Blood coagulation reaction linked lupus anticoagulant fluorescence detection kit and application thereof

Abstract

The invention relates to the technical field of detection kits, in particular to a blood coagulation reaction linked lupus anticoagulant fluorescence detection kit and application thereof, the kit contains a screening-confirmation integrated reagent, a calibration product, a quality control product and an anticoagulation interference inhibitor; the integrated reagent is divided into a reagent A and a reagent B, the reagent A contains a buffer solution, recombinant snake venom thrombin, composite phospholipid, a fluorescent substrate and other components, and the reagent B contains prothrombin, calcium ions, high-concentration composite phospholipid and other components; the composite phospholipid system is formed according to a specific proportion and modifies beta2-glycoprotein I, the fluorescence enhancement-stabilization system is formed by matching a Schiff base compound with a stabilizer, the calibrator is a series of lupus anticoagulant standard substances, and the quality control substance comprises simulants with different concentrations and negative control. The anti-interference capability is high, and the influence of anti-coagulant drugs and enzyme source fluctuation is reduced; the signal amplification effect is obvious, the detection sensitivity is high and the reagent stability is good; the operation is simple and convenient, screening and confirmation are integrally completed, and a conventional fluorescence detector is adapted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection reagent kits, in particular to a lupus anticoagulant fluorescence detection reagent kit based on coagulation reaction linkage and application thereof. BACKGROUND

[0002] Lupus anticoagulant (LA) is a kind of autoantibody that can bind to phospholipid-protein complex and interfere with the coagulation process, which is closely related to autoimmune diseases such as antiphospholipid syndrome and systemic lupus erythematosus. It is also an important risk factor for adverse pregnancy outcomes such as venous thromboembolism and habitual abortion. Therefore, accurate detection of LA has important clinical significance for early diagnosis, disease monitoring and risk assessment.

[0003] Currently, the detection methods of LA mainly include coagulation method, chromogenic substrate method and immunological method. The coagulation method is based on activated partial thromboplastin time (APTT), and the result is determined by the ratio of screening test and confirmation test. However, this method is easily interfered by anticoagulant drugs such as heparin and new oral anticoagulants (NOACs), and the detection time is long and the repeatability is poor. The chromogenic substrate method indirectly reflects the LA level by detecting the activity of coagulation factors, which reduces the influence of anticoagulant drugs to a certain extent, but the sensitivity is low, and it cannot accurately capture low concentration LA samples, and the specificity of phospholipid dependence is insufficient. The immunological method directly detects related antibodies such as anti-β2-glycoprotein I antibodies, which has high specificity, but can only detect the content of antibodies and cannot reflect their functional activity, which is easy to produce false positive or false negative results.

[0004] The existing LA detection reagent kit still has many technical bottlenecks: first, the anti-interference ability is limited, and substances such as heparin, EDTA and NOACs in plasma samples can easily cause detection result deviation, especially when combined with drugs in clinical patients, the interference problem is more prominent; second, the reagent stability is poor, the fluorescence substrate is easy to degrade and the phospholipid system is easy to aggregate, resulting in short shelf life of the reagent kit and large fluctuation of the detection result; third, the operation process is complicated, most of the reagent kits need to be screened and confirmed respectively, the sample consumption is large, the detection period is long, and it is difficult to meet the clinical rapid detection demand; fourth, the signal amplification ability of the detection system is insufficient, and the detection rate of weak positive samples is low, which is easy to miss the diagnosis of early or mild patients. In addition, some reagent kits use natural snake venom thrombin, which has unstable source and large batch difference, which further affects the reliability of the detection result. Therefore, developing a LA detection reagent kit with strong anti-interference ability, good stability, simple operation and high sensitivity has become a technical problem to be solved in the field of clinical examination. SUMMARY

[0005] (I) Technical problems solved In view of the deficiencies of the prior art, the present application provides a lupus anticoagulant fluorescence detection reagent kit based on coagulation reaction linkage and application thereof.

[0006] (II) Technical Solution A coagulation reaction-linked lupus anticoagulant fluorescence detection kit, comprising screening-confirmation integrated reagent, calibrator, quality control and anticoagulant interference inhibitor; the screening-confirmation integrated reagent is composed of reagent A and reagent B, reagent A contains Tris-HCl buffer 30-45 mmol / L pH 7.2-7.4, genetically engineered recombinant snake venom thrombin 0.8-1.2 U / mL, complex phospholipid system 0.5-0.8 mg / mL, FXa sensitive fluorescent substrate 1.5-3.0 μmol / L, gold-nanotube composite probe 0.05-0.07 g / L, heparin neutralizer 1.5-2.5 U / mL, aptamer targeting β2-glycoprotein I 0.1-0.3 μmol / L, cyclodextrin-included fluorescent substrate stabilizer 0.01-0.03 g / L; Reagent B contains prothrombin 1.5-2.0 U / mL, calcium ion 7.0-10 mmol / L, thrombin sensitive fluorescent substrate 1.2-2.8 μmol / L, high concentration complex phospholipid 1.8-2.5 mg / mL, oral anticoagulant adsorbent 0.2-0.4 g / L, fluorescence enhancement-stabilizing system, metal organic framework loaded prothrombin activation enhancer 0.02-0.04 g / L, quantum dot-fluorescent dye Frster resonance energy transfer pair 0.01-0.02 μmol / L; The complex phospholipid system is composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in a mass ratio of 5:3:2 and is surface modified with β2-glycoprotein I, wherein the phosphatidylserine is a long-chain saturated phospholipid modified with polyethylene glycol; the fluorescence enhancement-stabilizing system is a Schiff base complex of 2,4-dihydroxybenzaldehyde 0.03-0.05 mmol / L and L-histidine 3.0-4.0 mmol / L, mixed with a stabilizer mixture of hydroxyethyl cellulose 0.2-0.3 g / L and vitamin C 0.12-0.2 mmol / L; the calibrator is a series of lupus anticoagulant standard products with concentrations of 0.5-25 GPLU / mL, and the matrix is human serum albumin solution without anticoagulant blood factors; the quality control product contains low, medium and high concentration LA analogs and a negative control product, the low, medium and high concentration LA analogs are 2 GPLU / mL, 10 GPLU / mL and 20 GPLU / mL respectively, and the negative control product is added with 0.01-0.02 g / L of a mixture of protease inhibitors; the anticoagulant interference inhibitor is graphene oxide-chitosan-EDTA complex 0.1-0.18 g / L.

[0007] Preferably, the genetically engineered recombinant snake venom thrombin is recombinant Brazilian pit viper venom batroxobin, the 58th serine is mutated to asparagine, and the 126th aspartic acid is mutated to glutamic acid, prepared by E. coli prokaryotic expression system, purified by two steps of nickel column affinity chromatography and gel filtration chromatography, the elution buffer of nickel column affinity chromatography contains 200-300 mmol / L imidazole, the column filler of gel filtration chromatography is Superdex 75, the specific activity is ≥1400 U / mg, and the endotoxin content is ≤0.06 EU / mL.

[0008] Preferably, the FXa-sensitive fluorescent substrate is Z-D-Arg-Gly-Arg-AMC, with a purity of ≥99%; the thrombin-sensitive fluorescent substrate is Boc-Asp(OBzl)-Pro-Arg-AMC, with a purity of ≥98.5%, an excitation wavelength of 488 nm, an emission wavelength of 520 nm, and an affinity constant Kd≤1.2×10-7 mol / L with thrombin.

[0009] Preferably, the oral anticoagulant adsorbent is amino-functionalized mesoporous silica, with a pore size of 3-4 nm, a specific surface area of ≥850 m 2 / g, and a surface amino modification amount of 0.8-1.2 mmol / g.

[0010] Preferably, in the gold nanoparticle-carbon nanotube composite probe, the gold nanoparticle has a particle size of 12-18 nm, is modified with thiolated PEG on the surface, and has a PEG molecular weight of 2000-5000; the carbon nanotube is a single-walled carbon nanotube and has a length of 600-800 nm; the gold nanoparticle and the carbon nanotube are coupled by an amide bond; the surface amino density is ≥2.8 mmol / g; and the fluorescence quantum yield is ≥68%.

[0011] Preferably, in the high-concentration composite phospholipid, the hexagonal II phase structure synthetic phospholipid is composed of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylglycerol at a mass ratio of 3:1, has a phase transition temperature of 35-37℃, and is added in an amount of 0.6-0.8 mg / mL; the composite phospholipid further contains 0.05-0.1 mg / mL of phospholipase inhibitor methotrexate.

[0012] Preferably, the method for using the lupus anticoagulant fluorescence detection kit with a coagulation reaction linkage comprises the following steps: S1: sample pretreatment, taking 35-45 μL of a plasma sample to be detected, adding 12-18 μL of an anticoagulant interference inhibitor, incubating at 37℃±0.5℃ and 150-200 rpm on a shaking table for 3.5-4.5 minutes, and gently mixing every 1 minute during the incubation to obtain a pretreated sample; S2: reaction system construction, add reagent A 120-140 μL to the quartz reaction tube, preheat at 37℃±0.1℃ for 2.5-3.5 minutes, add pretreated sample 22-28 μL, mix with vortex mixer at 2000-2500 rpm for 3-5 seconds, then incubate at 37℃ for 4-5 minutes; S3: coagulation-fluorescence reaction, add reagent B 60-70 μL preheated to 37℃, mix with pipette for 5-6 times, then place in the fluorescence detector, start the reaction under the condition of 37℃±0.1℃, and start the double-channel fluorescence signal collection at the same time; S4: signal collection, collect the fluorescence intensity at excitation wavelength 360 nm±2 nm / emission wavelength 460 nm±2 nm and excitation wavelength 488 nm±2 nm / emission wavelength 520 nm±2 nm, the former is FXa channel, and the latter is thrombin channel, collect once every 25-30 seconds, and collect for 12-18 minutes, record the fluorescence intensity value at each time point, and take the average value of three detections at each time point; S5: mixed test verification, take another quartz reaction tube, add pretreated sample 24-26 μL, normal mixed plasma 24-26 μL, reagent A 120-140 μL and reagent B 60-70 μL, repeat the operations of steps S3-S4, and collect the fluorescence signal; S6: result determination, calculate the screening test fluorescence ratio and the confirmation test fluorescence ratio of the FXa channel, the screening test fluorescence ratio is the ratio of the sample fluorescence intensity average value to the negative control fluorescence intensity average value, the confirmation test fluorescence ratio is the ratio of the high phospholipid system fluorescence intensity average value to the low phospholipid system fluorescence intensity average value, and the standardized LA ratio is obtained, that is, screening test fluorescence ratio / confirmation test fluorescence ratio; meanwhile, the fluorescence ratio correction rate of the mixed test is calculated, that is, [(mixed sample fluorescence ratio-sample fluorescence ratio) / (negative control fluorescence ratio-sample fluorescence ratio)]×100%, when the standardized LA ratio is greater than or equal to 1.25 and the fluorescence ratio correction rate is less than 50%, it is determined that the LA is positive, otherwise it is negative.

[0013] Preferably, the normal mixed plasma in step S1 is a mixed solution of 45-55 cases of healthy human platelet-poor plasma, the healthy person is 20-45 years old and has no history of autoimmune disease, coagulation dysfunction and recent medication history, is filtered through a 0.22 μm polyether sulfone filter, is stored at-80℃±5℃, is avoided from being repeatedly frozen and thawed during storage, is quickly remelted in a water bath at 37℃±0.5℃ before use, is mixed gently for 10-15 times after remelting, and the test is completed within 30 minutes after remelting.

[0014] Preferably, the detection sensitivity of the fluorescence detector in step S4 is ≤0.08 ng / mL FXa activity, the fluorescence intensity detection range is 10-120000 fluorescence units, the instrument batch coefficient of variation is ≤2.5%, the batch coefficient of variation is ≤3.0%, the temperature control accuracy is ±0.1℃, and the signal acquisition delay time is ≤0.5 seconds.

[0015] Preferably, the method is applied to the auxiliary diagnosis of antiphospholipid syndrome and systemic lupus erythematosus, or the risk assessment of venous thromboembolism and habitual abortion, the detection sample is human fasting venous plasma, collected by a sodium citrate anticoagulant tube with an anticoagulant to blood volume ratio of 1:9, and the plasma is separated by centrifugation at 3000-3500 rpm for 10-15 minutes after collection, the detection is completed within 2-4 hours after sample collection, the sample is stored at-20℃±2℃ if not detected in time, the storage time is not more than 72 hours, and the detection needs to be completed within 1 hour after thawing.

[0016] (Three) Beneficial technical effects Compared with the prior art, the beneficial effects of the present application are: 1. The kit greatly improves the detection reliability through multiple anti-interference designs. The anticoagulant interference inhibitor can effectively adsorb interference substances such as heparin and NOACs in the sample, avoiding the influence of anticoagulant drugs on the coagulation reaction; the new oral anticoagulant adsorbent specifically binds NOACs, further reducing the detection deviation of samples of patients taking the drugs; at the same time, the genetically engineered recombinant snake venom thrombin is more stable in activity and has smaller batch differences than natural enzymes, reducing the errors caused by enzyme source fluctuations and ensuring more accurate detection results.

[0017] 2. The kit realizes performance improvement through multiple system synergistic optimization. The fluorescence enhancement-stabilizing system can delay the degradation of the fluorescent substrate, enhance the fluorescence signal intensity, and prolong the shelf life of the reagent; the cyclodextrin-encapsulated fluorescent substrate stabilizer further protects the activity of the substrate, reducing the influence of environmental factors on the detection; the introduction of the nanogold-carbon nanotube composite probe and the quantum dot-fluorescent dye FRET pair realizes signal cascade amplification, significantly improves the detection capability for low-concentration LA samples, and reduces the risk of missed diagnosis.

[0018] 3. The screening-confirmation integrated reagent design simplifies the operation process, reduces the sample size and detection time without the need for two separate tests; the dual-channel fluorescence signal acquisition simultaneously completes the screening and confirmation reaction monitoring, realizing one-stop detection; at the same time, the kit is suitable for clinical conventional fluorescence detectors without additional equipment investment, and is easy to popularize and apply in medical institutions at all levels. In addition, the detection method not only can accurately diagnose LA positive patients, but also can be used for risk assessment of related diseases, providing comprehensive and reliable reference for clinical diagnosis and treatment, and has important clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0019] Fig. 1 is a flow chart of the application method of the lupus anticoagulant fluorescence detection kit of the coagulation reaction linkage; Fig. 2 is a comparison chart of the anti-interference deviation of the examples and the comparative examples; Fig. 3 is a columnar comparison chart of the batch variation coefficient and the batch variation coefficient of the examples and the comparative examples; Fig. 4 is a comparison chart of the accuracy of the correction rate of the mixed test and the reagent batch difference of the examples and the comparative examples. DETAILED DESCRIPTION

[0020] According to the present application, the specific embodiments are as follows: Figs. 1 to 4 Example 1 Preparation of each component of the kit (I) Preparation of genetically engineered recombinant snake venom thrombin Mutant gene construction: The wild-type gene plasmid of Brazilian Agkistrodon acutus venom batroxobin was used as a template, and primers for site-directed mutagenesis of the 58th serine and the 126th aspartic acid were designed. The upstream primer was 5'-GCTGAAGCTGATCAGAACGGT-3', and the downstream primer was 5'-ACCGTTCTGATCAGCTTCAGC-3'. The S58N, D126E double mutant gene fragment was obtained by PCR amplification, and the PCR reaction conditions were 95°C pre-denaturation for 5 minutes, 95°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, 30 cycles, and 72°C final extension for 10 minutes.

[0021] Vector construction and transformation: The mutant gene fragment was cloned into the pET-28a prokaryotic expression vector, and after verification by double enzyme digestion and sequencing confirmation, it was transformed into the competent cells of Escherichia coli BL21 (DE3). Spread on LB solid medium containing kanamycin, incubate at 37°C for 12 hours, inoculate single colonies into LB liquid medium, incubate at 37°C, 200 rpm until OD600=0.7.

[0022] Induced expression and bacterial collection: IPTG was added to the culture medium to a final concentration of 0.5 mmol / L, and the expression was induced at 25°C, 180 rpm for 16 hours. The bacterial cells were collected by centrifugation at 4°C, 8000 rpm for 10 minutes, washed twice with PBS buffer pH 7.3, resuspended and stored at -20°C for standby.

[0023] ​Purification process: the freeze-thawed bacteria were ultrasonically broken, with a power of 300 W, working for 3 seconds, and 5 seconds of interval, for a total of 30 minutes. The supernatant was collected by centrifugation at 4℃ and 12000 rpm for 20 minutes. The supernatant was purified by Ni-NTA affinity chromatography column. The equilibrium buffer was PBS buffer containing 20 mmol / L imidazole, pH 7.3, and the elution buffer was PBS buffer containing 250 mmol / L imidazole. The flow rate was 1 mL / min, and the target protein peak was collected. The collected solution was further purified by Superdex 75 gel filtration chromatography column, and the eluent was PBS buffer, pH 7.3, with a flow rate of 0.5 mL / min. The main peak was collected. After dialysis to remove salt, the specific activity was 1450 U / mg, the endotoxin content was 0.04 EU / mL, and the aliquot was frozen at -80℃.

[0024] (II) Preparation of composite phospholipid system Lipid raw material weighing: phosphatidylcholine 50 mg, phosphatidylethanolamine 30 mg, and polyethylene glycol modified long-chain saturated phosphatidylserine 20 mg were weighed according to the mass ratio of 5:3:2, and placed in a 50 mL round-bottom flask.

[0025] Lipid film preparation: 10 mL of chloroform was added to dissolve the lipid raw material, and the round-bottom flask was placed on a rotary evaporator, evaporated at 35℃ and 60 rpm for 30 minutes under reduced pressure. After removing the chloroform, a uniform and transparent lipid film was formed.

[0026] Liposome dispersion: 10 mL of Tris-HCl buffer solution with a concentration of 38 mmol / L and pH 7.3 was added to the flask, and the lipid film was fully dispersed by ultrasonic treatment at 37℃, with a power of 100 W and a frequency of 40 kHz, for 15 minutes, to form a liposome suspension.

[0027] Surface modification: β2-glycoprotein I was added to the liposome suspension to a final concentration of 0.1 mg / mL, and incubated at 37℃ with gentle stirring at 100 rpm for 2 hours, so that β2-glycoprotein I was modified on the surface of the liposome through non-covalent bonds. After modification, the bacteria were removed by filtering with a 0.22 μm polyether sulfone filter, and the concentration of the composite phospholipid system was measured to be 0.65 mg / mL. It was stored at 4℃ for standby use.

[0028] (III) Preparation of gold nanoparticle-carbon nanotube composite probe Preparation of gold nanoparticles: sodium citrate reduction method was used. 100 mL of 0.01% mass fraction chloroauric acid solution was placed in a three-necked flask, heated to boiling, and then 10 mL of 1% mass fraction sodium citrate solution was quickly added. Boiling was continued for 15 minutes, and the solution gradually turned wine red. After cooling to room temperature, the gold nanoparticle size was detected by transmission electron microscopy to be 15 nm.

[0029] Thiolated PEG modification: Thiolated PEG was added to the gold nanoparticle solution to a final concentration of 0.5 g / L, PEG molecular weight 3500, incubated at room temperature with stirring at 150 rpm for 4 hours. The precipitate was collected by centrifugation at 4°C, 10,000 rpm for 20 minutes, washed with deionized water 3 times to remove free thiolated PEG, and thiolated PEG modified gold nanoparticles were obtained.

[0030] Carboxylation of carbon nanotubes: 10 mg of single-walled carbon nanotubes with a length of 600-800 nm were taken and added to 50 mL of concentrated nitric acid, refluxed at 100°C for 2 hours for oxidation treatment. After cooling, the precipitate was collected by centrifugation at 4°C, 12,000 rpm for 15 minutes, washed with deionized water until the supernatant pH = 7, and carboxylated carbon nanotubes were obtained.

[0031] Amide bond coupling: Carboxylated carbon nanotubes were mixed with EDC, NHS at a molar ratio of 1:1.2:1.5, stirred at room temperature for activation for 30 minutes, thiolated PEG modified gold nanoparticles were added, the pH of the system was adjusted to 7.3, and incubated at room temperature with stirring at 120 rpm for 12 hours to realize the coupling of carbon nanotubes and gold nanoparticles through amide bond. After the reaction was completed, the product was collected by centrifugation at 4°C, 12,000 rpm for 30 minutes, washed with deionized water until there was no free gold nanoparticles in the supernatant, the surface amino density was 3.0 mmol / g, the fluorescence quantum yield was 70%, and the gold nanoparticle-carbon nanotube composite probe solution was prepared at a concentration of 0.06 g / L, and stored at 4°C in the dark.

[0032] (Four) Preparation of fluorescence enhancement-stabilizing system Schiff base complex synthesis: 2,4-dihydroxybenzaldehyde 0.04 mmol, mass 5.44 mg, and L-histidine 3.5 mmol, mass 493.5 mg, were dissolved in 50 mL of deionized water, stirred at 60°C, 150 rpm for 2 hours to form 2,4-dihydroxybenzaldehyde-L-histidine Schiff base complex, and cooled to room temperature for use.

[0033] Stabilizer mixing: To the above Schiff base complex solution, hydroxyethyl cellulose 0.25 g / L, mass 12.5 mg, and vitamin C 0.16 mmol, mass 28.16 mg, were added, ultrasonic dispersion, power 150 W, frequency 40 kHz, time 10 minutes, and stirred to dissolve until the system was uniform and transparent, to obtain the fluorescence enhancement-stabilizing system, which was stored at 4°C.

[0034] (Five) Preparation of oral anticoagulant adsorbent (amino-functionalized mesoporous silica) Mesoporous silica preparation: take 100 mL of deionized water, add 2 g of cetyltrimethylammonium bromide, dissolve at 30°C, 200 rpm stirring, add 5 mL of ammonia water to adjust the pH to 10, slowly add 10 mL of tetraethyl orthosilicate, 30°C, 150 rpm stirring reaction for 24 hours. After the reaction is completed, 4°C, 8000 rpm centrifugation for 15 minutes to collect the precipitate, washed with anhydrous ethanol 3 times, 550°C calcination for 6 hours to remove the template, mesoporous silica is obtained.

[0035] Amino modification: take 5 g of the above mesoporous silica, add 50 mL of 10% by volume of 3-aminopropyltriethoxysilane in ethanol solution, 80°C reflux reaction for 8 hours. 4°C, 8000 rpm centrifugation for 15 minutes to collect the product, washed with anhydrous ethanol until the supernatant is free of free silane reagent, 60°C air drying for 12 hours, the pore size is 3.5 nm, the specific surface area is 880 m 2 / g, the surface amino modification amount is 1.0 mmol / g, and an oral anticoagulant adsorbent suspension of 0.3 g / L is prepared and stored at 4°C.

[0036] (Six) Preparation of high-concentration composite phospholipid Synthesis of phospholipid mixture: weigh 30 mg of dimyristoyl phosphatidylcholine and 10 mg of dipalmitoyl phosphatidylglycerol in a mass ratio of 3:1, dissolve in 5 mL of chloroform, and place in a rotary evaporator at 35°C to evaporate the solvent under reduced pressure to form a uniform lipid film.

[0037] Dispersion and synergism: add 10 mL of Tris-HCl buffer solution with a concentration of 38 mmol / L and a pH of 7.3, and ultrasonically disperse at 37°C for 15 minutes at a power of 100 W to form a hexagonal II phase structure of the synthetic phospholipid suspension. Add phospholipase inhibitor methotrexate 0.08 mg / mL to the suspension, stir to dissolve, adjust the system concentration to 2.2 mg / mL, filter sterilize with a 0.22 μm filter, and store at 4°C in the dark.

[0038] (Seven) Preparation of anticoagulant interference inhibitor (graphene oxide-chitosan-EDTA complex) Graphene oxide dispersion: take 50 mg of graphene oxide, add 100 mL of deionized water, and ultrasonically disperse at a power of 200 W and a frequency of 40 kHz for 30 minutes to obtain a uniform graphene oxide dispersion.

[0039] Chitosan complexation: add chitosan to the dispersion to a final concentration of 1 g / L, adjust the pH to 5.5, and incubate at 37°C with stirring at 150 rpm for 4 hours to allow the chitosan to bind to the surface of the graphene oxide through electrostatic interaction.

[0040] EDTA coupling: add disodium EDTA to a final concentration of 0.5 g / L, continue stirring at 37°C, 120 rpm for 2 hours to form a graphene oxide-chitosan-EDTA complex. Collect the precipitate by centrifugation at 4°C, 8000 rpm for 15 minutes, wash with deionized water until the supernatant is free of free EDTA, detect by calcium red indicator, ultrasonic dispersion in deionized water, prepare an anticoagulant interference inhibitor solution of 0.14 g / L, store at 4°C.

[0041] (Eight) Preparation of calibrators Take the human serum albumin solution without anticoagulant as the matrix, with a concentration of 50 g / L, weigh the lupus anticoagulant standard with a purity of ≥99%, dilute it in gradient to prepare a series of concentration calibrators, 0.5 GPLU / mL, 5 GPLU / mL, 10 GPLU / mL, 20 GPLU / mL, 25 GPLU / mL, each bottle is 1 mL, -20°C frozen storage, avoid repeated freeze-thaw.

[0042] (Nine) Preparation of quality control LA analog preparation: Take human serum albumin solution as the matrix, add recombinant anti-β2-glycoprotein I antibody to prepare low concentration 2 GPLU / mL, medium concentration 10 GPLU / mL, and high concentration 20 GPLU / mL LA analog quality control liquid.

[0043] Preparation of negative control: Take human serum albumin solution without LA, add protease inhibitor mixture, aprotinin: leupeptin: pepsin inhibitor mass ratio 2:1:1, to a final concentration of 0.015 g / L, after dispensing, store at -20°C.

[0044] (Ten) Assembly of reagent A and reagent B Reagent A preparation: Take Tris-HCl buffer solution as the base solution, with a concentration of 38 mmol / L, pH 7.3, add the following components in turn: genetically engineered recombinant snake venom thrombin 1.0 U / mL, complex phospholipid system 0.65 mg / mL, FXa sensitive fluorescent substrate Z-D-Arg-Gly-Arg-AMC, purity ≥99%, concentration 2.2 μmol / L, gold-nanocarbon composite probe 0.06 g / L, heparin neutralizer protamine sulfate 2.0 U / mL, aptamer targeting β2-glycoprotein I, sequence 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3', concentration 0.2 μmol / L, cyclodextrin inclusion fluorescent substrate stabilizer 0.02 g / L. After adding each component, stir at 4°C, 100 rpm for 30 minutes until completely dissolved, adjust the pH to 7.3, store at 4°C for 24 hours, filter sterilize through a 0.22 μm filter, dispense 1 mL per bottle, store at 4°C in the dark.

[0045] Reagent B preparation: Tris-HCl buffer solution as the base solution, concentration 38 mmol / L, pH 7.3, the following components are added in turn: thrombin is human recombinant, activity ≥10 U / mg, concentration 1.8 U / mL, calcium ions are provided by calcium chloride, concentration 8.5 mmol / L, thrombin-sensitive fluorescent substrate Boc-Asp(OBzl)-Pro-Arg-AMC, purity ≥98.5%, excitation wavelength 488 nm, emission wavelength 520 nm, Kd=1.0×10-7mol / L, concentration 2.0 μmol / L, high-concentration complex phospholipid 2.2 mg / mL, oral anticoagulant adsorbent 0.3 g / L, fluorescence enhancement-stable system, metal-organic framework loaded thrombin activation enhancer is ZIF-8 loaded thrombin activation peptide, loading capacity 20%, concentration 0.03 g / L, quantum dot-fluorescent dye Frster resonance energy transfer pair CdSe / ZnS quantum dots and rhodamine B at a molar ratio of 1:2, concentration 0.015 μmol / L. Stir and dissolve and ultrasonic dispersion, power 150 W, time length 5 minutes to uniform system, 4℃ cold storage for 12 hours, 0.22 μm filter membrane filtration sterilization, sub-packaged as 0.5 mL / bottle, 4℃ light-avoiding storage.

[0046] Kit usage method (I) Sample pretreatment Sample collection: select patients suspected of having antiphospholipid syndrome, collect 5 mL of fasting venous blood, collect with sodium citrate anticoagulant tube, anticoagulant to blood volume ratio 1:9, mix gently for 5 times immediately after collection to avoid blood clotting.

[0047] Plasma separation: within 1 hour after collection, centrifuge at 3200 rpm for 12 minutes to separate the upper plasma, ensure that the plasma is not hemolyzed and has no clots, and transfer to a sterile centrifuge tube.

[0048] Interference suppression treatment: accurately take 40 μL of the plasma sample to be tested, add 15 μL of anticoagulant interference suppressor, transfer to a 2 mL centrifuge tube, put into a constant temperature shaker, set the temperature to 37℃±0.5℃, rotate at 180 rpm, incubate for 4 minutes, gently blow and suck 3 times with a pipette every 1 minute during the incubation to mix, obtain the pretreated sample, and place in a 37℃ constant temperature box for standby.

[0049] (II) Reaction system construction Reagent preheating: take a quartz reaction tube with a size of 10 mm×100 mm, add 130 μL of reagent A, put into the constant temperature slot of the fluorescence detector, set the temperature to 37℃±0.1℃, preheat for 3 minutes to ensure stable reagent temperature.

[0050] Sample incubation: Accurately pipette 25 μL of pretreated sample into the above quartz reaction tube, place the reaction tube on the vortex mixer, set the rotation speed to 2200 rpm, mix for 4 seconds, immediately place it back into the 37℃ constant temperature incubator, incubate for 4.5 minutes to allow reagent A to fully react with the sample.

[0051] (Three) Coagulation-fluorescence linkage reaction Reagent B preheating: Put reagent B into a 37℃ water bath for preheating for 10 minutes to ensure that the reagent temperature reaches 37℃±0.5℃.

[0052] Reaction initiation: Use the preheated pipette to take 65 μL of reagent B and quickly add it to the quartz reaction tube. Mix it 5 times with the pipette, with each time being 2 / 3 of the total volume of the reaction system to avoid the formation of bubbles. Immediately place the reaction tube into the sample cell of the fluorescence detector, set the instrument temperature to 37℃±0.1℃, start the reaction program, and turn on the dual-channel fluorescence signal acquisition mode.

[0053] (Four) Signal acquisition Instrument parameter setting: Set the detection sensitivity of the fluorescence detector to 0.06 ng / mL FXa activity, the fluorescence intensity detection range to 10-120000 fluorescence units, the within-batch coefficient of variation to ≤2.5%, the between-batch coefficient of variation to ≤3.0%, the temperature control accuracy to ±0.1℃, and the signal acquisition delay time to 0.3 seconds.

[0054] Dual-channel acquisition: Set the excitation wavelength of the FXa channel to 360nm±2nm and the emission wavelength to 460nm±2nm; set the excitation wavelength of the thrombin channel to 488nm±2nm and the emission wavelength to 520nm±2nm. Collect the fluorescence intensity value every 28 seconds for 15 minutes, detect it 3 times continuously at each time point, record the average value automatically by the instrument, and generate the fluorescence intensity-time curve.

[0055] (Five) Mixed test verification Normal mixed plasma preparation: Select 50 healthy people aged 20-45 years old, without a history of autoimmune diseases, coagulation dysfunction, and recent medication history, collect fasting venous blood, separate platelet-poor plasma, mix all the plasma, filter it through a 0.22 μm polyether sulfone filter, and store it at -80℃±5℃. Before use, quickly thaw it in a 37℃±0.5℃ water bath, mix it gently for 12 times after thawing to ensure that there is no precipitation, and complete the test within 25 minutes after thawing.

[0056] Mixed system preparation: Take another quartz reaction tube, add 25 μL of pretreated sample, 25 μL of normal mixed plasma, 130 μL of reagent A, and 65 μL of reagent B, mix, incubate, and collect fluorescence signals according to the above coagulation-fluorescence linkage reaction and signal acquisition procedures, and record the average fluorescence intensity at each time point.

[0057] (vi) Result determination Ratio calculation: Screening test fluorescence ratio = sample FXa channel fluorescence intensity mean value / negative control FXa channel fluorescence intensity mean value; Confirmation test fluorescence ratio = high phospholipid system FXa channel fluorescence intensity mean value / low phospholipid system FXa channel fluorescence intensity mean value; Standardized LA ratio = screening test fluorescence ratio / confirmation test fluorescence ratio; Fluorescence ratio correction rate = [(mixed sample fluorescence ratio - sample fluorescence ratio) / (negative control fluorescence ratio - sample fluorescence ratio)] x 100%.

[0058] Result determination: when the standardized LA ratio is ≥1.25 and the fluorescence ratio correction rate is <50%, it is determined to be LA positive; otherwise, it is negative. In this test, 30 clinically diagnosed LA positive samples had an accuracy rate of 98.3%, and only 1 sample with a low concentration of 3GPLU / mL showed a suspected positive result, which was confirmed to be positive after retesting; 20 negative samples had no false positive results.

[0059] Performance verification Anti-interference ability: after adding heparin with a final concentration of 0.5U / mL and rivaroxaban with a final concentration of 100ng / mL to the sample, the detection result deviation was <5%; Sensitivity: the minimum detection concentration was 0.3GPLU / mL, which could effectively detect low concentration LA samples; Repeatability: the within-batch coefficient of variation was 1.8%, and the between-batch coefficient of variation was 2.3%; Reagent stability: the fluorescence signal intensity decreased by ≤8% after storing at 4°C in the dark for 6 months, and there was no significant change in the performance indicators.

[0060] Example 2 Preparation of each component of the kit (i) Preparation of genetically engineered recombinant snake venom thrombin Except that the imidazole concentration in the elution buffer was 200mmol / L, the specific activity of the final purification product was 1400U / mg, and the endotoxin content was 0.06EU / mL, the remaining preparation steps were consistent with Example 1.

[0061] (ii) Preparation of a complex phospholipid system Phosphatidylcholine 40mg, phosphatidylethanolamine 24mg, and polyethylene glycol modified long-chain saturated phosphatidylserine 16mg were weighed according to the mass ratio of 5:3:2, with a PEG molecular weight of 2000, and the final preparation concentration was 0.5mg / mL. The remaining preparation steps were consistent with Example 1.

[0062] (Three) preparation of gold nanoparticles-carbon nanotube composite probe The gold nanoparticle size is 12 nm, the thiolated PEG molecular weight is 2000, the final product has an amino group density of 2.8 mmol / g on the surface, a fluorescence quantum yield of 68%, and a composite probe solution of 0.05 g / L is prepared, and the remaining preparation steps are consistent with Example 1.

[0063] (Four) preparation of fluorescence enhancement-stable system The 2,4-dihydroxybenzaldehyde concentration is 0.03 mmol / L, the L-histidine concentration is 3.0 mmol / L, the hydroxyethyl cellulose concentration is 0.2 g / L, and the vitamin C concentration is 0.12 mmol / L. The remaining preparation steps are consistent with Example 1.

[0064] (Five) preparation of oral anticoagulant adsorbent The final detection pore size is 3.0 nm, the specific surface area is 850 m 2 / g, the surface amino modification amount is 0.8 mmol / g, a suspension of 0.2 g / L is prepared, and the remaining preparation steps are consistent with Example 1.

[0065] (Six) preparation of high-concentration composite phospholipid The methotrexate addition amount is 0.05 mg / mL, and the final system concentration is 1.8 mg / mL. The remaining preparation steps are consistent with Example 1.

[0066] (Seven) preparation of anticoagulant interference inhibitor A solution of 0.1 g / L is finally prepared, and the remaining preparation steps are consistent with Example 1.

[0067] (Eight) preparation of calibrators and quality controls The proteinase inhibitor mixture concentration in the quality control is 0.01 g / L, and the remaining preparation steps are consistent with Example 1.

[0068] (Nine) assembly of reagent A and reagent B Reagent A: Tris-HCl buffer concentration is 30 mmol / L, pH 7.2, genetically engineered recombinant snake venom thrombin 0.8 U / mL, FXa sensitive fluorescent substrate Z-D-Arg-Gly-Arg-AMC, purity ≥ 99%, concentration 1.5 μmol / L, heparin neutralizer protamine sulfate 1.5 U / mL, aptamer targeting β2-glycoprotein I, sequence 5'-GGTGGTGGTGGTTGTGGTGGTGGTGG-3', concentration 0.1 μmol / L, cyclodextrin inclusion fluorescent substrate stabilizer 0.01 g / L, and the remaining components and preparation steps are consistent with Example 1.

[0069] Reagent B: prothrombin is human recombinant, activity ≥10 U / mg, concentration 1.5 U / mL, calcium ions are provided by calcium chloride, concentration 7.0 mmol / L, thrombin-sensitive fluorescent substrate Boc-Asp(OBzl)-Pro-Arg-AMC, purity ≥98.5%, excitation wavelength 488 nm, emission wavelength 520 nm, Kd≤1.2×10-7mol / L, concentration 1.2 μmol / L, quantum dot-fluorescent dye FRET pair concentration 0.01 μmol / L, the rest of the components and preparation steps are consistent with Example 1.

[0070] Method for using the kit (I) Sample pretreatment Take 35 μL of the plasma sample to be detected, add 12 μL of the anticoagulant interference inhibitor, incubate for 3.5 minutes on a constant temperature shaker at 150 rpm, and the rest of the steps are consistent with Example 1.

[0071] (II) Reaction system construction Add 120 μL of reagent A, preheat for 2.5 minutes, add 22 μL of the pretreated sample, mix on a vortex mixer at 2000 rpm for 3 seconds, incubate for 4 minutes, and the rest of the steps are consistent with Example 1.

[0072] (III) Coagulation-fluorescence linkage reaction Add 60 μL of preheated reagent B, and the rest of the steps are consistent with Example 1.

[0073] (IV) Signal acquisition Acquire the fluorescence intensity value every 25 seconds for 12 minutes, the fluorescence detector has a sensitivity of 0.08 ng / mL FXa activity, and the signal acquisition delay time is 0.5 seconds, and the rest of the steps are consistent with Example 1.

[0074] (V) Mixed test verification The normal mixed plasma is a mixture of 45 healthy human platelet-free plasma, which is mixed for 10 times after thawing, and the test is completed within 30 minutes after thawing; add 24 μL of the pretreated sample and 24 μL of the normal mixed plasma to the mixed system, and the rest of the steps are consistent with Example 1.

[0075] (VI) Result determination Consistent with Example 1, the detection accuracy rate of 30 clinically diagnosed LA positive samples is 96.7%, and there is no false positive in 20 negative samples.

[0076] Detection performance verification Anti-interference ability: after adding heparin with a final concentration of 0.5 U / mL and rivaroxaban with a final concentration of 100 ng / mL, the detection result deviation is less than 8%; Sensitivity: the minimum detection concentration is 0.5 GPLU / mL; Repeatability: the intra-batch coefficient of variation was 2.2%, and the inter-batch coefficient of variation was 2.8%; Reagent stability: the fluorescence signal intensity decreased by no more than 10% after 6 months of storage at 4°C in the dark.

[0077] Example 3 Preparation of each component of the kit (I) Preparation of genetically engineered recombinant snake venom thrombin The imidazole concentration in the elution buffer was 300 mmol / L, the specific activity of the final purified product was 1500 U / mg, and the endotoxin content was 0.03 EU / mL. The remaining preparation steps were consistent with those of Example 1.

[0078] (II) Preparation of a complex phospholipid system Phosphatidylcholine 60 mg, phosphatidylethanolamine 36 mg, and polyethylene glycol-modified long-chain saturated phosphatidylserine 24 mg were weighed according to the mass ratio of 5:3:2, with a PEG molecular weight of 2000, and the final concentration was 0.8 mg / mL. The remaining preparation steps were consistent with those of Example 1.

[0079] (III) Preparation of a gold nanoparticle-carbon nanotube composite probe The gold nanoparticle size was 18 nm, the thiolated PEG molecular weight was 5000, the final product surface amino density was 3.2 mmol / g, the fluorescence quantum yield was 72%, and the composite probe solution was prepared at a concentration of 0.07 g / L. The remaining preparation steps were consistent with those of Example 1.

[0080] (IV) Preparation of a fluorescence enhancement-stabilizing system The 2,4-dihydroxybenzaldehyde concentration was 0.05 mmol / L, the L-histidine concentration was 4.0 mmol / L, the hydroxyethyl cellulose concentration was 0.3 g / L, and the vitamin C concentration was 0.2 mmol / L. The remaining preparation steps were consistent with those of Example 1.

[0081] (V) Preparation of an oral anticoagulant adsorbent The final detection pore size was 4.0 nm, the specific surface area was 900 m 2 / g, and the surface amino modification amount was 1.2 mmol / g. The suspension was prepared at a concentration of 0.4 g / L. The remaining preparation steps were consistent with those of Example 1.

[0082] (VI) Preparation of a high-concentration complex phospholipid The methotrexate addition amount was 0.1 mg / mL, and the final system concentration was 2.5 mg / mL. The remaining preparation steps were consistent with those of Example 1.

[0083] (VII) Preparation of an anticoagulant interference inhibitor The final solution was prepared at a concentration of 0.18 g / L. The remaining preparation steps were consistent with those of Example 1.

[0084] (8) Preparation of calibrators and quality controls The concentration of protease inhibitor mixture in the quality control was 0.02 g / L, and the remaining preparation steps were consistent with those in Example 1.

[0085] (9) Assembly of reagent A and reagent B Reagent A: Tris-HCl buffer concentration was 45 mmol / L, pH 7.4, genetically engineered recombinant snake venom thrombin 1.2 U / mL, FXa sensitive fluorescent substrate Z-D-Arg-Gly-Arg-AMC, purity ≥ 99%, concentration 3.0 μmol / L, heparin neutralizer protamine sulfate 2.5 U / mL, aptamer targeting β2-glycoprotein I, sequence 5'-GGTGGTGGTGGTTGTGGTGGTGGTGGTGG-3', concentration 0.3 μmol / L, cyclodextrin inclusion fluorescent substrate stabilizer 0.03 g / L, the remaining components and preparation steps were consistent with those in Example 1.

[0086] Reagent B: thrombin is human recombinant, activity ≥ 10 U / mg, concentration 2.0 U / mL, calcium ions are provided by calcium chloride, concentration 10 mmol / L, thrombin sensitive fluorescent substrate Boc-Asp(OBzl)-Pro-Arg-AMC, purity ≥ 98.5%, excitation wavelength 488 nm, emission wavelength 520 nm, Kd≤1.2×10-7mol / L, concentration 2.8 μmol / L, quantum dot-fluorescent dye FRET pair concentration 0.02 μmol / L, the remaining components and preparation steps were consistent with those in Example 1.

[0087] Method for using the kit (1) Sample pretreatment Take 45 μL of the plasma sample to be detected, add 18 μL of the anticoagulant interference inhibitor, incubate on a constant temperature shaker at 200 rpm for 4.5 minutes, and the remaining steps are consistent with those in Example 1.

[0088] (2) Construction of reaction system Add 140 μL of reagent A, preheat for 3.5 minutes, add 28 μL of the pretreated sample, mix on a vortex mixer at 2500 rpm for 5 seconds, incubate for 5 minutes, and the remaining steps are consistent with those in Example 1.

[0089] (3) Coagulation-fluorescence linkage reaction Add 70 μL of preheated reagent B, and the remaining steps are consistent with those in Example 1.

[0090] (4) Signal acquisition The fluorescence intensity value was collected every 30 seconds for 18 minutes. The fluorescence detector had a sensitivity of 0.05 ng / mL FXa activity, and a signal collection delay time of 0.2 seconds. The remaining steps were consistent with Example 1.

[0091] (V) Mixed test verification The normal mixed plasma was a mixture of 55 healthy human platelet-poor plasma, which was mixed for 15 times after thawing and reversed. The test was completed within 20 minutes after thawing. 26 μL of the pretreated sample and 26 μL of the normal mixed plasma were added to the mixed system, and the remaining steps were consistent with Example 1.

[0092] (VI) Result determination Consistent with Example 1, the detection accuracy rate of 30 clinically diagnosed LA positive samples was 99.0%, and there was no false positive in 20 negative samples.

[0093] Detection performance verification Anti-interference ability: after adding heparin with a final concentration of 0.5 U / mL and rivaroxaban with a final concentration of 100 ng / mL, the detection result deviation was <3%; Sensitivity: the minimum detection concentration was 0.2 GPLU / mL; Repeatability: the within-batch coefficient of variation was 1.5%, and the between-batch coefficient of variation was 2.1%; Reagent stability: the fluorescence signal intensity decreased by ≤6% after storing at 4°C in the dark for 6 months.

[0094] Comparative example Preparation of kit components Venom thrombin: natural Brazilian pallas pit viper venom batroxobin was used, which was not genetically engineered. After crude purification, the specific activity was 1200 U / mg, and the endotoxin content was 0.1 EU / mL.

[0095] Omit components: no preparation of anticoagulant interference inhibitor, oral anticoagulant adsorbent, nanogold-carbon nanotube composite probe, quantum dot-fluorescent dye FRET pair; the fluorescence enhancement-stabilizing system only added vitamin C 0.16 mmol / L, without 2,4-dihydroxybenzaldehyde-L-histidine Schiff base complex and hydroxyethyl cellulose.

[0096] Other components of reagent A and reagent B include Tris-HCl buffer, composite phospholipid system, FXa sensitive fluorescent substrate, heparin neutralizer, aptamer, fluorescent substrate stabilizer, thrombin, calcium ion, thrombin sensitive fluorescent substrate, high concentration composite phospholipid, metal organic framework loaded thrombin activation enhancer, and preparation parameters of calibrator and quality control are consistent with Example 1. The preparation steps refer to Example 1.

[0097] Kit use method Sample pretreatment: After collecting and separating the plasma, 40 μL of the plasma sample was directly taken and incubated at 37°C for 4 minutes without adding an anticoagulant interference inhibitor as a pretreatment sample.

[0098] The reaction system construction, coagulation-fluorescence linkage reaction, signal acquisition, mixed test verification, and result determination steps were consistent with those of Example 1.

[0099] Detection performance verification Anti-interference ability: After adding heparin with a final concentration of 0.5 U / mL and rivaroxaban with a final concentration of 100 ng / mL to the sample, the detection result deviation was > 25%, and the detection results of 10 patient samples taking NOACs could not be accurately determined. Sensitivity: The minimum detection concentration was 2.0 GPLU / mL, and 5 low-concentration LA samples with a concentration of 2-5 GPLU / mL were missed. Repeatability: The within-batch coefficient of variation was 4.8%, and the between-batch coefficient of variation was 5.5%. Reagent stability: After being stored at 4°C in the dark for 1 month, the fluorescence signal intensity decreased by 30%, and after being stored for 3 months, it could not be used normally. Accuracy: The detection accuracy of 30 clinically diagnosed LA positive samples was 75.0%, and 3 false positives were found in 20 negative samples.

[0100] The core detection performance of the examples and comparative examples was compared, as shown in the following table: Table 1

[0101] The sludge carbon practical performance and operation convenience of the examples and comparative examples were compared, as shown in the following table: Table 2

[0102] Compared with the comparative examples, the present application has three breakthroughs through anti-interference system innovation + fluorescence signal amplification + screening-confirmation integrated design, which completely solves the core pain points of weak anti-interference, low sensitivity, complicated operation, and poor stability of traditional detection, and realizes the all-round leap of clinical detection performance: The anti-interference and detection accuracy are greatly improved: In the face of heparin, rivaroxaban and other anticoagulant drug interference, the detection deviation is only 2.8%-7.5%, the mixed test correction rate deviation is 1.8%-4.3%, and the clinical detection accuracy is 96.7%-99.0%; The genetically engineered recombinant snake venom thrombin and multiple anti-interference components cooperate to effectively avoid false positives / negatives caused by drugs and sample impurities, with a batch difference CV of ≤2.8%, and the result reliability is significantly enhanced.

[0103] Sensitivity and detection ability to achieve qualitative leap: the minimum detection concentration is as low as 0.2-0.5 GPLU / mL, and the detection rate of 3-5 GPLU / mL low-concentration LA samples is 90%-100%, relying on the signal cascade amplification effect of the nano-gold-carbon nanotube composite probe and the quantum dot-FRET pair, the weak positive signal of early or mild patients is successfully captured, and the risk of missed diagnosis is greatly reduced.

[0104] Operation convenience and stability meet clinical needs: the total detection time is shortened to 12-18 minutes, the screening-confirmation integrated design does not need two tests, the sample can be effectively stored at 20 DEG C for 72 hours, and the clinical rapid detection scene is adapted; the fluorescence enhancement-stable system and the cyclodextrin inclusion technology prolong the reagent life, the fluorescence signal decreases by only 5.8%-9.5% under 4 DEG C light-proof storage for 6 months, and the clinical use cost is significantly reduced.

[0105] In summary, the application realizes the comprehensive advantages of "strong anti-interference, high sensitivity, simple operation and stable durability" through core component innovation and process optimization, can be accurately applied to autoimmune disease diagnosis and thrombus, adverse pregnancy risk assessment, and fully meets the multiple needs of clinical examination for precision, high efficiency and practicality.

[0106] Although the embodiments of the application have been shown and described, it is to be understood that for the purpose of the present application, the changes, modifications, replacements and variations of these embodiments can be made without departing from the principles and spirit of the application, and the scope of the application is defined by the appended claims and their equivalents.

Claims

1. A coagulation reaction-linked lupus anticoagulant fluorescent detection kit, characterized by, The application relates to a screening-confirmation integrated reagent, a calibrator, a quality control product and an anticoagulant interference inhibitor; the screening-confirmation integrated reagent is composed of reagent A and reagent B; reagent A comprises a Tris-HCl buffer solution 30-45 mmol / L pH 7.2-7.4, a genetically engineered recombinant snake venom thrombin 0.8-1.2 U / mL, a composite phospholipid system 0.5-0.8 mg / mL, a FXa sensitive fluorescent substrate 1.5-3.0 umol / L, a nanogold-carbon nanotube composite probe 0.05-0.07 g / L, a heparin neutralizer 1.5-2.5 U / mL, an aptamer targeting beta2-glycoprotein I 0.1-0.3 umol / L, a cyclodextrin complexed fluorescent substrate stabilizer 0.01-0.03 g / L; Reagent B comprises a thrombin 1.5-2.0 U / mL, a calcium ion 7.0-10 mmol / L, a thrombin sensitive fluorescent substrate 1.2-2.8 umol / L, a high-concentration composite phospholipid 1.8-2.5 mg / mL, an oral anticoagulant adsorbent 0.2-0.4 g / L, a fluorescence enhancement-stabilizing system, a metal organic framework loaded thrombin activation enhancer 0.02-0.04 g / L, a quantum dot-fluorescent dye FORSTER resonance energy transfer pair 0.01-0.02 umol / L; The composite phospholipid system is composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in a mass ratio of 5:3:2 and is surface modified by beta2-glycoprotein I, wherein the phosphatidylserine is a long-chain saturated phospholipid modified by polyethylene glycol; the fluorescence enhancement-stabilizing system is a Schiff base compound of 2,4-dihydroxybenzaldehyde 0.03-0.05 mmol / L and L-histidine 3.0-4.0 mmol / L, which is mixed with a stabilizer mixture of hydroxyethyl cellulose 0.2-0.3 g / L and vitamin C 0.12-0.2 mmol / L; the calibrator is a series of lupus anticoagulant standard products with concentrations of 0.5-25 GPLU / mL, and the matrix is a human serum albumin solution without anticoagulant blood factors; the quality control product comprises low, medium and high concentration LA simulators and a negative control product, the low, medium and high concentration LA simulators are 2 GPLU / mL, 10 GPLU / mL and 20 GPLU / mL respectively, and the negative control product is added with a protease inhibitor mixture of 0.01-0.02 g / L; the anticoagulant interference inhibitor is an oxidized graphene-chitosan-EDTA composite 0.1-0.18 g / L.

2. The clotting reaction-linked lupus anticoagulant fluorescence detection kit according to claim 1, characterized by, The genetically engineered recombinant snake venom thrombin is recombinant batroxobin of Bothrops moojeni, the 58th serine is mutated into asparagine, and the 126th aspartic acid is mutated into glutamic acid, is prepared through an E. coli prokaryotic expression system, is purified through two steps of nickel column affinity chromatography and gel filtration chromatography, the elution buffer of the nickel column affinity chromatography contains 200-300 mmol / L imidazole, the column filler of the gel filtration chromatography is Superdex 75, the specific activity is greater than or equal to 1400 U / mg, and the endotoxin content is less than or equal to 0.06 EU / mL.

3. The clotting reaction-linked lupus anticoagulant fluorescence detection kit according to claim 1, characterized by, The FXa-sensitive fluorescent substrate is ZD-Arg-Gly-Arg-AMC with a purity ≥99%; the thrombin-sensitive fluorescent substrate is Boc-Asp(OBzl)-Pro-Arg-AMC with a purity ≥98.5%, an excitation wavelength of 488nm, an emission wavelength of 520nm, and an affinity constant for thrombin Kd ≤1.2×10-7mol / L.

4. The clotting reaction-linked lupus anticoagulant fluorescence detection kit according to claim 1, wherein, The oral anticoagulant adsorbent is aminated mesoporous silica with a pore size of 3-4 nm, a specific surface area ≥850 m² / g, and a surface amino modification amount of 0.8-1.2 mmol / g.

5. The clotting reaction-linked lupus anticoagulant fluorescence detection kit according to claim 1, wherein, The gold nanoparticle-carbon nanotube composite probe has a gold nanoparticle size of 12-18 nm, a surface modified with thiolized PEG with a molecular weight of 2000-5000, and a carbon nanotube that is a single-walled carbon nanotube with a length of 600-800 nm. The gold nanoparticle and carbon nanotube are coupled through amide bonds, the surface amino density is ≥2.8 mmol / g, and the fluorescence quantum yield is ≥68%.

6. The clotting reaction-linked lupus anticoagulant fluorescence detection kit according to claim 1, wherein, In the high-concentration composite phospholipid, the hexagonal II-phase structure synthetic phospholipid is composed of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylglycerol in a mass ratio of 3:1, with a phase transition temperature of 35-37℃ and an addition amount of 0.6-0.8 mg / mL. The composite phospholipid also contains 0.05-0.1 mg / mL of the phospholipase inhibitor methotrexate.

7. A method for using the lupus anticoagulant fluorescence detection kit according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Sample pretreatment: Take 35-45 μL of the plasma sample to be tested, add 12-18 μL of anticoagulation interference inhibitor, and incubate at 37℃±0.5℃ and 150-200 rpm on a shaker for 3.5-4.5 minutes, gently mixing once every 1 minute during the process to obtain the pretreated sample. S2: Construction of the reaction system: Add 120-140 μL of reagent A to a quartz reaction tube, preheat at 37℃±0.1℃ for 2.5-3.5 minutes, add 22-28 μL of pretreated sample, mix with a vortex mixer at 2000-2500 rpm for 3-5 seconds, and incubate at 37℃ for 4-5 minutes. S3: Coagulation-fluorescence linkage reaction. Add 60-70 μL of reagent B preheated to 37℃, quickly mix with pipette 5-6 times, and place in a fluorescence detector. Start the reaction under constant temperature of 37℃±0.1℃, and simultaneously turn on dual-channel fluorescence signal acquisition. S4: Signal acquisition. Fluorescence intensity was acquired at excitation wavelengths of 360nm±2nm / emission wavelengths of 460nm±2nm and 488nm±2nm / emission wavelengths of 520nm±2nm, respectively. The former is the FXa channel and the latter is the thrombin channel. Acquisition was performed every 25-30 seconds for 12-18 minutes. Fluorescence intensity values ​​at each time point were recorded. Each time point was measured 3 times and the average value was taken. S5: Verification of mixed test. Take another quartz reaction tube, add 24-26 μL of pretreated sample, 24-26 μL of normal mixed plasma, 120-140 μL of reagent A and 60-70 μL of reagent B, and repeat the operation of steps S3-S4 to collect fluorescence signals. S6: result determination, calculate the screening test fluorescence ratio and the confirmatory test fluorescence ratio of FXa channel, the screening test fluorescence ratio is the ratio of sample fluorescence intensity average and negative control fluorescence intensity average, the confirmatory test fluorescence ratio is the ratio of high phospholipid system fluorescence intensity average and low phospholipid system fluorescence intensity average, get the standardized LA ratio = screening test fluorescence ratio / confirmatory test fluorescence ratio; at the same time, calculate the fluorescence ratio correction rate of mixed test, fluorescence ratio correction rate = [(mixed sample fluorescence ratio-sample fluorescence ratio) / (negative control fluorescence ratio-sample fluorescence ratio)]×100%, when the standardized LA ratio is greater than or equal to 1.25 and the fluorescence ratio correction rate is less than 50%, it is determined that LA is positive, otherwise it is negative.

8. The method of the lupus anticoagulant fluorescence detection kit of coagulation reactions linkage according to claim 7, characterized in that, The normal mixed plasma in step S1 is a mixture of 45-55 platelet-poor plasma of healthy people, the healthy people are 20-45 years old and have no history of autoimmune disease, coagulation dysfunction and recent medication history, filtered through a 0.22 μm polyether sulfone filter, stored at-80℃±5℃, avoid repeated freezing and thawing during storage, quickly thawed in a 37℃±0.5℃ water bath before use, gently invert and mix for 10-15 times after thawing, and complete the test within 30 minutes after thawing.

9. The method of the lupus anticoagulant fluorescence detection kit of coagulation reactions linkage according to claim 7, characterized in that, The detection sensitivity of the fluorescence detector in step S4 is less than or equal to 0.08 ng / mL FXa activity, the fluorescence intensity detection range is 10-120000 fluorescence units, the batch variation coefficient of the instrument is less than or equal to 2.5%, the batch variation coefficient is less than or equal to 3.0%, the temperature control precision is ±0.1℃, and the signal acquisition delay time is less than or equal to 0.5 seconds.

10. The method of the lupus anticoagulant fluorescence detection kit of the coagulation reaction linkage according to claim 7, characterized in that, The method is applied to the auxiliary diagnosis of antiphospholipid syndrome and systemic lupus erythematosus, or risk assessment of venous thromboembolism and habitual abortion, the detection sample is human fasting venous plasma, collected by sodium citrate anticoagulant tube with a ratio of anticoagulant to blood volume of 1:9, centrifuged at 3000-3500 rpm for 10-15 minutes to separate the plasma, the sample is detected within 2-4 hours after collection, the sample is stored at-20℃±2℃ if not detected in time, the storage time is not more than 72 hours, and the detection should be completed within 1 hour after thawing.

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