A kit for detecting lupus anticoagulants based on SCT method

Abstract

The present application discloses a kit for detecting lupus anticoagulants based on the SCT method, the kit comprising a screening reagent, a confirmation reagent and a calcium chloride aqueous solution, the screening reagent or the confirmation reagent being composed of silicon dioxide, phospholipids, a buffer, a protective agent, an ionic strength regulator, a heavy metal ion chelator, a preservative and water; the phospholipid content in the screening reagent is lower than the phospholipid content in the confirmation reagent; the calcium chloride aqueous solution contains a heparin neutralizer. The kit of the present application adds a heparin neutralizer to the calcium chloride aqueous solution, forming a kit different from the existing system, the kit has a long stable period, a high positive detection rate, can effectively resist the interference of heparin / low molecular weight heparin and oral anticoagulants, and can be used for screening and confirmation detection of systemic lupus erythematosus and antiphospholipid syndrome diseases.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to a kit for detecting lupus anticoagulants based on the SCT method. Background Art

[0002] Lupus anticoagulation (LA) is a heterogeneous immunoglobulin that can be produced naturally in the body or due to autoimmunity. It is an autoantibody against negatively charged phospholipids and mainly binds to β 2 Glycoprotein I (β 2 -glycoproteinⅠ,β 2 -GPⅠ), human prothrombin (PT) and other negatively charged phospholipid complexes to interfere with the phospholipid-dependent coagulation process and prolong the coagulation time. Lupus anticoagulants are important indicators for the diagnosis, efficacy evaluation and prognosis of autoimmune diseases such as systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS).

[0003] Indications for lupus anticoagulant testing include unexpected prolongation of coagulation screening tests (APTT, PT, TT) and evaluation of patients with arterial and venous thrombosis or morbid pregnancy. According to different coagulation pathways, there are three ways to detect LA: exogenous pathway, intrinsic pathway, and common pathway. The detection methods of the intrinsic pathway include APTT, SCT, KCT, and CSCT; the exogenous pathway includes dPT; and the common pathway includes dRVVT, ECT, and Textarin. In order to improve the detection accuracy and detection rate of LA, two different pathways are usually used. LA detection includes screening tests, mixed tests, and confirmation tests. The basic test principles are: (1) prolongation of phospholipid-dependent coagulation tests; (2) confirmation that the prolongation of coagulation time is not caused by the deficiency of one or more coagulation factors; (3) confirmation that circulating inhibitors are phospholipid-dependent. The screening reagent for LA is a reagent containing a low concentration of phospholipids, and the confirmation reagent is a reagent containing a high concentration of phospholipids. The ratio of the coagulation times obtained by each reagent is used to confirm the presence of LA in the sample to be tested. The screening reagent and confirmation reagent are used in the screening test stage and the confirmation test stage, respectively. The mixing test is to observe whether the prolonged screening time is corrected by mixing healthy normal plasma with the test plasma in a certain ratio (1:1 or 1:4). If the test plasma lacks one or more coagulation factors, the prolonged coagulation time after mixing will be corrected; on the contrary, if anticoagulants such as LA are present in the test plasma, it cannot be corrected.

[0004] According to the different activation pathways and reagent sensitivities, most of the kits used to detect LA on the market are divided into two categories: dRVVT and SCT. Compared with the dRVVT method, the SCT method has higher sensitivity, which can greatly improve the detection accuracy and reduce false negative results. In addition, in order to maintain the long-term stability of the reagents, the dRVVT method is mostly freeze-dried preparations. The freeze-dried powder preparations need to be re-dissolved when used. This process is affected by the quality of the re-dissolution water, the re-dissolution operation process, etc., and it is easy to cause false positives or false negatives in the test results. Therefore, the SCT method has broad prospects in clinical applications.

[0005] According to the CLSI H60-A guideline, LA monitoring should be conducted after the patient stops taking anticoagulants. However, some patients need to take oral anticoagulants or receive heparin / low molecular weight heparin anticoagulant treatment frequently due to their hypercoagulable state. In the process of detecting LA, anticoagulant interference often exists in the samples to be tested. In order to resist the interference of heparin / low molecular weight heparin and oral anticoagulants, heparin neutralizers are added to the existing screening reagents and confirmation reagents. Although adding heparin neutralizers to screening reagents and confirmation reagents can effectively resist the interference of heparin / low molecular weight heparin and oral anticoagulants, the applicant found that the SCT liquid reagent with heparin neutralizer added often has instability problems when conducting relevant experiments, mainly manifested in turbidity and precipitation, which will seriously affect the promotion and use of SCT reagents. Summary of the invention

[0006] In view of the defect of instability of existing SCT liquid reagents, the applicant aims to provide a liquid SCT kit, which has a long stable period, a high positive detection rate, and can effectively resist the interference of heparin / low molecular weight heparin and oral anticoagulants. It can be used for screening and confirmation detection of systemic lupus erythematosus and antiphospholipid syndrome diseases.

[0007] In the process of conducting experiments using the SCT method, the applicant unexpectedly discovered that the heparin neutralizer affects the long-term stability of the SCT liquid reagent. In order to overcome this defect, a large number of single-factor experiments on the components of the kit were conducted, and it was finally found that the components that affect the long-term stability of the reagent are silicon dioxide and phospholipids. Mixing the heparin neutralizer with silicon dioxide or phospholipids in liquid state will produce turbidity and sedimentation. This is because the heparin neutralizer is an adsorbent cationic polymer, and silicon dioxide and phospholipids are both anionic mixtures. When the heparin neutralizer is mixed with silicon dioxide and phospholipids, anionic and cationic polymerization reactions are very likely to occur, resulting in turbidity and sedimentation. In order to prolong the stability of the SCT liquid reagent, the applicant does not add heparin neutralizer to the screening reagent and confirmation reagent. At the same time, in order to make it have a stronger anti-interference ability, the heparin neutralizer is added to the separately packaged calcium chloride solution, and the heparin neutralizer is separated from the screening reagent or confirmation reagent, thereby prolonging its stability and maintaining its anti-interference ability.

[0008] In order to achieve the technical purpose of this application, this application is specifically implemented through the following technical solutions:

[0009] The present application provides a kit for detecting lupus anticoagulants based on the SCT method, the kit comprising a screening reagent, a confirmation reagent and a calcium chloride aqueous solution;

[0010] The screening reagent or confirmation reagent consists of silicon dioxide, phospholipids, a buffer, a protective agent, an ionic strength regulator, a heavy metal ion chelating agent, a preservative and water; the phospholipid content in the screening reagent is lower than that in the confirmation reagent; and the calcium chloride aqueous solution contains a heparin neutralizer.

[0011] The kit of the present application adds a heparin neutralizer to a calcium chloride aqueous solution. During storage, the heparin neutralizer is prevented from directly mixing with phospholipids or silica to produce anionic and cationic polymerization reactions to produce turbidity and sedimentation, thereby ensuring the stability of the screening reagent and confirmation reagent in the kit. When the kit of the present application is used, the sample to be tested is mixed with phospholipids and silica, and then calcium ions are added. With the participation of calcium ions, silica can activate the coagulation factors in the sample, thereby stimulating the coagulation process and observing the time required for the sample to coagulate. The screening reagent is used to detect coagulation experiments containing a small amount of phospholipids, and the confirmation reagent is used to detect coagulation experiments containing excessive phospholipids. When lupus anticoagulants are present, the screening coagulation time is prolonged and the confirmation coagulation time is normal, and the ratio increases.

[0012] As an embodiment of the present application, in order to ensure the uniformity of the calcium chloride aqueous solution and the accuracy of the detection reagent, the calcium chloride aqueous solution is composed of the following components by weight: 3‰ to 5‰ calcium chloride dihydrate, 1% to 3% heparin neutralizer, 0.2‰ to 1‰ preservative, and water makes up the balance.

[0013] In the above scheme, the test value of the quality control plasma of the kit of the present application can fall within the quality control range, and the negative / positive quality control test values ​​are close to the target value, showing extremely high accuracy.

[0014] Preferably, in order to further improve the detection accuracy and stability of the kit, the calcium chloride aqueous solution is composed of the following components by weight: calcium chloride (containing dicrystalline water) 3.6‰, heparin neutralizer 2.5%, preservative 0.8‰, and water makes up the balance.

[0015] As an embodiment of the present application, the screening reagent is composed of the following components by weight: silicon dioxide 0.5‰~1.5‰, phospholipids 0.08‰~0.2‰, buffer 0.5%~3%, protective agent 8%~12%, ionic strength regulator 0.8‰~3‰, heavy metal ion chelator 1.5‰~7.5‰, preservative 0.2‰~1‰, and water makes up the balance.

[0016] Preferably, the screening reagent is composed of the following components by weight: 1‰ silicon dioxide, 0.1‰ phospholipids, 1.5% buffer, 10% protective agent, 1.2‰ ionic strength regulator, 1.8‰ heavy metal ion chelator, 0.8‰ preservative, and water makes up the balance.

[0017] As an embodiment of the present application, the confirmation reagent is composed of the following components by weight: silicon dioxide 0.5‰~1.5‰, phospholipids 0.1‰~8‰, buffer 0.5%~3%, protective agent 8%~12%, ionic strength regulator 0.8‰~3‰, heavy metal ion chelator 1.5‰~7.5‰, preservative 0.2‰~1‰, and water makes up the balance.

[0018] Preferably, the confirmation reagent is composed of the following components by weight: 1‰ silicon dioxide, 6‰ phospholipids, 1.5% buffer, 10% protective agent, 1.2‰ ionic strength regulator, 1.8‰ heavy metal ion chelator, 0.8‰ preservative, and water makes up the balance.

[0019] In the above-mentioned screening reagent and confirmation reagent scheme, the use of protective agents and heavy metal ion chelators can improve the stability of the screening reagent without affecting the sensitivity of the reaction system. Among them, the protective agent can inhibit nonspecific adsorption and aggregation in the reagent solution and reduce the occurrence of precipitation. The heavy metal ion chelator can chelate the positively charged heavy metal ions in the reagent and reduce the polymerization reaction of the heavy metal ions in the solution with the anions and cations of the active ingredients. The combined synergistic effect of the protective agent and the heavy metal ion chelator greatly improves the appearance characteristics and functional stability of the reagent.

[0020] As an embodiment of the present application, the heparin neutralizer is selected from one of polyacrylamide, polycondensation amine or protamine.

[0021] As an embodiment of the present application, the phospholipid is selected from one of rabbit cephalin, egg choline, porcine cephalin, bovine cephalin, soybean lecithin, peanut lecithin or synthetic lecithin.

[0022] As one embodiment of the present application, the buffer is selected from one of Tris buffer, Hepes buffer, PBS buffer, MES buffer, imidazole buffer, MOPS buffer or citric acid buffer.

[0023] As an embodiment of the present application, the pH value of the screening reagent and the confirmation reagent is adjusted to 7.4-7.6 by the buffer.

[0024] As an embodiment of the present application, the protective agent is glycine, which is a non-polar amino acid and does not undergo polymerization reaction with the anionic mixture (silicon dioxide, phospholipids), thereby ensuring the efficacy of the active ingredient and avoiding the occurrence of precipitation.

[0025] As an embodiment of the present application, the ionic strength regulator is selected from NaCl, KCl or MgCl 2 One of the.

[0026] As an embodiment of the present application, the heavy metal ion chelating agent is aluminum chloride.

[0027] As an embodiment of the present application, the preservative is selected from one of sodium azide, sodium benzoate, sodium thimerosal, PC950, potassium sorbate, gentamicin, sodium lactate or nitrite.

[0028] The beneficial effects of this application are:

[0029] The present application provides a kit for detecting lupus anticoagulants based on the SCT method. By adding a heparin neutralizer to an aqueous calcium chloride solution, the anionic and cationic polymerization reaction of the heparin neutralizer with phospholipids and silica is avoided to produce turbidity and precipitation, so as to improve the storage stability of the kit. At the same time, protective agents and heavy metal ion chelators are added to the screening reagents and confirmation reagents, and the stability of the screening reagents and confirmation reagents is further improved through their synergistic effect. Finally, the kit of this application has excellent stability, and the shelf life stability, bottle opening stability, and in-machine stability are all better than similar kits.

[0030] In addition, a reagent system in which a heparin neutralizer is added to a calcium chloride aqueous solution is established to ensure the anti-interference performance of the kit. Experiments have shown that the concentration of anti-ordinary heparin in the kit of the present application can reach 1.2U / mL, the concentration of anti-low molecular weight heparin can reach 2.0U / mL, the concentration of anti-rivaroxaban can reach 160ng / mL, and the concentration of anti-dabigatran can reach 140ng / mL, and the anti-interference performance is better than that of similar kits.

[0031] Therefore, the present application provides a reliable detection method for the clinical diagnosis and treatment of systemic lupus erythematosus and antiphospholipid syndrome diseases, which can achieve import substitution and is conducive to further promotion and use in the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the correlation curves of the shelf life, bottle opening and in-machine stability of the reagent of the present invention and similar commercial reagents;

[0033] Figure 2 It is a schematic diagram of the interference correlation curve between the reagent of the present invention and similar commercial reagents against ordinary heparin, low molecular weight heparin, rivaroxaban and dabigatran. DETAILED DESCRIPTION

[0034] The following will be combined with the specific embodiments of the present application to clearly and completely describe the technical solution of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0035] The present application provides a kit for detecting lupus anticoagulants based on the SCT method, the kit comprising a screening reagent, a confirmation reagent and a calcium chloride aqueous solution. In order to overcome the poor stability of the existing SCT reagents, no heparin neutralizer is added to the screening reagent and confirmation reagent system of the kit, which avoids the anionic and cationic polymerization reaction of the heparin neutralizer with silica and phospholipids, so that the screening reagent and confirmation reagent can be stably stored for a long time. At the same time, in order to make the kit also have a strong anti-interference ability, the applicant adds the heparin neutralizer to the calcium chloride aqueous solution to form a new system for detecting lupus anticoagulants based on the SCT method.

[0036] In a specific embodiment of the present application, the screening reagent is composed of silica, phospholipids, buffers, protective agents, heavy metal ion chelators and water. After the sample to be tested and the screening reagent are mixed, a calcium chloride aqueous solution is added. The phospholipids and silica in the screening reagent can activate the coagulation factors of the endogenous coagulation system of the sample to be tested, and then trigger the coagulation process under the condition of calcium ions, thereby detecting the coagulation time of the sample to be tested. Since the screening reagent contains a small amount of phospholipids, when lupus anticoagulant is present in the sample to be tested, the lupus anticoagulant, as an immunoglobulin, can bind to the phospholipid complex, thereby inhibiting the coagulation mechanism on the phospholipid surface and interfering with the coagulation process dependent on phospholipids, resulting in an extension of the screening coagulation time.

[0037] Among them, adding protective agents to screening reagents can inhibit nonspecific adsorption and aggregation in the reagent solution, slow down the reaction, maintain chemical equilibrium, reduce surface tension, prevent photothermal decomposition or oxidative decomposition, etc., increase the stability of the solution, so that the performance of the screening reagent system is stable for a long time and the accuracy of the detection is improved. Heavy metal ion chelators can chelate heavy metal ions in the reagent system, reduce the polymerization reaction of heavy metal ions in the aqueous solution with the anions and cations of the active ingredients, and also reduce the denaturation of enzymes and other proteins caused by heavy metal ions, thereby improving the stability of the reagent system.

[0038] Preferably, the phospholipid can be one of rabbit cephalin, egg choline, porcine cephalin, bovine cephalin, soybean lecithin, peanut lecithin or synthetic lecithin, etc. More preferably, in this embodiment, the phospholipid is rabbit cephalin, and the applicant has found that rabbit cephalin helps to enhance the stability of the screening reagent and prolong the shelf life.

[0039] Preferably, the protective agent is glycine, which is a non-polar amino acid and does not undergo polymerization reaction with anionic mixtures (silicon dioxide, phospholipids), thereby ensuring the efficacy of the active ingredients and avoiding the occurrence of precipitation. Compared with other protective agents, such as polyethylene glycol 2000, Span-80, BSA, HSA, gelatin, Tween-20, trehalose, glucose, β-cyclodextrin or mannitol, it exhibits good stability.

[0040] Preferably, aluminum chloride is selected as a heavy metal ion chelating agent. Adding aluminum chloride to the screening reagent and confirmation reagent can chelate the heavy metal ions in the reagent, reducing the polymerization reaction between the heavy metal ions contained in the reagent and silicon dioxide and phospholipids, thereby further improving the stability of the screening reagent and confirmation reagent. In addition, the use of aluminum chloride as a chelating agent can prevent the chelating agent from chelating or complexing the ionic strength regulator present in the solution, thereby not affecting the activity of the reagent, and helping to improve the accuracy of the detection.

[0041] In this embodiment, the confirmation reagent system is the same as the screening reagent, except that the phospholipid content in the screening reagent is lower than the phospholipid content in the confirmation reagent.

[0042] Preferably, the weight percentage of the phospholipids in the screening reagent is 0.08‰ to 0.2‰, and the weight percentage of the phospholipids in the confirmation reagent is 0.1‰ to 8‰. More preferably, the weight percentage of the phospholipids in the screening reagent is 0.1‰, and the weight percentage of the phospholipids in the confirmation reagent is 6‰. By setting the phospholipid content of the screening reagent and the confirmation reagent, the detection rate of positive samples is greatly improved, the false negative test results are reduced, and it is helpful for clinical promotion and application.

[0043] In this embodiment, the calcium chloride aqueous solution contains a heparin neutralizer, and the concentration of the heparin neutralizer is 1% to 3%. A detection system for adding a heparin neutralizer to a calcium chloride aqueous solution is established. At this concentration, the kit of the present application has good performance in resisting interference from heparin / low molecular weight heparin, rivaroxaban, and dabigatran, and exhibits excellent anti-interference ability.

[0044] Preferably, the heparin neutralizer may be one of polyacrylamide, polycondensate or protamine.

[0045] In this embodiment, further, a buffer, an ionic strength regulator and a preservative are added to the screening reagent and the confirmation reagent. A preservative is added to the calcium chloride aqueous solution.

[0046] The buffer may be one of Tris buffer, Hepes buffer, PBS buffer, MES buffer, imidazole buffer, MOPS buffer or citric acid buffer. In this embodiment, the buffer is imidazole buffer, and the screening reagent or confirmation reagent is adjusted to a pH value of 7.4 to 7.6 using imidazole buffer, and the reagent system maintains good appearance characteristics and stable performance characteristics.

[0047] The ionic strength regulator can be NaCl, KCl or MgCl 2 In this embodiment, the ionic strength regulator is sodium chloride. By adding the ionic strength regulator, the solution is kept at a high ionic strength. The use of the ionic strength regulator in combination with the imidazole buffer helps to control the pH value, and can also mask interfering ions to a certain extent. At the same time, it is used to stabilize the ion activity coefficient in the solution and improve the accuracy of the detection.

[0048] The preservative can be one of sodium azide, sodium benzoate, sodium thimerosal, PC950, potassium sorbate, gentamicin, sodium lactate or nitrite. In this embodiment, the preservative is PC950. The addition of the preservative can achieve the antiseptic effect on the reagent system, prevent the contamination of the reagent by microorganisms caused by the repeated opening of the reagent cover, and help to extend the shelf life of the reagent kit. It also has a certain effect on the stability of the reagent.

[0049] In this embodiment, the component functional units of the screening reagent and the confirmation reagent are simplified, and each functional unit only includes a single component, namely, a buffer, a protective agent, an ionic strength regulator, a heavy metal ion chelator, and a preservative. For other similar reagents, the component functional units mostly include a buffer, an antifreeze, a stabilizer, a protective agent, a coagulation factor protective agent, a surfactant, an ion regulator, an antioxidant, a preservative, and lysozymes, etc., and each functional unit often contains multiple components. The kit of the present invention simplifies the functional units and corresponding components, simplifies the preparation process, and reduces the production cost.

[0050] In this embodiment, preferably, the screening agent is composed of the following components by weight: silicon dioxide 0.5‰~1.5‰, phospholipid 0.08‰~0.2‰, buffer 0.5%~3%, protective agent 8%~12%, ionic strength regulator 0.8‰~3‰, heavy metal ion chelator 1.5‰~7.5‰, preservative 0.2‰~1‰, and water makes up the balance. More preferably, the screening agent is composed of the following components by weight: silicon dioxide 1‰, phospholipid 0.1‰, buffer 1.5%, protective agent 10%, ionic strength regulator 1.2‰, heavy metal ion chelator 1.8‰, preservative 0.8‰, and water makes up the balance. Most preferably, the screening agent is composed of the following components by weight: 1‰ silicon dioxide, 0.1‰ phospholipids, 1.5% imidazole, 10% glycine, 1.2‰ sodium chloride, 1.8‰ aluminum chloride, 0.8‰ PC950, pH 7.4-7.6, and the balance is water.

[0051] Preferably, the confirmation reagent is composed of the following components by weight: silicon dioxide 0.5‰~1.5‰, phospholipid 0.1‰~8‰, buffer 0.5%~3%, protective agent 8%~12%, ionic strength regulator 0.8‰~3‰, heavy metal ion chelator 1.5‰~7.5‰, preservative 0.2‰~1‰, and water makes up the balance. More preferably, the confirmation reagent is composed of the following components by weight: silicon dioxide 1‰, phospholipid 6‰, buffer 1.5%, protective agent 10%, ionic strength regulator 1.2‰, heavy metal ion chelator 1.8‰, preservative 0.8‰, and water makes up the balance. Most preferably, the confirmation reagent is composed of the following components by weight: 1‰ silicon dioxide, 0.6% phospholipids, 1.5% imidazole, 10% glycine, 1.2‰ sodium chloride, 1.8‰ aluminum chloride, 0.8‰ PC950, pH 7.4-7.6, and the balance is water.

[0052] Preferably, the calcium chloride aqueous solution is composed of the following components by weight: calcium chloride (containing dicrystalline water) 3‰~5‰, heparin neutralizer 1%~3%, preservative 0.2‰~1‰, and water makes up the balance. More preferably, the calcium chloride aqueous solution is composed of the following components by weight: calcium chloride (containing dicrystalline water) 3.6‰, heparin neutralizer 2.5%, preservative 0.8‰, and water makes up the balance. Most preferably, the calcium chloride aqueous solution is composed of the following components by weight: calcium chloride (containing dicrystalline water) 3.6‰, heparin neutralizer 2.5%, PC950 0.8‰, and the balance is water.

[0053] As another specific embodiment of the present application, a method for detecting lupus anticoagulant of the above kit is provided, wherein the volume ratio of the sample to be tested (the mass volume concentration of the component to be tested is 90%) to the activated preparation prepared by the reagent (the mass volume concentration of the reagent provided by the present invention is 4.98%) is 1:1, comprising the following steps:

[0054] ①Prepare the sample for testing: centrifuge the sample at 1500g for 10 minutes, transfer the supernatant, repeat the centrifugation at 1500g for 10 minutes, and keep the supernatant for testing;

[0055] ② Preparation of normal human mixed plasma (NPP): plasma from 40 apparently normal persons (half male and half female, aged 17-70 years old), each sample was centrifuged twice at 1500 g for 10 minutes, the supernatant was collected, and finally the 40 centrifuged plasma samples were mixed.

[0056] ③ Take the sample to be tested and the SCT screening reagent in a volume ratio of 1:1, preheat at 37°C for 180s, and then mix with an equal volume of calcium chloride solution to detect the screening time of the sample to be tested (S t ).

[0057] ④ Mix the sample to be tested with the SCT confirmation reagent in a volume ratio of 1:1, preheat at 37°C for 180s, and then mix with an equal volume of calcium chloride solution to detect the confirmation time of the sample to be tested (C t ).

[0058] ⑤ Mix the NPP and SCT screening reagent in a volume ratio of 1:1, preheat at 37°C for 180s, and then mix with an equal volume of calcium chloride solution to detect the NPP screening time (S nt ).

[0059] ⑥ Mix the NPP and the SCT confirmation reagent in a volume ratio of 1:1, preheat at 37°C for 180s, and then mix with an equal volume of calcium chloride solution to detect the NPP confirmation time (C nt ).

[0060] ⑦Calculation:

[0061] Screening ratio (SR) = screening time of the sample to be tested (S t ) / NPP screening time (S nt );

[0062] Confirmation ratio (CR) = Confirmation time of the sample to be tested (C t ) / NPP confirmation time(C nt );

[0063] Normalized ratio (NR) = screening ratio (SR) / confirmation ratio (CR).

[0064] The technical solution and effects of the present application are further described below in conjunction with specific test kits and experiments.

[0065] Examples 1 to 6

[0066] Add phospholipids, protective agents, ionic strength regulators, heavy metal ion chelating agents, and preservatives to the silica solution and stir evenly, then add a buffer to adjust the pH to 7.4-7.6 and then fix the volume to obtain a screening reagent or a confirmation reagent. Weigh calcium chloride dihydrate, heparin neutralizer, and preservative in water, mix and stir to fix the volume to obtain a calcium chloride aqueous solution. Separately package the quantitative screening reagent, confirmation reagent, and calcium chloride aqueous solution, and assemble to obtain a test kit.

[0067] Table 1 Composition of screening reagents, confirmation reagents and calcium chloride aqueous solution in the kit

[0068]

[0069]

[0070]

[0071] After the reagent systems of Examples 1 to 6 with different concentration ratios were allowed to stand for 72 hours, the corresponding standardized ratios were detected with quality control plasma. The reagents of each embodiment were detected three times and the average value was taken to compare the NR conditions of the reagent systems of different embodiments. The negative quality control selected in this experiment was HemosIL LA Negative Control, with a target value and range of 0.98 (<1.16), and the positive quality control selected was HemosIL LA Positive Control, with a target value and range of 3.20 (≥1.16). The relevant experimental data are shown in Table 2 below.

[0072] Table 2 NR results of quality control blood samples tested with kits of different concentrations in Examples 1 to 6

[0073]

[0074]

[0075] Combining Table 1 and Table 2, it can be seen that after the reagent kit systems of Examples 1 to 6 were left standing for a period of time, the appearance of the reagents all appeared as a uniform solution, indicating that the reagent system of the kit has excellent stability. The detection of quality control plasma showed that the quality control test values ​​of the reagent kit systems of Examples 1 to 6 were all within the quality control range, and the negative / positive quality control test values ​​were close to the target value, indicating that the kit of this application has good accuracy, and also indirectly proves that it has good stability. Among them, the deviation between the test value of the reagent system of Example 1 and the target value is the smallest, indicating that the reagent ratio of Example 1 is the best implementation scheme of this application.

[0076] Example 7 Effect of adding different concentrations of glycine and aluminum chloride on the stability of the reagent system Based on the reagent system of Example 1, experimental groups without adding glycine and / or aluminum chloride and adding different concentrations of glycine and / or aluminum chloride were set up respectively. Three parallel control groups were set up for each experimental group. After standing for 72 hours, the standardized ratio corresponding to the reagent system under different glycine and / or aluminum chloride concentration ratios was detected with quality control plasma, and the average value was taken to compare the NR of the reagent under different glycine and / or aluminum chloride concentration conditions. The negative quality control selected in this experiment is HemosIL LA Negative Control, and the target value and range are 0.98 (<1.16), and the positive quality control selected is HemosIL LA Positive Control, and the target value and range are 3.20 (≥1.16). The experimental results are shown in Table 3 below.

[0077] Table 3 Effect of adding different concentrations of glycine and aluminum chloride on the stability of the reagent system

[0078]

[0079]

[0080]

[0081] As shown in Table 3, the reagents in experimental group 1 did not add glycine and aluminum chloride, and after 72 hours, the appearance of the reagents produced different degrees of precipitation, and when the reagent was used to detect the quality control plasma, the test value exceeded the range of the quality control plasma, which seriously affected the accuracy of the detection reagent. However, after 72 hours of standing, the appearance of experimental groups 2 and 3, which added (8% to 12%) glycine and (1.5‰ to 7.5‰) aluminum chloride, remained good. After testing the quality control plasma, the test values ​​fell within the quality control range, and the negative / positive quality control test values ​​were close to the target value. Experimental group 4 only added amino acids, but no aluminum chloride. After standing for 72 hours, the appearance was slightly turbid, indicating that the addition of aluminum chloride helps to improve the stability of the reagent. This is because aluminum chloride can chelate a small amount of positively charged heavy metal ions in the reagent. Due to the strong chelation of aluminum chloride, the polymerization reaction of heavy metal cations with silica and phospholipids is reduced, thereby achieving the purpose of stabilizing the reagent. The test results of the quality control plasma in Experimental Group 4 showed that the deviation from the target value was smaller than that in Example 1, but larger than that in Examples 2 and 3, which indirectly proved the stabilizing effect of aluminum chloride on the reagent system.

[0082] Example 8 Effect of Heparin Neutralizer on the Stability of Screening Reagents and Confirmation Reagents

[0083] Based on the reagent system of Example 1, experimental groups were set up in which different concentrations of heparin neutralizers were added to the screening reagent, confirmation reagent and calcium chloride solution, and three parallel control groups were set up for each experimental group. After standing for 72 hours, the standardized ratio corresponding to the reagent system under different glycine and / or aluminum chloride concentration ratios was detected with quality control plasma, and the average value was taken to compare the NR of the reagent under different glycine and / or aluminum chloride concentration conditions. The negative quality control selected in this experiment is HemosIL LA Positive Control, and the target value and range are 0.98 (<1.16), and the positive quality control selected is HemosIL LA Positive Control, and the target value and range are 3.20 (≥1.16). The experimental results are shown in Table 4 below.

[0084] Table 4 Effect of heparin neutralizer on the stability of screening reagents and confirmation reagents

[0085]

[0086]

[0087] As can be seen from the data in Table 4, when the heparin neutralizer is added to the screening reagent and the confirmation reagent, an anionic and cationic polymerization reaction will occur to produce precipitation, and the test value of the quality control plasma is not within the quality control range. After (1% to 3%) heparin neutralizer is added to the calcium chloride solution, the appearance of the screening reagent and the confirmation reagent remains uniform, indicating that the heparin neutralizer is the main reason for the precipitation of the screening reagent and the confirmation reagent. This is because the heparin neutralizer is an adsorbent cationic polymer, and silicon dioxide and phospholipids are anionic mixtures. If the above three substances are mixed in the screening reagent and the confirmation reagent, an anionic and cationic polymerization reaction is very likely to occur, resulting in turbidity and sedimentation.

[0088] At the same time, experimental groups 6 and 7 added different amounts of heparin neutralizer to the calcium chloride solution, proving that within the weight content of 1% to 3%, the quality control test results of the kit remained within the quality control range, and the negative / positive quality control test values ​​were close to the target values.

[0089] Example 9 Stability test of the kit of this application

[0090] According to the actual situation of clinical testing, the reagents in Example 1 and commercially available test kits were tested for 29 days of accelerated stability at 37°C, 130 days of refrigeration at 2°C to 8°C after opening the bottle, and 15 days of stability at 16°C after opening the bottle. The test method was to use negative quality control plasma to detect coagulation time. The negative quality control selected in this experiment was HemosIL LA Negative Control. Each test was repeated three times and the average value was taken to compare the stability performance of the reagents in Example 1 and similar commercial reagents. The enterprise standard requires that the test value deviation of the reagent during the monitoring period does not exceed ±2s, and it can be judged as qualified.

[0091] 1) Stability of the reagent during the shelf life simulated by 37℃ accelerated test

[0092] Table 5 Test values ​​of reagents placed at 37°C / 29 days

[0093]

[0094] As shown in Table 5, the deviation of the coagulation time of the reagents in the kit of the present application from the 1st to the 23rd day to the coagulation time of the first day at 37°C was kept within 1s, and the maximum deviation within 29 days was 1.6s; while the deviation of the commercially available screening reagents exceeded 1s on the third day, reached 15.6 on the 23rd day, and the maximum deviation within 29 days was 32.7s. Figure 1 Figure A in the figure proves that the stability of the kit of the present application at 37°C is much better than that of commercially available kits.

[0095] 2) Stability of the simulated reagent after opening the bottle at 2-8°C

[0096] Table 6 Test values ​​of reagents after opening the bottle and placing at 2~8℃ / 130 days

[0097]

[0098] As shown in Table 6, after the reagents of the kit of the present application are opened and stored at 2-8°C, the deviation between the coagulation time on the 130th day and the coagulation time on the first day is kept within 1s, with a maximum deviation of 0.7s; while the deviation of the commercially available screening reagents exceeds 1s on the 30th day, and the maximum deviation within 130 days is 6.4s. Figure 1 In B, it is proved that the stability of the kit of the present application at 2-8°C is much better than that of the commercially available kit.

[0099] 3) Stability of the simulated reagent at 16°C in the machine after opening the bottle

[0100] Table 7 Test values ​​of reagents after opening the bottle and placing at 16℃ / 15 days

[0101]

[0102]

[0103] As shown in Table 7, after the reagents in the kit of the present application are opened and placed in a 16°C incubator, the deviation between the coagulation time on the 15th day and the coagulation time on the first day is kept within 1 second, with a maximum deviation of 0.6 seconds; while the deviation of the commercially available screening reagents exceeds 1 second on the 7th day, and the maximum deviation within 15 days is 6.6 seconds. Figure 1 Figure C shows that the stability of the kit of the present application at 16°C in the machine is much better than that of the commercially available kit.

[0104] In summary, the test results of the reagents of the present application during the monitoring period all met the enterprise standard requirements (the test value was no more than 2.0s before and after), and were comprehensively judged to be qualified. In addition, the test value deviations of the reagents of the present application for 29 days of accelerated stability at 37°C, 130 days of refrigerated stability at 2°C to 8°C after opening the bottle, and 15 days of stability in the machine at 16°C after opening the bottle were all much smaller than the test value deviations of commercially available reagents, that is, the stability of the reagents of the present application is better than that of similar commercially available reagents.

[0105] Example 10 Comparison of the positive sample detection rate of the kit of this application and the commercially available kit The reagent of Example 1 was compared with the commercially available similar reagents for lupus anticoagulant-positive clinical samples to evaluate the response of the corresponding reagent system to lupus anticoagulant. This experiment was divided into a commercially available reagent group and an Example 1 reagent group, and 30 lupus anticoagulant-positive clinical samples were tested, each sample was tested twice, and the mean value of each group of tests was recorded.

[0106] Table 8 Lupus anticoagulant positive detection rate of Example 1 and similar commercial kits

[0107]

[0108]

[0109] As can be seen from Table 8, the detection rate of the reagent in Example 1 for clinically positive samples of lupus anticoagulant is 96.7%, and the correlation (r) with the original clinical value can reach 0.99, which is closer to the original clinical detection value than the detection value of similar commercially available reagents, indicating that the reagent system of the present invention has a good response to lupus anticoagulant and can be used for clinical lupus anticoagulant detection.

[0110] Example 11 Anti-interference ability of the reagent of the present application

[0111] The experimental groups were commercially available reagents and the reagent group of Example 1. Two reagents were randomly selected from each group to detect negative control plasma containing different concentrations of interfering substances, and the effects of different concentrations of interfering substances on the NR of the reagents were compared. Among them, the negative control was HemosIL LA Negative Control, and the target value and range were 0.98 (<1.16). Each reagent was tested twice, and the mean of the test values ​​of each group and the deviation of the mean of each group from the untreated group were calculated, thereby evaluating the anti-interference ability of anticoagulants.

[0112] Table 9 Anti-interference ability of Example 1 and commercially available reagents against anticoagulants

[0113]

[0114]

[0115] As shown in Table 9, the anti-interference ability of the reagent in Example 1 to heparin reaches 1.0 U / mL, the anti-interference ability to low molecular weight heparin reaches 2.0 U / mL, the anti-interference ability to rivaroxaban reaches 160 ng / mL, and the anti-interference ability to dabigatran reaches 140 ng / mL. Figure 2 It can be proved that the kit of the present application has excellent anti-interference ability for ordinary heparin, low molecular weight heparin, rivaroxaban and dabigatran compared with the commercially available kit.

Claims

1. A kit for detecting lupus anticoagulants based on the SCT method, the kit comprising a screening reagent, a confirmation reagent and a calcium chloride aqueous solution, Features: The screening reagent is composed of the following components by weight: 1‰ silicon dioxide, 0.1‰ phospholipid, 1.5% buffer, 10% protective agent, 1.2‰ ionic strength regulator, 1.8‰ heavy metal ion chelator, 0.8‰ preservative, and the balance is made up of water; the confirmation reagent is composed of the following components by weight: 1‰ silicon dioxide, 6‰ phospholipid, 1.5% buffer, 10% protective agent, 1.2‰ ionic strength regulator, 1.8‰ heavy metal ion chelator, 0.8‰ preservative, and the balance is made up of water; the calcium chloride aqueous solution is composed of the following components by weight: 3.6‰ calcium chloride, 2.5% heparin neutralizer, 0.8‰ preservative, and the balance is made up of water; The phospholipids in the screening reagent and the confirmation reagent are rabbit cephalin; The buffer in the screening reagent and the confirmation reagent is an imidazole buffer; The protective agent in the screening reagent and the confirmation reagent is glycine; The ionic strength regulator in the screening reagent and confirmation reagent is sodium chloride; The heavy metal ion chelating agent in the screening reagent and the confirmation reagent is aluminum chloride; The preservative in the screening reagent, confirmation reagent, and calcium chloride aqueous solution is PC950; The heparin neutralizer is one of polyacrylamide, polycondensate and protamine.

2. A kit for detecting lupus anticoagulants based on the SCT method according to claim 1, Features: The pH value of the screening reagent or confirmation reagent is 7.4-7.6.

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