

[0090] First, normal human mixed plasma (AT content in plasma is 80% to 100%) was used as a blank sample, and the blank sample was tested using 8 to 10 reagents of the experimental group 1 and the control group according to the detection method of Example 2. The measurement was repeated 20 times, and the OD of the sample was calculated. 405nm mean The blank mean value minus twice the standard deviation was substituted into the calibration curve equation of LMWH to calculate the minimum detection limit. The results are shown in Table 6.

[0091] Next, rivaroxaban, edoxaban, and apixaban were dissolved in DMSO to prepare 0.6 mg / mL stock solutions. These stock solutions were then diluted to 30 ng / mL with normal human plasma to obtain three test samples at the clinical cutoff. The three samples were tested using 1 test group and 8-10 test reagents in the control group, respectively, according to the detection method of Example 2. The assays were repeated three times, and the mean anti-Xa activity values ​​were calculated. The results are shown in Table 7.

[0092] The results in Tables 6 and 7 demonstrate that magnesium chloride and NP-40, when acting synergistically, can effectively enhance the detection sensitivity of the present kit, and are also effective against direct FXa inhibitors. The present kit has a minimum detection limit of 0.02 U / mL for heparin, which is lower than the minimum detection limit (0.04 U / mL) stated in the instructions for commercially available heparin detection kits (chromogenic substrate method).

[0093] Table 6 Analysis results of minimum detection limit

[0094]

[0095]

[0096] Table 7 Sensitivity validation results for direct FXa inhibitors at clinical cutoff values

[0097]

[0098] (2) Anti-interference capability

[0099] Hemoglobin, bilirubin, triglycerides, and platelet factor PF4 were dissolved into stock solutions of a certain concentration according to the instructions for each sample. These stock solutions were then diluted to the desired concentrations using a human plasma standard containing UFH to obtain different concentration gradients of test samples containing hemoglobin, bilirubin, triglycerides, or platelet factor PF4 (the theoretical content of UFH in samples containing hemoglobin, bilirubin, and triglycerides is 0.50 U / mL, and the theoretical content of UFH in samples containing PF4 is 1.0 U / mL. The concentrations of the interfering substances are shown in Table 8). The test samples were tested using the reagents of experimental group 1 and control groups 9 and 11-12, respectively, according to the detection method of Example 2. The measurements were repeated three times, and the mean values ​​of their anti-Xa activity were calculated. The results are shown in Table 8. The results showed that the synergistic effect of magnesium chloride, TritonX-100 and NP-40 can effectively improve the anti-interference ability of the reagent of the present invention. When the hemoglobin content in the sample plasma is lower than 3.5g / L, the bilirubin content is lower than 0.3g / L, the triglyceride content is lower than 9g / L, and the PF4 is lower than 80ng / mL, the kit of the present invention will not affect the detection of the heparin content in the sample.

[0100] Table 8 Detection structure of anti-interference ability

[0101]

[0102]

[0103] (3) Stability

[0104] (I) Screening of R1 reagent stabilizers

[0105] The reagents of experimental group 1 and control group 12 to 16 were placed in an electric constant temperature incubator at 37 ° C and taken out for testing on days 0, 7 and 14. At each test time interval, the five quality control products of HYPHEN (respectively: UFH Control level 1, UFH Control level 2, LWMH Control level 1, LWMH Control level 2, LWMH Control level 3) were tested using the test method of Example 2 using the reagents of experimental group 1 and control group 12 to 16. The measurement was repeated 3 times, and the relative deviation of the mean of the test results on day 7 or day 14 from the mean of the test results on day 0 was calculated based on the mean of the test results. The results are shown in Table 9. The results show that the synergistic effect of lysine and Triton X-100 can effectively improve the stability of the R1 reagent. When other amino acids are used to replace lysine and synergize with Triton X-100, the stability effect is not as good as the kit of the present invention.

[0106] Table 9 Screening results of R1 reagent stabilizers

[0107]

[0108]

[0109] (II) Screening of R2 reagent stabilizers

[0110] The reagents of experimental group 1 and control group 17 to 21 were placed in an electric constant temperature incubator at 37 ° C and taken out for testing on days 0, 7 and 14. At each test time interval, the five quality control products of HYPHEN (respectively: UFH Control level 1, UFH Control level 2, LWMH Control level 1, LWMH Control level 2, LWMH Control level 3) were tested using the test method of Example 2 using the reagents of experimental group 1 and control group 17 to 21, respectively. The measurement was repeated 3 times, and the relative deviation of the mean of the test results on day 7 or day 14 from the mean of the test results on day 0 was calculated based on the mean of the test results. The results are shown in Table 10. The results show that the stability of the R2 reagent can be effectively improved when arginine and NP-40 act synergistically. When other amino acids are used to replace arginine and NP-40, the stability effect is not as good as the kit of the present invention.

[0111] Table 10 Screening results of R1 reagent stabilizers

[0112]

[0113]

[0114] (III) Stability testing of the kit of the present invention

[0115] The reagents of experimental group 1 were placed in an electric constant temperature incubator at 37°C and taken out for testing on days 0, 7, 14, 21, 28, and 38. At each test time interval, the reagents of experimental group 1 were used to test HYPHEN's five quality control products (respectively: UFH Control level 1, UFH Control level 2, LWMH Control level 1, LWMH Control level 2, LWMH Control level 3) according to the detection method of Example 2. The test was repeated 3 times, and the mean of the test results was calculated. The results are shown in Table 11. The results show that the test results of the quality control products at different concentration levels of the kit of the present invention were almost unchanged within 35 days at 37°C, that is, the kit of the present invention can be effectively stable for 35 days at 37°C.

[0116] Table 11 Stability of the kit of the present invention at 37°C

[0117]

[0118] (III) Stability test of the reconstitution of the freeze-dried product of the kit of the present invention

[0119] The reagents of experimental group 2 and control group 22 to control group 27 were first tested before lyophilization according to the method of Example 2, and freeze-dried after the test results met the requirements. After lyophilization, the lyophilized product was reconstituted with distilled water and tested after lyophilization according to the method of Example 2. The test results of the reagents of experimental group 2 after lyophilization were used as the test results on day 0. At the same time, the reconstituted reagents of experimental group 2 were placed at room temperature (18-25°C) and taken out for testing on days 5, 10, 20 and 30 respectively. The test method for each test was to use the reagents of experimental group 1 to test HYPHEN's two quality control products (respectively: UFH Control level 2 and LWMH Control level 2) according to the test method of Example 2. The test was repeated 3 times, the mean of the test results was calculated, and the significance test results of the slope of the linear regression analysis were performed using SPSS 17.0 software as shown in Tables 12 and 13. The results showed that compared with the combination of other amino acids with Triton X-100 or NP-40, the combination of lysine and Triton X-100 contained in the R1 reagent of the kit of the present invention and arginine and NP-40 contained in the R2 reagent can effectively maintain the stability of the enzyme activity, facilitate the freeze-drying of the reagent, and further extend the shelf life of the reagent; the results in Table 13 show that the freeze-dried product of the kit of the present invention can be stable at room temperature for 30 days after reconstitution, which is longer than the period (14 days) specified in the instructions of the commercially available heparin detection kit (chromogenic substrate method).

[0120] Table 12 Stability of reagents before and after lyophilization with different stabilizers

[0121]

[0122] Table 13 Stability of the freeze-dried product of the kit of the present invention after reconstitution at room temperature

[0123]

[0124]

[0125] Comparison of Example 4 with marketed products

[0126] A total of 170 blood samples were randomly collected from inpatients and outpatients of Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine and Longhua Hospital affiliated to Shanghai University of Traditional Chinese Medicine. The blood was mixed with 0.109 mol / L sodium citrate anticoagulant at a ratio of 9:1 and centrifuged at 2500 rpm for 15 minutes to separate the plasma. The kit of the present invention (experimental group 1) obtained in Example 1 and the STAGO -Liquid Anti-Xa kit (Cat. No. 00311) was used to measure the samples respectively, calculate the correlation coefficient between the two, and perform linear regression. The results showed that the correlation coefficient of the two kits was r = 0.9908, and the linear regression equation was y = 1.0039x-0.0046, see Figure 1 .

[0127] According to the requirements of the U.S. Clinical Laboratory Standards Institute (CLSI) document (r>0.975), the test data of the kit of the present invention and the kit imported from France STAGO Company have good consistency.

[0128] Example 5 Concentration range of main components

[0129] First, the reagents of experimental groups 16 to 17 (formulas shown in Table 14) were used to test the samples with the highest concentrations of the interfering components (hemoglobin, bilirubin, triglycerides and PF4) described in (2) of Example 3 and the samples without interfering components (i.e., samples with a concentration of 0) according to the detection method of Example 2. The measurements were repeated three times, and the mean of the test results of each sample was calculated. The results are shown in Table 15.

[0130] Secondly, experimental groups 4 to 17 (formulas are shown in Table 14) were placed in an electric constant temperature incubator at 37°C. On day 0 and day 20, two quality control products (UFH Control level 2 and LWMH Control level 2) were taken out and tested according to the detection method of Example 2. The measurement was repeated three times, and the relative deviation of the mean test results on day 20 and day 0 was calculated based on the mean of the test results. The results are shown in Table 16.

[0131] The results in Tables 15 and 16 show that the R1 reagent in the kit of the present invention, with the main component S-2732 content ranging from 1.2 to 3.0 mg / mL, the lysine content ranging from 1% to 10%, and the Triton X-100 content ranging from 0.2 to 0.5 mL / L, still met the stability requirements after 20 days at 37°C. Similarly, the R2 reagent in the kit of the present invention, with the main components porcine FXa content ranging from 0.1 to 0.45 U / mL, arginine content ranging from 1% to 10%, NP-40 content ranging from 0.5 to 2.0 mL / L, and magnesium chloride content ranging from 0.05 to 0.25 mol / L, also met the stability requirements after 20 days at 37°C. Furthermore, the anti-interference ability was still good when the magnesium chloride content was within the range of 0.05 to 0.25 mol / L.

[0132] Table 14 Experimental group formula of concentration range of main components

[0133]

[0134]

[0135] Table 15 Verification results of the anti-interference ability of magnesium chloride at different concentrations

[0136]

[0137] Table 16 Stability verification results of experimental group formulas in different concentration ranges

[0138]

[0139]

[0140] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A heparin assay kit, characterized in that: The kit includes R1 reagent and R2 reagent, and the working concentration of each component in the R1 reagent is: 1.5 mg / mL S-2732, 0.5 U / mL heparin hydrolase, 5% lysine, 0.3 mL / L Triton X-100, 50 mM Tris-HCl, 10 mM HEPES, and 5% mannitol; The working concentrations of the components in the R2 reagent are: 0.25 U / mL FXa, 5% arginine, 0.1 mL / L NP-40, 0.2 mol / L magnesium chloride, 50 mM Tris-HCl, 2% mannitol, and 0.5% BSA; Heparin hydrolase is heparinase II or acharan sulfate lyase; In the R2 reagent, FXa is porcine FXa or bovine FXa.

2. The heparin assay kit according to claim 1, wherein The R1 reagent or the R2 reagent is a liquid reagent or a freeze-dried reagent.

3. A method for determining heparin content for non-diagnostic purposes, characterized in that: The heparin assay kit according to claim 1 is used to assay a sample, comprising the following steps: ① Mix R1 reagent with the sample and incubate at 37°C for 0.5-2 minutes; ② Add R2 reagent to the mixture obtained in step ①, incubate at 37°C for 3-4 minutes, and read the absorbance of the reaction product at 405 nm; ③ According to the known standard curve, the heparin content in the sample is obtained.

4. The heparin content determination method according to claim 3, characterized in that: The sample is a liquid sample, R1 reagent and R2 reagent are liquid reagents, or R1 reagent and R2 reagent are reconstituted freeze-dried reagents, and the volume ratio of R1 reagent, sample and R2 reagent is (1~2):(1~2):(1~2).

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