Lupus anticoagulant detection kit

Abstract

The invention relates to the technical field of biochemical detection, in particular to a lupus anticoagulant detection kit. The invention provides a lupus anticoagulant detection kit based on a dRVVT method. The lupus anticoagulant detection kit comprises a screening reagent and a confirmation reagent. The screening reagent comprises a buffer solution, bovine cephalin (low concentration), a viper venom activator RVV-X, calcium, a heparin neutralizer, a preservative and a stabilizer. The confirmation reagent comprises a buffer solution, bovine cephalin (high concentration), a viper venom activator RVV-X, calcium, a heparin neutralizer, a preservative and a stabilizer. The reagent is good in sensitivity and more stable after being redissolved, and false negative results possibly caused after sample freezing treatment are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical detection, in particular to a lupus anticoagulant detection kit. Background Art

[0002] Lupus anticoagulants (LA) are phospholipid-dependent pathological circulating anticoagulants, which are heterogeneous immunoglobulins of IgG or IgM. They mainly interfere with the phospholipid-dependent coagulation process by binding to β2-glycoprotein I (β2-GPI), human prothrombin (PT), and other phospholipid complexes, thereby prolonging the coagulation time. LA is named after it was first discovered in patients with systemic lupus erythematosus (SLE). It can be seen in a variety of autoimmune diseases such as antiphospholipid antibody syndrome (APS), SLE, and connective tissue diseases. It is closely related to arterial and venous thrombosis and morbid pregnancy in APS patients. LA detection can not only predict the occurrence of thrombosis, but also determine the prognosis. Therefore, the detection of LA content in plasma has important clinical significance for the early diagnosis of thrombotic diseases, monitoring of the disease course, and monitoring of treatment with thrombolytic drugs. The indications for LA detection mainly include the following categories:

[0003] High-risk patients include: patients under 50 years old with unexplained arteriovenous thrombosis, thrombosis in rare sites, miscarriage in late pregnancy, and patients with autoimmune diseases (SLE, rheumatoid arthritis, autoimmune thrombocytopenia, autoimmune hemolytic anemia, etc.) with thrombosis or morbid pregnancy.

[0004] Moderate-risk patients include those with occasional prolonged APTT, recurrent miscarriage in early pregnancy, and thrombosis in young patients;

[0005] Low-risk patients include: Elderly patients with arteriovenous thrombosis.

[0006] Due to antibody heterogeneity, different coagulation assays can detect different antibody populations in the same patient. The type of lupus anticoagulant assay reagent, phospholipid composition and concentration, different detection principles (such as magnetic beads versus optical methods, scattered light versus transmitted light), and different endpoint determination methods can all affect test results. In recent years, a variety of LA detection methods have been developed, of which the coagulation method is the most widely used. Based on the different coagulation pathways, coagulation tests are divided into the following three pathways: the extrinsic pathway (dilute prothrombin time (dPT), tissue thromboplastin inhibition (TTI), activated seven lupus anticoagulant (ASLA)), the intrinsic pathway (activated partial thromboplastin time (APTT), and APTT-based tests such as kaolin clotting time (KCT), silica clotting time (SCT), platelet neutralization procedure (PNP), and hexagonal phase phospholipid neutralization test (HPNT)), and the shared pathway (dRVVT, ecarin time, and textarin time).

[0007] According to external quality assessment reports from the Royal College of Pathologists of Australasia (RCPA) and the College of American Pathologists (CAP), the intrinsic / common pathway-based aPTT and the common pathway-based modified viper venom phospholipid time (dRVVT) are the two most commonly used tests in clinical laboratories worldwide. EQA (external quality assessment) survey results have found that dRVVT provides more consistent results in LA specimens compared to other tests. In APS screening, dRVVT is more closely associated with thrombosis than kcal CT because it detects the majority of β2GPI-dependent LA antibodies. dRVVT is more specific than aPTT, kcal CT, and dPT because it only involves common pathway factors (coagulation factors X, V, II, and fibrinogen). Therefore, it is insensitive to deficiencies in coagulation cascade factors beyond factor X or specific inhibitors of these factors (such as factors VII, VIII, IX, XI, and contact factors). High levels of factor VIII (eg, pregnancy) may shorten the aPTT result and may mask patients with borderline LA, whereas dRVVT is unaffected.

[0008] The most recent guidelines published by the Clinical and Laboratory Standards Institute (CLSI) in 2014 state that there is currently no "gold standard" (international standard) for evaluating and comparing LA assays. Due to the heterogeneity of LA antibodies, different coagulation assays can detect different antibody populations in the same patient. Therefore, the diagnostic specificity and sensitivity of different assays are particularly important. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a lupus anticoagulant detection kit, which has good stability and good accuracy in detecting frozen samples.

[0010] The lupus anticoagulant detection kit provided by the present invention comprises a screening reagent and a confirmation reagent;

[0011] The screening reagents include: Hepes buffer, bovine cephalin, viper venom activator RVV-X, calcium chloride, heparin neutralizer, preservative, BSA, Tween 80, histidine, serine and aspirin;

[0012] The confirmation reagents include: Hepes buffer, bovine cephalin, viper venom activator RVV-X, calcium chloride, heparin neutralizer, preservative, BSA, Tween 80 and PEG6000.

[0013] The principle of the dRVVT method for detecting lupus anticoagulants is that in the presence of calcium, the RVV-X activator can directly activate factor X, leading to plasma coagulation. The phospholipid content in the screening reagent is low, and the presence of LA prolongs the coagulation time of the screening test. The high concentration of phospholipids in the confirmation reagent can neutralize LA, restoring the coagulation time to normal.

[0014] The screening and confirmation reagents for the dRVVT method generally include a buffer, a phospholipid, a viper venom activator RVV-X, and calcium. The type and concentration of the phospholipids affect the sensitivity of the assay. Commonly used phospholipids in the prior art include animal phospholipids, such as bovine cephalin, and synthetic phospholipids. The ratios of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in these phospholipids vary, leading to differences in sensitivity to lupus anticoagulants. The present invention compared soybean lecithin and bovine cephalin and found that bovine cephalin had greater sensitivity. Because bovine cephalin is rich in phosphatidylserine, it has a better neutralizing effect on antibodies, restoring clotting time to normal and improving the detection sensitivity of positive samples. Preferably, the concentration of bovine cephalin in the screening reagent is 1 to 10 μg / mL, and the concentration of bovine cephalin in the confirmation reagent is 50 to 100 μg / mL.

[0015] The present invention screens buffer solutions. Preferably, the buffer solution is selected from any one or more of a pH 6.5-8.0 Tris (trishydroxymethylaminomethane)-HCl buffer, a Hepes (4-(2-hydroxyethyl)piperazine-1-ylethanesulfonic acid) buffer, and an imidazole buffer. Experimental verification has shown that 20 mM Hepes buffer at pH 7.30 provides the most stable and sensitive buffer solution. To further ensure a certain ionic strength for the buffering effect, a certain concentration of an inorganic salt, such as 100-200 mM NaCl, preferably 150 mM, can be added to the buffer solution.

[0016] Preferably, the concentration of viper venom coagulation factor X activator RVV-X ranges from 1 to 10 mU / mL, with a preferred concentration of 5 mU / mL.

[0017] In the present invention, the source of calcium ions can be calcium gluconate, calcium lactate, or calcium chloride, with a concentration of 10 to 40 mM. Experiments have shown that calcium chloride is more conducive to improving the sensitivity of detection than other calcium sources. Further preferably, 25 mM calcium chloride is used in both the screening reagent and the confirmation reagent.

[0018] The primary function of a heparin anticoagulant is to eliminate interference from heparin in the sample with test results, ensuring test accuracy. In the reagent of the present invention, the heparin neutralizer is 1 to 5 μg / mL of protamine sulfate or polybrene, more preferably 3 μg / mL of protamine sulfate. After selecting an appropriate neutralizer and optimizing its concentration, the reagent of the present invention can detect plasma samples containing up to 1 U / mL of heparin.

[0019] In the present invention, at least one of the screening reagent and the confirmation reagent in the kit further comprises a preservative, which can be 0.1% Proclin 300 or sodium azide, preferably 0.1% sodium azide.

[0020] Because the viper venom factor X activator RVV-X in the kit has poor stability, its stability is enhanced by the use of a protective agent and a surfactant. Experiments screened BSA, sodium alginate, glycine, dextran, ascorbic acid, BHT, or BHA alone showed little stabilizing effect on the kit. However, the addition of a surfactant significantly improved stability. Tween 80 showed the most significant improvement. When used with the aforementioned protective agent in the kit of the present invention, Tween 80 not only stabilized the reagent but also protected it during lyophilization. The most effective combination was BSA and Tween 80, with a preferred concentration of 0.5% BSA and 0.2% Tween 80.

[0021] The confirmation reagent in the kit of the present invention also contains 1% PEG6000. Experiments show that the addition of PEG6000 helps to suspend high-concentration phospholipids, effectively reduces precipitation generated by the confirmation reagent after being placed, and improves the practicality of the reagent.

[0022] In the kit of the present invention, a sensitivity modifier is also added to the screening reagent. After screening numerous components, it was determined that histidine and serine can significantly improve the detection sensitivity of weakly positive samples and reduce the false positive rate of interference samples. Therefore, the screening reagent of the present invention also includes 5mg / mL histidine and 3mg / mL serine as sensitivity modifiers.

[0023] The kit of the present invention also contains a platelet inhibitory agent, which can reduce the activation of platelets that may remain during the freezing and thawing process of the sample, thereby reducing false negative test results. For example, the platelet inhibitor can be at least one of aspirin, clopidogrel, ticagrelor, tirofiban, dipyridamole, or cilostazol. After screening, the present invention selected aspirin as the platelet inhibitor in the screening reagent from the perspectives of both efficacy and composition, with 100 μg / mL aspirin being selected.

[0024] The screening reagents and confirmation reagents provided by the kit provided by the present invention are both lyophilized powders. Due to their rational composition, especially the good coordination of the components in the added composite stabilizer, the stability period after reconstitution is prolonged. The addition of a composite sensitivity modifier to the screening reagent improves the detection sensitivity of both weakly positive and moderately positive samples. The addition of a platelet inhibitor to the screening reagent reduces residual platelet activation caused by the freezing process of some samples, thereby reducing the probability of false negatives. The screening reagent and confirmation reagent are paired for detection simultaneously, which improves the sensitivity and accuracy of the kit detection.

[0025] In some embodiments, the screening reagents include:

[0026] Hepes buffer at pH 6.5-8.0, 1-10 μg / mL bovine cephalin, 1-10 mU / mL viper venom activator RVV-X, 10-40 mM calcium chloride, 1-5 μg / mL protamine sulfate, 0.1 wt% sodium azide, 0.1 wt%-1 wt% BSA, 0.1 wt%-1 wt% Tween 80, 0.1 wt%-1 wt% histidine, 0.1 wt%-1 wt% serine and 80 μg / mL-120 μg / mL aspirin.

[0027] In some specific embodiments, the screening reagent consists of water and the following components:

[0028] 20 mM Hepes buffer, pH 7.3, 5 μg / mL bovine cephalin, 5 mU / mL viper venom activator RVV-X, 25 mM calcium chloride, 10 μg / mL protamine sulfate, 0.1% sodium azide, 0.5% BSA, 0.2% Tween 80, 0.5% histidine, 0.3% serine, and 100 μg / mL aspirin.

[0029] In some embodiments, the confirmation reagent comprises:

[0030] Hepes buffer at pH 6.5-8.0, 50-100 μg / mL bovine cephalin, 1-10 mU / mL viper venom activator RVV-X, 10-40 mM calcium chloride, 1-5 μg / mL protamine sulfate, 0.1 wt% sodium azide, 0.1 wt%-1 wt% BSA, 0.1 wt%-1 wt% Tween 80 and 0.5 wt%-2 wt% PEG6000.

[0031] In some specific embodiments, the confirmation reagent consists of water and the following components:

[0032] 20 mM Hepes buffer at pH 7.3, 80 μg / mL bovine cephalin, 5 mU / mL viper venom activator RVV-X, 25 mM calcium chloride, 3 μg / mL protamine sulfate, 0.1 wt% sodium azide, 0.5% BSA, 0.2% Tween 80 and 1 w% tPEG6000.

[0033] Furthermore, the screening reagent and / or confirmation reagent of the present invention also includes NaCl.

[0034] In some embodiments, the concentration of NaCl in the screening reagent and / or confirmation reagent is 100-200 mM NaCl, preferably 150 mM.

[0035] The present invention also provides a method for detecting dRVVT of lupus anticoagulant, which comprises detecting a sample using the detection kit as described above.

[0036] The test of the present invention includes mixing a blood sample with a screening reagent and recording the coagulation time. If the result is normal, no further testing is performed. If the result is abnormal, the blood sample is mixed with a confirmation reagent and the coagulation time is measured again.

[0037] The present invention provides a lupus anticoagulant detection kit based on the dRVVT method, comprising a screening reagent and a confirmation reagent. The screening reagent comprises: a buffer solution, bovine cephalin (low concentration), a viper venom activator RVV-X, calcium, a heparin neutralizer, a preservative, and a stabilizer. The confirmation reagent comprises: a buffer solution, bovine cephalin (high concentration), a viper venom activator RVV-X, calcium, a heparin neutralizer, a preservative, and a stabilizer. The reagent of the present invention has good sensitivity, is more stable after reconstitution, and reduces the false negative results that may be caused by freezing of the sample. The positive compliance rate is 91.3%, the negative compliance rate is 95.8%, and the total compliance rate is 93.5%, all of which are above 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shows the effect of different amino acids on the sensitivity of the kit;

[0039] Figure 2 The effects of platelet inhibitors on the test results of frozen samples were demonstrated. DETAILED DESCRIPTION

[0040] The present invention provides a lupus anticoagulant detection kit. Those skilled in the art can refer to the contents herein 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. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0041] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0042] The terms "include," "comprising," and "having" are used interchangeably and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include," "comprising," and "having" in this document also provides a "consisting of" solution.

[0043] The term "and / or" describes the relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0044] The numerical ranges and parameters used in this disclosure are presented as precisely as possible in the specific examples. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure are subject to reasonable deviation within a certain range, for example, within ±10%, ±5%, ±1%, or ±0.5%.

[0045] The Examples and Comparative Examples of the present invention describe some examples. These examples illustrate certain implementations of the present invention. However, this does not necessarily mean that the effects of the present invention are achieved only in these examples. In fact, any concentration of the components between the two endpoints shown in the Examples can achieve good stability and good accuracy in detecting frozen samples.

[0046] In addition, each embodiment only shows some cases of comparison between preferred components and components with poor effects. In addition, many attempts were made during the research and development process, that is, there are still many components that were screened out during the research and development, which will not be repeated here.

[0047] The test materials used in the present invention are all common commercial products and can be purchased on the market. It should be understood that in the various embodiments of the present application, the size of the sequence number of each process does not mean the order of execution. Some or all steps can be executed in parallel or in sequence. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application. The present invention is further described below in conjunction with the embodiments:

[0048] Example 1: Effects of different phospholipids on the sensitivity of the kit

[0049] The present invention compares soybean lecithin and bovine cephalin. The results are shown in Table 1. The screening reagent formula is as follows:

[0050] 20 mM HEPES buffer (pH 7.30), 5 μg / mL soybean lecithin / bovine cephalin, 5 mU / mL viper venom activator RVV-X, 25 mM calcium chloride, 3 μg / mL protamine sulfate, 0.5% BSA, 0.2% Tween 80, 0.1% sodium azide;

[0051] Confirm the reagent formula as follows:

[0052] 20 mM HEPES buffer (pH 7.30), 80 μg / mL soybean lecithin / bovine cephalin, 5 mU / mL viper venom activator RVV-X, 25 mM calcium chloride, 3 μg / mL protamine sulfate, 0.5% BSA, 0.2% Tween 80, 0.1% sodium azide, 1% PEG6000;

[0053] Table 1

[0054]

[0055] As shown in Table 1, the bovine cephalin in the confirmation reagent can better bind to the antibodies in the positive sample, restore the coagulation time to normal, and improve the sensitivity of the test. Therefore, the bovine cephalin has a higher detection sensitivity than soybean lecithin.

[0056] Example 2: Effects of different lyoprotectants and stabilizers on the results

[0057] Control group:

[0058] Screening reagents: 20 mM HEPES buffer (pH 7.30), 5 μg / mL bovine cephalin, 5 mU / mL viper venom activator RVV-X, 25 mM calcium chloride, 3 μg / mL protamine sulfate, 0.1% sodium azide;

[0059] Confirmation reagents: 20 mM HEPES buffer (pH 7.30), 80 μg / mL bovine cephalin, 5 mU / mL viper venom activator RVV-X, 25 mM calcium chloride, 3 μg / mL protamine sulfate, 0.1% sodium azide, 1% PEG6000;

[0060] Experimental group 1: control group + 0.5% BSA

[0061] Experimental group 2: control group + 0.2% Tween 80

[0062] Experimental group 3: control group + 0.5% BSA + 0.2% Tween 80

[0063] Testing method: LA negative quality control and LA positive quality control were tested simultaneously. The freeze-drying attenuation results are shown in Table 2, and the stability test results are shown in Table 3.

[0064] Table 2

[0065]

[0066] Table 3

[0067]

[0068] As shown in Table 2, since the attenuation of the LA-positive quality control in the screening reagents before and after lyophilization was large, the addition of 0.5% and 0.2% Tween 20 to the mixture provided better lyophilization protection. In Table 3, the screening and confirmation reagents prepared with 0.5% BSA and 0.2% Tween 80 were reconstituted and placed at room temperature for 3 days. The relative deviations of the LA-negative and LA-positive quality controls were small, both of which were better than the effects of adding a single component. The combination of 0.5% BSA and 0.2% Tween 80 had a good effect on the stability and lyophilization attenuation of the reagents.

[0069] Example 3: Effect of adding different amino acids on the sensitivity of the kit

[0070] Figure 1 The results of the kit of the present invention in which different types and concentrations of amino acids were added to the screening reagent based on the optimized formula of Example 2 were respectively used to detect LA weak-positive and LA-positive samples, and compared with the results of simultaneous detection with the imported kit.

[0071] Depend on Figure 1 It can be seen that the combination of 0.5% histidine and 0.3% serine improves the detection sensitivity of LA weak-positive and LA moderate-strong-positive samples.

[0072] Example 4: Effect of adding platelet inhibitors on the test results of frozen samples

[0073] Based on the screening reagent formula of 0.5% histidine and 0.3% serine in Example 3, different concentrations of platelet activation inhibitor aspirin were added, and the results of the frozen sample test were shown in FIG. Figure 2 :

[0074] Depend on Figure 2 It can be seen that adding aspirin, a platelet activation inhibitor, to the screening reagent improves the detection sensitivity of frozen samples and reduces the false negative rate of sample detection near the critical value. The optimal added concentration of aspirin is 100 μg / mL.

[0075] Example 5: Comparison of clinical sample detection using the kit of the present invention and imported kits

[0076] Based on the reagent formula of 0.5% histidine and 0.3% serine in Example 4, 100 known LA-positive clinical samples, 5 samples treated with oral anticoagulation, and 5 samples treated with heparin were compared with the imported kit. The results are shown in Table 4:

[0077] Table 4

[0078] Kit of the present invention Imported test kits Known LA-positive samples 94％(94 / 100) 90％(90 / 100) Oral Anticoagulation Therapy Sample 0％(0 / 5) 20％(1 / 5) Heparin treatment samples 0％(0 / 5) 40％(2 / 5)

[0079] As shown in Table 4, the diagnostic sensitivity and anti-interference ability of the kit of the present invention are better than those of the imported kit.

[0080] 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. A detection kit for lupus anticoagulant, characterized in that: Includes screening reagents and confirmation reagents; The screening reagents include: Hepes buffer, bovine cephalin, viper venom activator RVV-X, calcium chloride, heparin neutralizer, preservative, BSA, Tween 80, histidine, serine and aspirin; The confirmation reagents include: Hepes buffer, bovine cephalin, viper venom activator RVV-X, calcium chloride, heparin neutralizer, preservative, BSA, Tween 80 and PEG6000.

2. The detection kit according to claim 1, characterized in that The heparin neutralizer is protamine sulfate or polybrene; The preservative is Proclin 300 or sodium azide.

3. The detection kit according to claim 1 or 2, characterized in that The screening reagents include: Hepes buffer at pH 6.5-8.0, 1-10 μg / mL bovine cephalin, 1-10 mU / mL viper venom activator RVV-X, 10-40 mM calcium chloride, 1-5 μg / mL protamine sulfate, 0.1 wt% sodium azide, 0.1 wt%-1 wt% BSA, 0.1 wt%-1 wt% Tween 80, 0.1 wt%-1 wt% histidine, 0.1 wt%-1 wt% serine and 80 μg / mL-120 μg / mL aspirin.

4. The detection kit according to claim 3, characterized in that The screening reagent consists of water and the following components: 20 mM Hepes buffer, pH 7.3, 5 μg / mL bovine cephalin, 5 mU / mL viper venom activator RVV-X, 25 mM calcium chloride, 10 μg / mL protamine sulfate, 0.1% sodium azide, 0.5% BSA, 0.2% Tween 80, 0.5% histidine, 0.3% serine, and 100 μg / mL aspirin.

5. The detection kit according to claim 1 or 2, characterized in that The confirmation reagents include: Hepes buffer at pH 6.5-8.0, 50-100 μg / mL bovine cephalin, 1-10 mU / mL viper venom activator RVV-X, 10-40 mM calcium chloride, 1-5 μg / mL protamine sulfate, 0.1 wt% sodium azide, 0.1 wt%-1 wt% BSA, 0.1 wt%-1 wt% Tween 80 and 0.5 wt%-2 wt% PEG6000.

6. The detection kit according to claim 5, characterized in that The confirmation reagent consists of water and the following components: 20 mM Hepes buffer at pH 7.3, 80 μg / mL bovine cephalin, 5 mU / mL viper venom activator RVV-X, 25 mM calcium chloride, 3 μg / mL protamine sulfate, 0.1 wt% sodium azide, 0.5% BSA, 0.2% Tween 80 and 1 w% t PEG6000.

7. The detection kit according to any one of claims 1 to 6, characterized in that The screening reagent and / or confirmation reagent also includes NaCl.

8. The detection kit according to claim 7, characterized in that In the screening reagent and / or confirmation reagent, the concentration of NaCl is 100-200 mM NaCl.

9. The detection kit according to claim 8, characterized in that The concentration of NaCl was 150 mM.

10. A method for detecting dRVVT of lupus anticoagulant, comprising detecting a sample using the detection kit according to any one of claims 1 to 9.

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