Immunoturbidimetric D-dimer assay

Abstract

The present invention relates to the field of detection technology, and in particular to an immunoturbidimetric detection reagent for D-dimer. The present invention improves the repeatability and stability of the reagent in the low value area by optimizing the formula of R1 and R2 reagents. Experiments show that when the reagent of the present invention is used to detect low-value samples, the repeatability CV value does not exceed 3%, and the reagent remains stable within 36 months.

Description

Technical Field

[0001] The invention relates to the field of detection technology, in particular to an immunoturbidimetric detection reagent for D-dimer. Background Art

[0002] D-Dimer is a specific degradation product produced by the hydrolysis of fibrin monomers after cross-linking with activated factor XIIIa and then by plasmin. D-Dimer originates from the cross-linked fibrin clots dissolved by plasmin. Under normal physiological conditions, the human body maintains a dynamic balance between coagulation and fibrinolysis to ensure the timely formation and removal of fibrin. If this balance is disrupted, the intravascular coagulation tendency is enhanced, fibrin aggregates, fibrin degradation products increase, and the D-dimer content increases. Therefore, the increase in D-dimer levels reflects the dual activation of the coagulation and fibrinolytic systems in the body, and is an indicator of the body's hypercoagulable state, thrombosis, and secondary hyperfibrinolysis.

[0003] Elevated D-dimer is commonly seen in clinical cases of disseminated intravascular coagulation (DIC), deep vein thrombosis (DVT), pulmonary embolism (PE), cerebral infarction, acute myocardial infarction, lung cancer, and pregnancy-induced hypertension. Negative D-dimer can be used to rule out deep vein thrombosis (DVT) or pulmonary embolism (PE).

[0004] Currently, the commonly used D-Dimer detection methods are mainly qualitative or semi-quantitative tests based on the principle of latex agglutination and quantitative determination based on the principle of ELISA. The classic latex agglutination method is simple and fast to operate, suitable for emergency detection, and is often used for screening. The ELISA method is accurate, quantitative, and highly sensitive, but the operation requirements are strict and time-consuming, and a standard curve needs to be made at the same time for each experiment, which is not suitable for the needs of emergency and clinical patients for timely diagnosis and treatment.

[0005] In recent years, latex immunoturbidimetry is used for D-Dimer detection, and this method has realized automated detection on coagulation analyzer, has the advantages such as easy and simple to operate, fast, quantitative and accurate, can meet the needs such as outpatient and emergency, and is more and more widely used in clinical research. There are mainly two types of D-dimer (D-Dimer) assay reagents (latex immunoturbidimetry) on the market, one is highly sensitive, and the detection range is relatively narrow, and one is relatively low in sensitivity, and the detection range is wider. But the above two reagents have the phenomenon of poor repeatability in the measured value of low value area. Therefore, improving the measured value repeatability of D-dimer immunoturbidimetry assay reagent in low value area is a problem to be solved in this area. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to improve the repeatability of the D-dimer immunoturbidimetric detection reagent in the low value area by enhancing the signal. At the same time, through experiments, the stability of the latex reagent is enhanced.

[0007] The immunoturbidimetric detection reagent for D-dimer provided by the present invention comprises R1 reagent and R2 reagent:

[0008] The R1 reagent contains Tris, NaCl, BSA, TritonX-100, cyclodextrin, Biolipidure and NaN3, and the pH value is 7.0-8.5;

[0009] The R2 reagent contains microspheres coated with D-dimer antibody, BSA, NaN3, PVA and storage buffer, and the pH value is 7.0.

[0010] The present invention has experimentally found that The addition of PVA has a positive effect on the repeatability and stability of the reagent in the low value area.

[0011] In the present invention, the R1 reagent is composed of water and the following components: 15mM~55mM Tris, 0.1M~0.3MNaCl, 0.2% (w / v)~0.8% (w / v) BSA, 0.0005% (w / v)~0.008% (w / v) TritonX-100, 0.005% (w / v)~1.2% (w / v) cyclodextrin, 0.002% (w / v)~0.01% (w / v) Biolipidure and 0.1% (w / v) NaN3.

[0012] Further, in the R1 reagent of the present invention, the concentration of Tris is 15-55 mM, preferably 20 mM; the concentration of NaCl is 0.1-0.3 M, preferably 0.15 M; the concentration of BSA is 0.2% (w / v)-0.8% (w / v), preferably 0.5% (w / v); the concentration of TritonX-100 is 0.0005% (w / v)-0.008% (w / v), preferably 0.005% (w / v); the concentration of cyclodextrin is 0.005% (w / v)-1.2% (w / v), preferably 0.5% (w / v); The pH value of the R1 reagent of the present invention is 7.0 to 8.5, and the preferred pH value of the R1 reagent is 7.5.

[0013] In the embodiment of the present invention, the Biolipidure is -103, -1002, -203 or -405. The embodiments of the present invention show that Biolipidure combined with other reagents in R1 can improve the repeatability of low value detection at appropriate concentrations. In some embodiments, the Biolipidure is -103. Experiments show that compared with other Biolipidure products, -103 is more conducive to the enhancement of detection signals, thereby improving the repeatability of low value detection by the reagent.

[0014] In some specific embodiments, the R1 is composed of water and 20 mM Tris, 0.15 M NaCl, 0.5% (w / v) BSA, 0.005% (w / v) Triton X-100, 0.5% (w / v) cyclodextrin, 0.005% (w / v) -103, 0.1% (w / v) NaN3, pH value is 7.5.

[0015] In the present invention, the R2 reagent is composed of a storage buffer and the following components: 1-3 mg / mL microspheres coated with D-dimer antibody, 0.2% (w / v)-1.0% (w / v) BSA, 0.05% (w / v)-0.1% (w / v) NaN3 and PVA 0.05% (w / v)-1.0% (w / v).

[0016] Generally, the particle size of the microspheres affects the sensitivity of the reagent. Small particle size means low sensitivity and a relatively wide linear range. Large particle size means high sensitivity and a relatively narrow linear range. In some embodiments, the microspheres in the microspheres coated with the D-dimer antibody are styrene microspheres with a particle size of 100 to 300 nm. Preferably, the particle size is 150 to 300 nm.

[0017] In the present invention, the preparation of the microspheres coated with the D-dimer antibody can be synthesized by physical adsorption or chemical coupling. In an embodiment of the present invention, the preparation of the microspheres coated with the D-dimer antibody adopts a physical adsorption method, specifically comprising: mixing a solution of the D-dimer antibody with a styrene microsphere suspension, incubating and sealing to obtain microspheres coated with the D-dimer antibody. Among them, the amount of the D-dimer antibody is 0.5 to 1.5 times the saturated adsorption amount of the styrene microspheres. The adsorption buffer is a buffer of pH 7.0 to 8.5, such as 10mM phosphate, 20mM boric acid, 50mMTris or 50mMHEPES, etc. The incubation conditions include 2 to 8°C reaction for 4h to 12h. The blocking agent used in the blocking is a 1% (w / v) BSA solution. Or the blocking solution uses a BSA analog or ethanolamine, etc., and the blocking conditions include 2 to 8°C reaction overnight. The prepared D-dimer antibody-coated microspheres are centrifuged and then resuspended in a storage buffer. In the present invention, the storage buffer is Tris, HEPES, MOPS or phosphate buffer.

[0018] Experiments show that the addition of PVA is beneficial to improving stability. PVA can also be used in combination with polyvidone (PVP), glycerol, hydroxyethyl cellulose, etc. However, compared with the use with other reagents, only adding PVA not only has lower costs, reduces the complexity of the solvent, but also has a better effect in maintaining stability. In the present invention, the PVA is any one of PVA0588, PVA1795, PVA1797, PVA1799, PVA1788, or a combination of two or more. In some embodiments, compared with other PVAs, the use of PVA1788 is more conducive to improving detection stability.

[0019] In some specific embodiments, the R2 reagent is composed of 5 mM MOPS pH 7.0 buffer and the following components: 2 mg / mL D-dimer antibody-coated microspheres, 0.5% (w / v) BSA, 0.1% (w / v) NaN3 and 0.5% (w / v) PVA1788.

[0020] The present invention enhances the signal value by optimizing the formula of R1 and R2 reagents, thereby improving the repeatability of the reagents in detecting low-value samples and improving the stability of the reagents. Experiments show that when the reagents of the present invention are used to detect samples, the repeatability CV value does not exceed 3%, and the reagents remain stable within 36 months. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of testing clinical samples in Example 1 and marketed products - ranking deviation diagram (numerical value) are shown;

[0022] Figure 2Example 1 shows the regression line of passing bablok for clinical samples tested with marketed products;

[0023] Figure 3 The calibration curve results of Example 1 and Comparative Example 1 are shown (showing the signal enhancement effect). DETAILED DESCRIPTION

[0024] The present invention provides an immunoturbidimetric detection reagent for D-dimer, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0025] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0026] Example 1

[0027] R1 reagent: 20mM Tris, 0.15M NaCl, 0.5% BSA, 0.005% TritonX-100, 0.5% cyclodextrin, 0.005% -103, 0.1% NaN3, pH 7.5 adjusted with HCl.

[0028] Table 1 Formula of R1 reagent per liter

[0029]

[0030] R2 reagent: 5 mM MOPS pH 7.0 buffer and the following components: 2 mg / mL D-dimer antibody-coated microspheres, 0.5% (w / v) BSA, 0.1% (w / v) NaN3 and 0.5% (w / v) PVA1788.

[0031] Table 2 R2 storage buffer recipe per liter

[0032] Components Addition amount MOPS 1.0464g BSA 5g PVA1788 5g <![CDATA[NaN3]]> 1g NaOH Appropriate amount (to adjust pH value) Total volume 1L

[0033] Among them, the preparation of microspheres coated with D-dimer antibodies is as follows: take 100 mg of uniformly distributed latex (particle size 200 nm), add adsorption buffer to the latex to 10 mL and mix, and vortex to disperse; dilute 7.14 mg of anti-D-Dimer monoclonal antibody to 10 mL with adsorption buffer; stir the antibody and add the diluted microspheres thereto, and stir gently for 4 hours at 2-8° C.; add 2 mL of 10% BSA solution (BSA is dissolved with adsorption buffer) to the adsorbed latex, that is, the final BSA concentration is 1%; stir overnight at 2-8° C.; transfer the latex to a centrifuge tube, centrifuge at 2-8° C., 15000 rpm, for 25 minutes, and carefully remove the supernatant; add storage buffer to the latex, vortex to disperse, 15000 rpm, 2-8° C., centrifuge for 25 minutes, and remove the supernatant (repeat the washing step with storage buffer once); add storage buffer to the latex, resuspend, and make the volume to 50 mL, and vortex to disperse.

[0034] Note: Adsorption buffer used here: 50 mM Tris-HCl pH 8.0. Blocking solution: 10% (w / v) BSA (prepared with adsorption buffer).

[0035] Comparative Example 1R1 does not contain -103

[0036] R1 reagent: 20 mM Tris, 0.15 M NaCl, 0.5% BSA, 0.005% TritonX-100, 0.5% cyclodextrin, 0.1% NaN3, pH adjusted to 7.5 with HCl.

[0037] Other reagents are the same as in Example 1.

[0038] Comparative Example 2 R2 reagent does not contain PVA1788

[0039] R2 reagent: 5 mM MOPS pH 7.0 buffer and the following components: 2 mg / mL D-dimer antibody-coated microspheres, 0.5% (w / v) BSA, 0.1% (w / v) NaN3.

[0040] Table 3 R2 storage buffer recipe per liter

[0041] Components Addition amount MOPS 1.0464g BSA 5g <![CDATA[NaN3]]> 1g NaOH Appropriate amount (to adjust pH value) Total volume 1L

[0042] Other reagents are the same as in Example 1.

[0043] Effect detection

[0044] 1. Correlation with products already on the market:

[0045] The reagents on the market were used as reference reagents, and the reference reagents were tested on their applicable test systems. The reagents in Example 1 were tested on a UP3000 fully automatic coagulation analyzer. The results of testing 92 clinical samples are shown in Table 4. The test results are analyzed as follows: Figure 1-2 The data fits the passing bablok regression, and the regression result is y=-35.452+1.073x, with a correlation coefficient of γ=0.995. The offset of the two systems at 500 ng / mL is 0.24%. The correlation between the two systems is good, and the offset at the medical decision level is small, so the test results of Example 1 are comparable to those of the reference reagent.

[0046] Table 4 Clinical sample test results

[0047]

[0048]

[0049]

[0050] Note: Absolute deviation = yx; Relative deviation = absolute deviation / average of two system tests × 100%.

[0051] 2. Stability test

[0052] The reagents in Example 1 were stored for 36 months, during which different standard products were sampled and tested for changes in signal values. The testing instrument was a UP3000 fully automatic coagulation analyzer. The results are shown in Table 5:

[0053] Table 5 Stability test results

[0054]

[0055] The results show that the reagent of Example 1 has good stability and the measured signal does not change much when stored at 2-8°C for 36 months.

[0056] 3. Repeatability test

[0057] Two clinical samples were tested with the reagent of Example 1, and the experiment was controlled with Comparative Example 1. The testing instrument was UP3000 fully automatic coagulation analyzer, and the results are shown in Tables 6-7. The results in Table 6 show that both the signal ratio and the signal difference are increased. Table 7 shows that the repeatability is significantly improved. Figure 3 The difference in signal values ​​of the calibration curves was tested for Example 1 and Comparative Example 1. The results showed that the signal value of Example 1 increased significantly.

[0058] Table 6 Signal comparison results

[0059]

[0060] Table 7 Repeatability test results

[0061]

[0062] The results show that, compared with Comparative Example 1, the reagent of Example 1 has better stability.

[0063] 3. Comparative Example 2: PVA1788 was not added, and the R2 reagent showed precipitation as the storage time increased. The results of visual observation are shown in Table 8.

[0064] Table 8 Observation results of R2 reagent status

[0065]

[0066] The above are only preferred embodiments 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 the scope of protection of the present invention.

Claims

1. An immunoturbidimetric detection reagent for D-dimer, characterized in that: Including R1 reagent and R2 reagent: The R1 is composed of water and 20 mM Tris, 0.15 M NaCl, 0.5% (w / v) BSA, 0.005% (w / v) TritonX-100, 0.5% (w / v) cyclodextrin, 0.005% (w / v) Biolipidure®-103, 0.1% (w / v) NaN3, with a pH value of 7.5; The R2 reagent is composed of 5 mM MOPS pH 7.0 buffer and the following components: 2 mg / mL microspheres coated with D-dimer antibody, 0.5% (w / v) BSA, 0.1% (w / v) NaN3 and 0.5% (w / v) PVA1788; The microspheres in the microspheres coated with the D-dimer antibody are styrene microspheres, and the particle size thereof is 200 nm.

2. The detection reagent according to claim 1, characterized in that The preparation of the microspheres coated with the D-dimer antibody adopts a physical adsorption method, which specifically includes: mixing a solution of the D-dimer antibody with a styrene microsphere suspension, incubating and sealing to obtain the microspheres coated with the D-dimer antibody; wherein the amount of the D-dimer antibody is 0.5 to 1.5 times the saturated adsorption amount of the styrene microspheres.

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