Lupus anticoagulant detection reagent based on synergistic stability of collagen peptide and phospholipid

Abstract

The invention provides a lupus anticoagulant detection reagent based on synergistic stability of collagen peptide and phospholipid, and relates to the technical field of biological detection. By systematically screening and optimally combining key components, namely the blood coagulation activator, the phospholipid and the stabilizer, a full-liquid reagent composition capable of realizing physical and chemical long-term stability by using the least necessary components is found, so that the problem that the detection result is influenced by insufficient reagent stability in the prior art is solved; the silicon dioxide in the reagent composition is derived from a purified silica sol solution, and the collagen peptide is fish-derived I-type collagen peptide, does not contain a nonionic surfactant and a saccharide stabilizer, and has an application value in preparation of an in-vitro diagnosis product for diagnosis or auxiliary diagnosis of the anti-phospholipid syndrome.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a detection reagent for lupus anticoagulants based on the synergistic stability of collagen peptides and phospholipids. Background Technology

[0002] Lupus anticoagulant (LA) detection is a crucial step in the diagnosis of thrombotic diseases and antiphospholipid syndrome (APS). The silica clotting time (SCT) test is one of the important functional methods for detecting LA. Its principle is based on the specific activation of contact factors (such as factor XII) in the intrinsic coagulation pathway by activated silica reagents (e.g., diatomaceous earth, synthetic silicates). The presence of LA is indirectly determined by measuring the clotting time of plasma samples. As a pathological autoantibody, LA does not directly cause bleeding, but rather interferes with the in vitro phospholipid-dependent coagulation reaction by binding to phospholipid-protein complexes, leading to prolonged clotting time. Therefore, the specificity, sensitivity, and stability of the SCT reagent are crucial to the accuracy of the test results.

[0003] Traditional SCT reagents are mostly in the form of lyophilized powder or concentrated solution, requiring reconstitution or dilution before use. This process not only increases the number of steps and time involved but also introduces the risk of human error and contamination. Furthermore, the key components of the reagents—phospholipids and activators (silica)—are prone to physical or chemical changes during long-term storage in a liquid environment. For example, silica particles may settle or aggregate due to gravity, leading to decreased reagent homogeneity and affecting the reproducibility of activation efficiency; phospholipid vesicles may undergo hydrolysis, oxidation, or fusion, altering their surface properties and thus affecting their ability to bind to LA antibodies. These instabilities directly lead to an increased coefficient of variation in SCT test results, a narrowed detection window, and the potential for false positives or false negatives, severely restricting the standardization of LA testing and the comparability of results between laboratories.

[0004] To improve reagent stability, existing technologies primarily focus on optimizing buffer systems (such as adjusting pH and ionic strength), adding surfactants (such as Tween-20) to prevent particle aggregation, or using sugars (such as sucrose, trehalose, and inulin) as stabilizers to protect protein and phospholipid structures. However, these methods have limited effectiveness in maintaining the long-term stability of all-liquid reagents. This is especially true for SCT detection systems, which rely on solid-liquid interface reactions (interactions between silica particles and plasma components), where stable particle dispersion is a key challenge. While physical stirring or ultrasonic treatment can temporarily resuspend particles, it may destroy reagent activity or accelerate degradation. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a fully liquid lupus anticoagulant silica coagulation time (LA-SCT) reagent. Through systematic screening of three core components—coagulation activators, collagen peptides, and phospholipids—a simplified golden ratio with collagen peptides as the key stabilizer was established. This solution utilizes fish-derived type I collagen peptides, which produce a synergistic stabilizing effect with specific synthetic phospholipids and purified silica sol, eliminating dependence on traditional surfactants and sugars and fundamentally solving the long-term stability problem of fully liquid LA-SCT reagents.

[0006] The first objective of this invention is to provide a reagent composition for detecting lupus anticoagulants, the reagent composition comprising a screening reagent, a confirmatory reagent, and a activating reagent, wherein: The screening and confirming reagents contain silica, synthetic phospholipids, and collagen peptides. The silica is derived from purified silica sol solution, the synthetic phospholipids are a mixture of phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine, and the collagen peptides are fish-derived type I collagen peptides. The initiating agent contains calcium chloride.

[0007] Collagen peptides (CPs), small molecular weight polypeptide fragments obtained from the enzymatic hydrolysis of collagen, possess excellent biocompatibility, water solubility, and amphiphilicity. Studies have shown that collagen peptides can interact with metal ions or the surface of inorganic materials in solution through their specific amino acid sequences (such as Gly-XY repeat sequences), forming stable steric hindrance layers or hydration layers, thereby effectively inhibiting the sedimentation and aggregation of nano- or micro-sized particles. Simultaneously, collagen peptides themselves possess certain antioxidant and membrane-stabilizing properties, which may help protect phospholipid components in reagents from oxidative damage.

[0008] Furthermore, the screening and confirmatory reagents also contain buffer solution, L-alanine, and preservatives.

[0009] In one embodiment of the present invention, the buffer solution is a HEPES buffer solution with a concentration of 50 mM / L.

[0010] In one embodiment of the present invention, the concentration of L-alanine is 3%.

[0011] In one embodiment of the present invention, the preservative is Proclin 300, and the concentration of the preservative is 0.05%.

[0012] Furthermore, the molecular weight of the fish-derived type I collagen peptide is 2000-3000 Da.

[0013] In one embodiment of the present invention, the molecular weight of the fish-derived type I collagen peptide is 2000 Da.

[0014] Furthermore, the concentration of the fish-derived type I collagen peptide is 1-2 mg / mL.

[0015] In one embodiment of the present invention, the concentration of the fish-derived type I collagen peptide is 1 mg / mL.

[0016] Furthermore, the molar ratio of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the synthesized phospholipid is (70-75):(20-25):(5-10).

[0017] In one embodiment of the present invention, the molar ratio of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the synthesized phospholipid is 70:25:5.

[0018] Furthermore, the initiating agent also contains a preservative and a heparin neutralizer, wherein the heparin neutralizer is agglutinin or protamine.

[0019] In one embodiment of the present invention, the preservative is Proclin 300, and the concentration of the preservative is 0.05%.

[0020] In one embodiment of the present invention, the heparin neutralizing agent is a coagulating amine, and the concentration of the coagulating amine is 20 μg / mL.

[0021] A second objective of this invention is to provide the application of the above-described reagent composition in the preparation of a lupus anticoagulant detection kit.

[0022] A third object of the present invention is to provide a kit comprising the above-described reagent composition.

[0023] Furthermore, the kit also includes a lupus anticoagulant control.

[0024] A fourth object of the present invention is to provide the use of the above-described reagent composition or the above-described kit in the preparation of in vitro diagnostic products for the diagnosis or auxiliary diagnosis of thrombotic diseases and / or antiphospholipid syndrome.

[0025] The beneficial effects of this invention are: (1) The present invention has reduced the number of components in conventional reagent compositions in the prior art, which greatly reduces the number of components, simplifies the formula, reduces costs and production complexity, and makes it easier to control quality, ensuring extremely high batch-to-batch consistency. (2) The present invention uses collagen peptides as stabilizers and screens the source and molecular weight of collagen peptides. The mechanism of collagen peptides can effectively prevent the physical sedimentation and aggregation of silica particles and protect the chemical integrity of phospholipids. The effect is significantly better than the existing technology that relies on the electrostatic repulsion of surfactants and the stability of sugars. (3) The reagent composition provided by the present invention remains uniformly suspended after accelerated stability testing (37°C, 14 days), opening stability (2-8°C, 35 days), and long-term storage (2-8°C, 4 months), with no visible sedimentation or loss of activity. Its physical and chemical stability meets or exceeds that of complex formulations, truly achieving "ready to use," without the need for shaking or vortexing before use, simplifying operation and reducing human error; (4) The improved uniformity of the reagent composition provided by the present invention significantly reduces the inter-batch coefficient of variation (CV) to below 5%, thereby improving the reliability and comparability of the test results and providing a more accurate basis for clinical diagnosis. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0027] The devices, samples, or reagent kits involved in the following embodiments are shown below: Magnetic stirrers, vortex mixers, mini centrifuges, pH meters, and Ci-300, Ci-310, Ci-320, Ci-330, Ci-120i, Ci-120x, Ci-120n, AutoCimo C6000, AutoCimo C6200, AutoCimo C6400, and AutoCimo C6600 fully automated coagulation analyzers manufactured by Hiken Medical Technology (Suzhou) Co., Ltd.

[0028] Normal pooled plasma (NPP): A mixture of sodium citrate anticoagulated plasma from at least 20 healthy volunteers, confirmed to be LA negative.

[0029] Commercial LA quality control products: HemoSIL Positive Control (lot number: N0330160) for commercially available lupus anticoagulant; HemoSIL Negative Control (lot number: E0159327) for commercially available lupus anticoagulant.

[0030] Lupus anticoagulant positive substance (LAP): WHO Reference Panel 1st International Reference Panel for Lupus Anticoagulant NIBSC code: 13 / 172.

[0031] HemoSIL LA-SCT Detection Kit: Contains SCT screening reagent (low phospholipids), SCT confirmatory reagent (high phospholipids), and 25 mM Ca2+.2+ . Example

[0032] The SCT test is based on the CLSI H60-A guidelines and is performed strictly in accordance with the kit instructions.

[0033] The conventional sugar-containing LA-SCT reagent composition (Y1) is shown in Table 1.

[0034] Table 1. Common Sugar-Containing LA-SCT Reagent Compositions

[0035] To address the problems existing in the prior art, this embodiment simplifies the LA-SCT reagent composition. The simplified components are shown in Table 2. In addition to clotting amine, protamine can also be selected as the heparin neutralizer. Activators (such as silica or kaolin) activate the intrinsic coagulation pathway in the presence of phospholipids and calcium ions. LA antibodies delay the formation of prothrombin complex by binding to phospholipids, thereby prolonging coagulation time. The presence of LA is determined by comparing the difference in coagulation time under low-phospholipid (screening reagent) and high-phospholipid (confirmatory reagent) conditions. The preparation method involves dissolving buffer, synthetic phospholipids, preservatives, etc., in water to form a base solution. Collagen peptides are dissolved in the above base solution; then gently mixed evenly; finally, the activator (silica solution) is added, and it is fully dispersed into a uniform suspension using gentle homogenization techniques (such as low-speed stirring and vortex mixing). The pH is adjusted to the physiological range of 7.4, and after volume adjustment, it is sterilized, filtered, and dispensed.

[0036] Table 2 Simplified LA-SCT reagent compositions

[0037] Next, some of the core components in the simplified LA-SCT reagent composition described above were screened.

[0038] I. Screening of Activators As a coagulation activator, the physical properties of silica are crucial. Three alternatives were selected: silica powder produced by Sinopharm Group, diatomaceous earth with a medium particle size of 13 μM, and commercially available purified silica sol solution, with specific concentrations shown in Table 3. The weighed silica and diatomaceous earth powders were slowly and gradually added in small batches to a beaker containing HEPES (pH=7.4) buffer solution. Simultaneously, a magnetic stirrer was turned on and continuously stirred at a low to medium speed (e.g., 500-1000 rpm) to a target concentration of 50 mg / mL. The initially dispersed slurry was transferred to the feed container of a high-pressure homogenizer. The homogenizer was circulated at a pressure of 500-1000 bar under ice bath or external cooling conditions for 3-5 cycles, or until the slurry appeared homogeneous and fine. After homogenization, let the suspension stand (mature) at 2-8°C for 12-24 hours. After maturation, the suspension may contain a very small number of particles that are not completely wetted or are too coarse. These impurities can be removed by low-speed centrifugation (e.g., 1000×g, 1-2 minutes) or by taking the supernatant after standing.

[0039] Table 3. Screening and Optimization Schemes for Silica

[0040] II. Screening of Synthetic Phospholipids Synthetic phospholipids provide targets for LA binding, and their ratio and purity affect reagent performance. Therefore, a mixture of synthetic phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) was chosen. Synthetic phospholipids exhibit high purity and good homogeneity, reducing batch-to-batch variability. For comparison, rabbit brain phospholipids and soybean phospholipids were selected for preparing the LA-SCT system.

[0041] Synthetic phospholipid mixtures with different percentages of PS / PE / PC in the total amount of synthetic phospholipids were prepared, with a fixed total concentration of 10 mg / mL. Details are shown in Table 4. Appropriate amounts of the synthetic phospholipids phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) (as a percentage of the total amount of synthetic phospholipids) were weighed and dissolved in chloroform. The mixture was dried under nitrogen and then vacuum-dried to remove residual chloroform (2 h). A certain amount of purified water was added, and the mixture was magnetically stirred at room temperature for 2 h to prepare a phospholipid liposome solution. This solution was stored at -20 ℃ protected from light for later use.

[0042] Table 4. Screening and Optimization Schemes for Synthetic Phospholipids

[0043] III. Screening of Collagen Peptides Collagen peptides, as core stabilizers, are crucial in their type and molecular weight. Fish collagen, primarily type I, is the most abundant collagen in the human body and is fundamental to healthy skin, connective tissue, and bones. Its peptides exhibit excellent biocompatibility and water solubility. Fish-derived collagen peptides, especially those from deep-sea cod, are preferred due to their high purity (up to 99.99%), low immunogenicity, and superior absorption properties. Therefore, type I fish-derived collagen peptides are the preferred choice.

[0044] Micropeptides with a molecular weight ≤1500 Da can be directly absorbed through the intestinal wall, exhibiting high bioavailability. Collagen peptides with a molecular weight of around 1700-2300 Da are another preferred range because they can form a protective hydration layer of ideal thickness on the particle surface, providing optimal steric hindrance.

[0045] Type I fish collagen peptides with different molecular weights (0.5 kDa, 2 kDa, 5 kDa) and 2000 Da collagen peptides from different sources (bovine and porcine) were prepared. The concentrations of each molecular weight peptide (fixed concentration 10 mg / mL) were used as the stock solution concentrations. Specific categories are shown in Table 5.

[0046] Table 5. Optimization scheme for molecular weight and concentration of collagenase peptides

[0047] IV. Screening for Detection Validity 1. Preheating: Place the SCT screening reagent, confirmatory reagent, reconstituted quality control / plasma sample, and CaCl2 solution in a 37°C water bath or instrument preheating station for at least 15 minutes.

[0048] 2. Sample addition and incubation: Add 50 μL of preheated quality control sample (or NPP) to the test cup or cuvette, then add 50 μL of preheated SCT screening reagent, and incubate the mixture precisely at 37°C for 180 seconds.

[0049] 3. Start the reaction: Add 50 μL of preheated CaCl2 solution to the mixture and incubate the mixture precisely at 37°C for 240 seconds.

[0050] 4. Record the results: Record the number of seconds required for plasma to coagulate, which is the screening time (Ts-sct).

[0051] 5. Validation test: Repeat steps 2-4, but replace the SCT screening reagent with the SCT validation reagent (high phospholipid), and record the solidification time, which is the validation time (Tc-sct).

[0052] The formula for calculating the SCT standardized ratio is: Screening ratio SR = Screening result of lupus anticoagulant to be tested (in seconds) / Average value of normal screening range (in seconds) Confirmation ratio (CR) = Confirmation result of the lupus anticoagulant being tested (in seconds) / Average of the confirmed normal range (in seconds) Standardized ratio (NR) = Screening ratio / Confirmation ratio Reference range for reagent ratios: like The ratio was between 1.2 and 1.5, indicating weakly positive LA. like The ratio was between 1.5 and 2.0, indicating moderately positive LA. like A ratio greater than 2.0 indicates a strong positive LA result.

[0053] The screening results of phospholipids are shown in Table 6. The best detection performance was achieved with LZ2, a molar ratio (70:25:5) of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Compared with extracted animal phospholipids and soybean phospholipids, synthetic phospholipids have a clear chemical structure, no risk of animal-derived pathogens, and can be accurately detected for both negative and positive quality controls. Rabbit brain phospholipids and soybean phospholipids showed weakly positive and negative NR values ​​for positive quality controls, respectively. Incorrect detection data were not accepted.

[0054] Table 6 Screening of Synthetic Phospholipid Types and Ratios

[0055] The screening results for silica are shown in Table 7. Untreated silica and diatomaceous earth powder exhibited excessively long detection times in the LA-SCT detection system for 20 mixed normal human plasma samples and negative controls, exceeding the normal range. Furthermore, the normalized ratio (NR) for positive controls was less than 1.2, rendering them undetectable. Therefore, the selected coagulation activator was a high-purity purified silica sol solution. Compared to natural diatomaceous earth and silica, the purified silica sol solution demonstrated a more uniform particle size distribution and higher chemical purity.

[0056] Table 7 Screening of Silica Types

[0057] V. Reagent Stability Screening (1) Accelerated stability at 37℃ To simulate the degradation rate of the reagent at high temperatures and quickly predict its shelf life under normal conditions, according to the Q10 rule (the reaction rate doubles for every 10°C increase in temperature), one day at 37°C is approximately equivalent to 4-5 days at 2-8°C. The reagent was aliquoted into sealed reagent bottles and placed in a 37°C incubator. Samples were taken on day 0 (baseline), day 7, day 14, and day 28. Using a calibrated coagulation analyzer, the coagulation time of normal mixed plasma and lupus anticoagulant-positive plasma was measured.

[0058] Key performance indicators: Reagent stability: The rate of change in normal plasma clotting time (relative deviation should be <7.5% compared to day 0).

[0059] Detection sensitivity stability: Whether the degree of prolongation of coagulation time in lupus anticoagulant positive plasma (i.e., lupus anticoagulant activity) remains stable (relative deviation should be <10% compared with day 0).

[0060] (2) Stability test after opening the bottle at 2-8℃ The stability of the reagents was evaluated during actual clinical use, after opening and storage in the instrument's reagent compartment (refrigerated at 2-8°C). This is a key indicator of the practicality of all-liquid reagents. The reagent bottles of each embodiment were opened to simulate the open state in routine testing and placed in a refrigerator at 2-8°C. Samples were taken from the reagent bottles at fixed intervals: day 0 (baseline), day 14, day 35, and day 40 for testing. After testing, the bottles were recapped. The testing period lasted 50 days, and the testing method was the same as described above, using normal and positive plasma samples.

[0061] Key performance indicators: consistent with the above.

[0062] (3) Long-term effectiveness test at 4℃ The actual shelf life of the evaluation reagent in its unopened state under recommended storage conditions (4°C) is assessed. This is a shelf-life indicator for the product. The reagents from each embodiment were sealed and stored long-term in a 4°C cold storage. Samples were taken for testing at 0, 2, 4, and 6 months. In addition to coagulation time testing, inter-batch validation was performed using clinically known lupus anticoagulant positive and negative samples to ensure the accuracy of its clinical diagnosis.

[0063] Key performance indicators: Whether the various test indicators (normal time, positive rate) remain within the preset acceptable range throughout the 4 months.

[0064] The test results showed no significant difference compared to those obtained with fresh reagents.

[0065] Using a relative deviation >7.5% as the failure criterion, Table 8 below shows the percentage change in the relative deviation of the solidification time of weakly positive LA quality control samples relative to T0. Experimental results show that after 14 days at 37℃, only formulations J2 and Y1 remained stable (relative deviation <2.0%), with J2 exhibiting the smallest change rate (only 1.2%), demonstrating excellent resistance to thermal degradation. Regarding open-bottle stability: after 35 days of opening, the change rate of formulation J2 was only 2.9%, far below the failure criterion, significantly better than the blank control group CTRL (12.5%) and other experimental groups, indicating its effective resistance to evaporation and microbial influence after opening. Long-term stability at 4℃ (4 months) is a key indicator for assessing the actual shelf life of the reagents. The change rate of the control group (CTRL) reached 8.5%, exceeding the 7.5% failure criterion, indicating that its actual shelf life may be much shorter than 4 months. The optimal formulation J2 has a relative deviation of only 1.5%, with negligible performance degradation, suggesting that this formulation has the potential to achieve a shelf life of up to 6 months or even longer, demonstrating a significant advantage.

[0066] The above results indicate that, compared with the original LA-SCT all-liquid reagent formulation Y1, the formulation J2, which adds collagen peptides at a concentration of 1.0 mg / mL and a molecular weight of 2 kDa to the silica-containing coagulation method reagent for lupus anticoagulants, exhibits better accelerated stability at 37 ℃ and long-term stability at 4 ℃ than the saccharide-containing formulation Y1. However, the two formulations show similar performance in terms of open-bottle stability at 4 ℃.

[0067] Table 8 Results of accelerated stability experiments based on molecular weight and concentration optimization of collagenase peptides

[0068] V. Detection of Positive Quality Control Results for Lupus Anticoagulant Tests were performed using a Heeken fully automated coagulation analyzer. Each test included the following samples (consistent with the above): (1) Normal mixed plasma (NPP): from at least 20 healthy individuals; (2) Lupus anticoagulant positive plasma (LAP): from at least 5 clinically confirmed samples with different antibody types / titers; (3) Commercial calibrators / controls: including normal and abnormal value controls.

[0069] Using the same batch of reagents, control plasma at both normal and abnormal levels was tested 10 times consecutively at the same time point. The standard deviation (SD) and coefficient of variation (CV%) of the clotting time (CT) of the 10 test results were calculated. A lower CV% indicates better repeatability (not exceeding 10%).

[0070] Where: CV—coefficient of variation; SD – Standard deviation of the measurement result; —The average value of the measurement results At each time point, using three different batches of the same group of reagents (e.g., three batches from Protocol 1), the test results for the three batches were calculated. SD and CV%. The smaller the CV%, the better the consistency between batches. The coefficient of variation of the obtained results should be CV% ≤ 10%.

[0071] A group of clinical samples (at least 20 cases, including negative and positive controls) were simultaneously tested using the test reagent, repeated three times. The screening test ratio (SR), confirmatory test ratio (CR), and normalized ratio (NR) were calculated for each sample, and the average of the normalized ratios from the three tests was obtained. The results should meet the criteria for positive or negative. Three batches of seven groups (including controls) were prepared simultaneously and subjected to accelerated and long-term stability tests, which greatly reduced the time required. At each stability time point, repeatability, batch-to-batch variation, and accuracy were tested in one go, resulting in efficient data acquisition. A quantitative failure criterion was set. Once the data indicated a significant decline in the performance of a group, subsequent testing of that group was stopped, and resources were concentrated on the high-performing groups. The test results are shown in Table 9 below.

[0072] Table 9. Results of performance testing for molecular weight and concentration optimization of collagenase peptides (reproducibility, batch-to-batch variation).

[0073] Table 9 shows the results regarding repeatability and batch-to-batch variation: the optimal formulation J2 had the lowest CV% values ​​for both groups, indicating the best precision and batch-to-batch consistency. Concentration effect: Comparing J2, J4, and J5, 1.0 mg / mL (J2) was the performance inflection point; neither increasing nor decreasing the concentration significantly improved performance, and concentrations above 3 mg / mL prolonged the second value. Molecular weight screening: When the molecular weight was below 1500 Da, the steric hindrance effect was insufficient, and the second value remained unchanged; above 3000 Da, the solution viscosity increased, potentially affecting reagent dispersibility and reaction kinetics, leading to an increase in the second value. Collagen peptides around 2000 Da could form a protective hydration layer of ideal thickness on the particle surface, providing the best steric hindrance effect. Source screening: Fish-derived collagen peptides were the optimal choice due to their lower CV% value for batch-to-batch repeatability variation.

[0074] This indicates that collagenase peptides with excessively small molecular weights (e.g., 500 Da) are more prone to degradation or polymerization in the liquid state, resulting in poor stability at 37°C and after-bottle stability. Formulations (J2) with moderate molecular weights (e.g., 2 kDa) and appropriate concentrations (1 mg / mL) achieve the best balance between molecular structural stability and effectiveness in binding to phospholipids and antibodies, exhibiting excellent long-term efficacy and after-bottle stability. High molecular weight (5 kDa) collagenase peptides may have greater steric hindrance, affecting their binding efficiency to antibodies, leading to a decrease in the detection sensitivity of lupus anticoagulants and an increase in the detection time.

[0075] Using the commercially available HemoSIL LA-SCT test kit as a control, the accuracy of the above-mentioned preferred scheme J2, which has optimized stability, repeatability, and batch-to-batch variation, was compared. The results are shown in Table 10 below. The average value of the standardized ratio of three tests for each sample was calculated for the preferred scheme J2, the blank control group, and the commercially available HemoSIL LA-SCT test kit. The results of the mixed sample of 20 normal individuals and the HemoSIL Negative / Positive Control product all met the criteria for positive and negative results, and the accuracy of the test results met the requirements.

[0076] Table 10 Results of performance testing for molecular weight and concentration optimization of collagenase peptides

[0077] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A reagent composition for detecting lupus anticoagulants, characterized by comprising, The reagent composition comprises a screening reagent, a confirming reagent and a starting reagent, wherein: The screening reagent and the confirming reagent comprise silica, synthetic phospholipid and collagen peptide, the silica is derived from a purified silica sol solution, the synthetic phospholipid is a mixture of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, and the collagen peptide is fish-derived type I collagen peptide; The starting reagent comprises calcium chloride.

2. The reagent composition of claim 1, wherein: The screening reagent and the confirming reagent further comprise a buffer, L-alanine and a preservative.

3. The reagent composition of claim 1, wherein: The fish-derived type I collagen peptide has a molecular weight of 2000-3000 Da.

4. The reagent composition of claim 1, wherein: The concentration of the fish-derived type I collagen peptide is 1-2 mg / mL.

5. The reagent composition of claim 1, wherein: The molar ratio of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the synthetic phospholipid is (70-75):(20-25):(5-10).

6. The reagent composition of claim 1, wherein: The starting reagent further comprises a preservative and a heparin neutralizer, and the heparin neutralizer is polybrene or protamine.

7. Use of the reagent composition of claims 1-6 in the preparation of a lupus anticoagulant detection kit.

8. A kit characterized in that: The kit comprises the reagent composition of any one of claims 1-6.

9. The kit of claim 8, wherein: The kit further comprises a lupus anticoagulant control sample.

10. Use of the reagent composition of any one of claims 1-6 or the kit of claims 8 or 9 in the preparation of an in vitro diagnostic product for the diagnosis or auxiliary diagnosis of thrombotic diseases and / or antiphospholipid syndrome.

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