Antigen binding protein specifically binding to beta2-glycoprotein I-cardiolipin complex and application thereof

Abstract

The invention relates to an antigen binding protein specifically binding to a beta2-glycoprotein I-cardiolipin complex and application of the antigen binding protein. The antigen binding protein capable of being specifically bound with the beta 2-glycoprotein I-cardiolipin compound is designed firstly, the antigen binding protein can be specifically bound with the beta 2-glycoprotein I-cardiolipin compound, and the sensitivity of the antigen binding protein to the beta 2-glycoprotein I-cardiolipin compound is 8 micrograms per milliliter; the strong binding capacity with a target antigen is shown in ELISA (Enzyme-Linked Immunosorbent Assay) detection. Furthermore, when the antigen binding protein is applied to preparation of the positive quality control product of the lupus anticoagulant, the antigen binding protein can better simulate the effect of the lupus anticoagulant and is not easily interfered by various factors in plasma compared with the lupus anticoagulant, and the obtained positive quality control product is stable in target specificity and high in specificity. And the evaluation is more accurate.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunoassay technology, and particularly to an antigen-binding protein specifically binding to β2-glycoprotein I-cardiolipin complex and application thereof. BACKGROUND

[0002] Lupus anticoagulant (LA) is an autoantibody targeting negatively charged phospholipid-protein complex, i.e. antiphospholipid antibody (APA), which is one of the core laboratory diagnostic markers of antiphospholipid syndrome (APS). LA detection is crucial for evaluating the risk of clinical events such as thrombosis and pathologic pregnancy. Its detection relies on coagulation function tests such as dilute Russell viper venom time (dRVVT) and silicon clotting time (SCT), and the presence of LA is confirmed by comparing the coagulation time difference between phospholipid-deficient and phospholipid-rich reagents.

[0003] The accuracy and standardization of lupus anticoagulant (LA) detection are key challenges faced by clinical laboratories. Due to the strong heterogeneity of LA antibodies (which can target different targets such as β2-glycoprotein I and prothrombin), and the interference of various factors in plasma (such as coagulation factor deficiency and anticoagulant drugs), reliable positive and negative quality control materials need to be strictly used in daily detection to monitor the effectiveness of the entire detection system.

[0004] Currently, the LA positive quality control commonly used internationally is mostly derived from mixed plasma of positive patients. However, this scheme has problems such as difficulty in collecting patient plasma, large batch difference, high potential bio-safety risk, unstable antibody titer and unclear target specificity. Patent No. CN115219728A uses a positive material made of bovine plasma mixed with dodecyltrimethylammonium chloride as a lupus anticoagulant positive quality control. However, the use of bovine plasma, a traditional animal plasma mixed positive material scheme, may differ from the complex reaction characteristics of polyclonal antibodies in real patient samples, which may cause deviations in the evaluation of the sensitivity of the detection kit. In addition, the use of dodecyltrimethylammonium chloride has problems such as easy degradation of chemicals and inability to simulate the complex reaction of polyclonal antibodies in real patient samples. Moreover, this scheme is only applicable to the dRVVT method, and when used for quality control of the SCT method, the batch difference is large, the reagent is very sensitive, and the repeatability is poor. Patent No. CN118191335A uses the high affinity of annexin and phospholipids to competitively inhibit the binding of LA to the solid phase. However, this scheme is only for the interference of annexin A5, which has a relatively single coverage. Patent No. CN118731378A contains human plasma, artificially screened viveridae snake venom and other anticoagulant modulating substances in its quality control. However, viveridae snake venom includes antibody proteins or anti-platelet substances that can bind to platelet membranes (phospholipids), which are the target of lupus anticoagulant in the body to cause thrombosis, not the target of in vitro detection (the pathophysiological basis and detection principle of lupus anticoagulant are completely opposite), and is not suitable for use as a positive quality control. Therefore, it is necessary to further seek a specific lupus anticoagulant quality control targeting β2-glycoprotein I-cardiolipin with high stability. SUMMARY

[0005] To this end, the technical problem to be solved by the present application is to overcome the problem of unstable target specificity of LA positive quality control in the prior art and the deviation in evaluation.

[0006] To solve the above technical problems, the present application provides an antigen binding protein specifically binding to β2-glycoprotein I-cardiolipin complex and its application. The present application first designs an antigen binding protein that can specifically bind to β2-glycoprotein I-cardiolipin complex. The antigen binding protein of the present application can specifically bind to β2-glycoprotein I-cardiolipin complex, and the sensitivity of the antigen binding protein of the present application to β2-glycoprotein I-cardiolipin complex is 8 μg / mL. Further, the present application provides a non-biological source synthetic LA positive quality control. The positive quality control of the present application can specifically simulate the interaction of LA antibody with phospholipid-protein target, exhibit stable anticoagulant activity in the LA detection system, and be used for daily quality control in the laboratory.

[0007] The design principle of the present application is that the anticoagulant effect of the antigen binding protein of the present application is consistent with that of lupus anticoagulant, so that the antigen binding protein of the present application can better simulate the interaction of lupus anticoagulant with phospholipid-protein target, exhibit stable anticoagulant activity in the LA detection system, and further serve as a positive quality control in the detection process of lupus anticoagulant, for daily quality control in the laboratory.

[0008] A first object of the present application is to provide an antigen binding protein specifically binding to a β2-glycoprotein I-cardiolipin complex, the antigen binding protein comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NO. 1-3 respectively, and the light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NO. 5-7 respectively, the β2-glycoprotein I-cardiolipin complex being a product of mixing β2-glycoprotein I with cardiolipin.

[0009] Further, the heavy chain variable region comprises a framework region VH-FR, wherein the framework region VH-FR comprises a sequence as shown in SEQ ID NO. 4 or a sequence with a homology of not less than 90% to the sequence.

[0010] Further, the light chain variable region comprises a framework region VL-FR, wherein the framework region VH-FR comprises a sequence as shown in SEQ ID NO. 8 or a sequence with a homology of not less than 90% to the sequence.

[0011] A second object of the present application is to provide a nucleic acid molecule encoding the antigen binding protein.

[0012] A third object of the present application is to provide an expression vector comprising the nucleic acid molecule.

[0013] A fourth object of the present application is to provide an application of the antigen binding protein, the nucleic acid molecule, the expression vector or the host cell in preparing a β2-glycoprotein I-cardiolipin complex binding product.

[0014] A fifth object of the present application is to provide an application of the antigen binding protein, the nucleic acid molecule, the expression vector or the host cell in preparing a lupus anticoagulant quality control.

[0015] A sixth object of the present application is to provide a lupus anticoagulant positive quality control, the lupus anticoagulant positive quality control comprising the antigen binding protein, plasma, a buffer system, a preservative and an excipient.

[0016] Further, the plasma is prepared by mixing blood with an anticoagulant.

[0017] Further, the buffer system is selected from one or more of Tris-HCl, imidazole, Hepes buffer, phosphate buffer.

[0018] Further, the preservative is selected from one or more of sodium azide, PC300, CMIT.

[0019] Further, the excipient is selected from one or more of mannitol, sorbitol.

[0020] The above technical solutions of the present application have the following beneficial effects compared with the prior art:

[0021] The antigen binding protein of the present application can specifically bind to the β2-glycoprotein I-cardiolipin complex, and the sensitivity of the antigen binding protein of the present application to the β2-glycoprotein I-cardiolipin complex is 8 μg / mL. Further, the antigen binding protein of the present application is applied to the preparation of positive quality control of lupus anticoagulant, the antigen binding protein of the present application has similar coagulation effect with the lupus anticoagulant, so it can better simulate the effect of the lupus anticoagulant, and compared with the lupus anticoagulant, it is not easily interfered by various factors in the plasma, the target specificity of the obtained positive quality control is stable, and the evaluation is more accurate. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings.

[0023] Figure 1 is the electropherogram of the antigen binding protein of the present application. DETAILED DESCRIPTION

[0024] The present application will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present application and implement it, but the embodiments are not limiting the present application.

[0025] Example 1: Expression and purification of recombinant antigen binding protein in 293T cells

[0026] 1. Materials and equipment

[0027] 293T cell strain (purchased from ATCC), FreeStyleTM 293 expression medium (Gibco), linear polyethyleneimine (PEI Max, Polysciences), heavy chain and light chain expression plasmid, Protein A pre-packed chromatography column (Cytiva), chromatography system (AKTA pure or similar products).

[0028] Equipment: cell culture room, biosafety cabinet, CO2 incubator, shaker / bioreactor, protein purification system, HPLC, SPR instrument;

[0029] 2. Method

[0030] 2.1 Cell culture and transfection

[0031] 2.1.1 293T cell culture

[0032] 293T cells were inoculated in a flask at a density of 0.8 x 10 6 cells / mL, and were cultured in suspension at 37°C, 8% CO2, 125 rpm constant temperature shaker. When the cell density reached 1.2 x 10 6 cells / mL, and the viability was higher than 95%, transfection was performed.

[0033] 2.1.2 Preparation of NTP-2 antigen binding protein

[0034] This example uses a strain of high-affinity antigen binding protein (NTP-2) targeting β2-glycoprotein I (β2-GPI) and cardiolipin complex. The heavy chain and light chain variable region amino acid sequences are shown in Table 1 below.

[0035] Table 1 Related sequences of NTP-2 antigen binding protein

[0036]

[0037] The framework region is a human sequence, which can minimize immunogenicity and is suitable for in vitro diagnostic reagents. The above CDR region sequences are optimized by computer-aided design and in vitro affinity maturation, so that they can specifically recognize the conformational neoepitope exposed after β2-GPI and cardiolipin binding, rather than β2-GPI or cardiolipin alone. This is the molecular basis for its ability to mimic the real LA activity.

[0038] 2.1.3 Construction and ligation method of expression plasmid

[0039] 1. Vector backbone: Use commercially available mammalian cell bi-directional expression vector pcDNA3.4-TOPO or similar high-efficiency expression vector.

[0040] 2. Gene synthesis and cloning

[0041] The gene sequence encoding the heavy chain variable region of mAb-LA01 (SEQ ID NO: 4) was spliced with the human IgG1 constant region gene sequence to form a complete heavy chain gene.

[0042] The gene sequence encoding the variable region of the light chain of mAb-LA01 (SEQ ID NO: 8) was spliced with the gene sequence of the constant region of human kappa light chain to form a complete light chain gene.

[0043] The complete heavy chain gene and the complete light chain gene were inserted into two independent CMV promoters on the same plasmid vector by Gibson Assembly or In-Fusion cloning technology to form a double expression vector, named pLV-mAb-NTP2, and the expression plasmid DNA had a molecular weight of 8500 bp.

[0044] 2.1.4 Preparation of DNA-PEI complex

[0045] PEI was used in the form of linearized polyethyleneimine (PEI Max) and mixed with the purified pLV-mAb-NTP2 plasmid at a ratio of DNA:PEI=1:2 (w / w) to form a complex, and then transfected into 293T cells in the logarithmic growth phase.

[0046] 2.2 Expression and Harvesting

[0047] Six hours after transfection, 6 mM sodium propionate and 1.5% Valproic acid (histone deacetylase inhibitor) were added to the culture solution to enhance protein expression, and the cell culture was harvested on the 6th day after transfection. The cells and cell debris were separated by centrifuge at 4,000 x g, 4°C for 30 minutes, the supernatant was collected and filtered with a 0.22 μm PES membrane to obtain a clear cell culture supernatant (CCF).

[0048] 2.3 Antibody Purification

[0049] 2.3.1 Protein A affinity chromatography capture:

[0050] The AKTA pure system and Protein A chromatography column were equilibrated with 5 column volumes (CV) of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4), and the filtered CCF was loaded at a linear flow rate of 150 cm / h. The chromatography column was washed with 10 CV of binding buffer until the ultraviolet absorption (UV 280 nm) signal returned to baseline to remove unbound impurities, and eluted with 5 CV of elution buffer (0.1 M Citric Acid, pH 3.0). The automatic collector collected the elution peak components according to the UV 280 nm absorption peak, and immediately neutralized the pH of the collected eluate to 7.0-7.5 with neutralization buffer (1 M Tris-HCl, pH 9.0) to prevent antibody denaturation.

[0051] 2.3.2 Buffer replacement and concentration:

[0052] The neutralized antibody pool was loaded into pretreated dialysis bags and dialyzed against PBS (pH 7.4) at 4°C overnight, with the dialysis solution being replaced every 4 hours;

[0053] The antibody concentration (A 280 nm).

[0054] 2.4 Analysis and activity verification

[0055] 2.4.1 Purity analysis:

[0056] The purity of the antibody was analyzed by SDS-PAGE (4-20% gradient gel) under reducing and non-reducing conditions. Clear bands of approximately 50 kDa heavy chains and 30 kDa light chains were observed, and the purity was >95%.

[0057] 2.4.2 Functional LA activity verification:

[0058] Purified antibodies of different concentrations (such as 0-30 μg / mL) were added to LA-negative normal mixed human plasma. The dRVVT and SCT detection kits were used for detection. The results showed that compared with the control group without antibody, the coagulation time of the experimental group was significantly prolonged, and the dRVVT ratio and SCT ratio were both greater than 1.2 (positive cut-off value), and showed a dose-dependent manner, confirming the functional LA activity.

[0059] Example 2: Performance test of NTP-2 antigen binding protein

[0060] Immune binding activity verification (ELISA)

[0061] Purpose of the experiment:

[0062] After preparing LA (lupus anticoagulant) positive antibodies in the 293 T cell expression system, an indirect enzyme-linked immunosorbent assay (ELISA) functional activity verification process is needed to confirm the binding specificity, affinity, and functional properties of the antibodies.

[0063] ELISA verification experiment steps:

[0064] Principle of the experiment: The indirect ELISA method is used to detect the binding of the antibody to the target antigen cardiolipin β2-GP I complex. Cardiolipin is coated on the microplate to simulate the phospholipid surface of the cell membrane. β2-GPI is added to bind to cardiolipin and undergo conformational changes, exposing the target epitope. Finally, the antibody to be tested is added. If the antibody can specifically recognize this conformational epitope, a signal is generated through the enzyme-labeled secondary antibody and substrate.

[0065] Key reagents and materials:

[0066] Coating antigens: 1. Experimental group (target antigen): Cardiolipin and β2-GPⅠ complex were selected as the coating antigen based on the specificity of LA antibody; 2. Negative control group: Bovine serum albumin (BSA) unrelated to the LA target was coated to assess the non-specific adsorption of the antibody; 4. Blank control group: Only coating buffer was added (to assess the non-specific adsorption background after coating and blocking of ELISA plate wells) and only β2-GPⅠ antigen was coated without cardiolipin antigen.

[0067] Antibodies to be tested: 1. LA positive antibody prepared in the 293 system; 2. Blank control (PBS); 3. Negative control (anti-green fluorescent protein GFP antibody of the same IgG type as LA antibody but with an unrelated target); 4. Positive control: known LA positive antibody (from Shanghai Chilue, catalog number CL901).

[0068] Enzyme-labeled secondary antibody: Horseradish peroxidase (HRP)-labeled anti-human IgG antibody—goat anti-human IgG-HRP—was selected based on the species origin of the primary antibody.

[0069] Other reagents: coating buffer (0.05 M carbonate buffer, pH 9.6), washing buffer (PBS with 0.05% Tween-20, pH 7.4), blocking buffer (PBS with 5% BSA), substrate (TMB), and stop solution (2 M H2SO4).

[0070] Equipment: 96-well polystyrene microplate, microplate reader, etc.

[0071] Detailed experimental steps:

[0072] Cardiolipin coating: Dilute the antigen to 50 μg / mL with anhydrous ethanol, add 100 μL to each well, and let the ELISA plate evaporate completely overnight at room temperature until an invisible dry film forms at the bottom of the well. After evaporation, add 150 μL of pre-cooled PBS to each well, gently shake once, and shake dry. This step can remove unadsorbed phospholipids. Set up a blank control group: add 100 μL of antigen dilution buffer to each well.

[0073] Blocking: Add 200 μL of blocking solution to each well and incubate overnight at 4 °C.

[0074] β2-GPⅠ antigen incubation:

[0075] Discard the blocking solution, pat dry on thick absorbent paper, wash the plate three times, soaking for 1 minute each time and then pat dry.

[0076] Experimental group sample addition: Add 50 μL of 10 μg / mL β2-GPⅠ solution diluted with PBS to each well;

[0077] Negative control group: Add 50 μL of 10 μg / mL BSA solution diluted with PBS to each well;

[0078] Blank control group: Add 50 μL of coating buffer to each well.

[0079] Blocking: Add 200 μL of blocking solution to each well and incubate at 37°C for 1-2 hours. Wash 3 times.

[0080] Add the primary antibody to be tested: Serially dilute the LA positive antibody 1.6-fold starting at 50 μg / mL and add it to the wells (100 μL / well). Incubate at 37°C for 2 hours. Wash 3 times.

[0081] Add enzyme-labeled secondary antibody: Add HRP-labeled secondary antibody diluted 1:10000 (100 μL / well) and incubate at 37°C for 1 hour. Wash 3-5 times.

[0082] Color development: Add 100 μL of TMB substrate solution to each well and develop color at 37°C in the dark for 15 minutes.

[0083] Termination of reaction and detection: Add 50 μL of stop solution (2M H2SO4) to each well. Immediately measure the OD value of each well at 450 nm using a microplate reader.

[0084] Data recording and results analysis:

[0085] Record the OD values ​​of each experimental group and control well in three repeated experiments. 450 A positive result is defined as an OD value ≥ 2.1 times that of the negative control. The relationship between antibody concentration and OD value is then visualized using a dose-response curve, and the half-maximal effective concentration (EC50) is calculated. 50 This demonstrates its affinity.

[0086] Table 2 Results of affinity assay between LA positive antibody and target antigen

[0087]

[0088] Table 3. Specificity detection of LA positive antibodies and target antigens

[0089]

[0090] This invention critically establishes an ELISA method for verifying the functional activity of the antibody. Through rigorous antigen coating, blocking, antibody incubation, and color development steps, it confirms that the antibody exhibits a significant binding signal compared to the negative control (e.g., an OD value ≥ 2.1 times that of the negative control, and an LA antibody concentration greater than 2.0 μg / mL is detectable), and demonstrates a clear dose-response relationship. The antibody exhibits a strong binding capacity to the target antigen (cardiolipin + β2-GPI) in ELISA detection, significantly superior to irrelevant control antibodies. This antibody and its verification method provide a novel core tool and solution for the diagnosis, pathological research, and targeted therapy development of lupus anticoagulant-related autoimmune diseases.

[0091] Example 3: Preparation of positive control material for lupus anticoagulant

[0092] The lupus anticoagulant positive control material provided in this embodiment includes: normal canine plasma, buffer system, purified NTP-2 antigen-binding protein, preservatives and lyophilized excipients, etc.

[0093] Normal canine plasma processing procedure: Collect sufficient plasma sample by arterial puncture, add 2-5% trisodium citrate anticoagulant at a blood-to-anticoagulant volume ratio of 9:1, centrifuge for 30 minutes at a relative centrifugal force of 2000g, and carefully separate and aspirate the supernatant plasma.

[0094] Buffer system: any one of Tris-HCl, imidazole, Hepes buffer, or phosphate buffer, preferably Tris-HCl buffer with pH 7.5-7.8;

[0095] The concentration range of recombinant phospholipid antibodies is 6 μg / mL to 50 μg / mL, preferably 8 to 30 μg / mL.

[0096] Preservatives: one or more of the following: sodium azide 0.01-0.05%, PC300 0.1-0.6%, CMIT 0.1-0.6%;

[0097] Stabilizers: one or more of the following: glycine 10mM-20mM, BSA 0.5%-5%, and sucrose 0.5%-5%.

[0098] Freeze-drying excipients: one or more of mannitol 1%-5% and sorbitol 2%-10%.

[0099] Table 4. Relevant Formulas for Positive Control Products

[0100]

[0101] Example 4: Performance Validation of Positive Quality Control Product for Lupus Anticoagulant – dRVVT & SCT Detection

[0102] 1. Purpose

[0103] This embodiment aims to demonstrate in detail how to use the dilute viper venom time (dRVVT) method and silica coagulation time (SCT) method to verify the performance of the lupus anticoagulant (LA) positive control product (hereinafter referred to as "this product") prepared in this invention, so as to prove its effectiveness, stability and reliability on different testing platforms.

[0104] 1.1 Materials and Methods

[0105] Lupus anticoagulant test kits: Silica gelation assay (LA-SCT) kit manufacturer: HemoSIL, screening reagent batch number N0946201, confirmatory reagent batch number N0845136; Dilution viper venom time (LA-dRVVT) kit manufacturer: SIEMENS, screening reagent batch number 574175A, confirmatory reagent batch number 574257A; Commercially available lupus anticoagulant positive control: HemoSILPositiveControl, catalog number: 0020012500, batch number: N1248432 2026.12.

[0106] 1.2 Main Instruments and Equipment

[0107] Sysmex CS5100 fully automated coagulation analyzer; HemoSIL ACLTOP750 fully automated coagulation analyzer.

[0108] 2. Experimental Materials

[0109] (1) LA positive control sample prepared in Example 2: lyophilized product, reconstituted with deionized water before use, after reconstitution, the cap is tightly closed and placed at room temperature of 20-25℃. Take one bottle of completely reconstituted sample at 0.5, 4 and 8 hours respectively, and test with imported SIEMENS LA-dRVVT reagent and imported HemoSIL LA-SCT reagent, and record the obtained data.

[0110] (2) Normal pooled plasma (NPP): A mixture of sodium citrate anticoagulated plasma from at least 20 healthy volunteers, confirmed to be LA negative.

[0111] (3) Commercial LA positive control: HemoSIL Positive Control, batch number: 0020012500

[0112] (4) Test kit: SIEMENS LA-dRVVT test kit: contains dRVVT screening reagent (low phospholipid) and dRVVT confirmatory reagent (high phospholipid).

[0113] HemoSIL LA-SCT Detection Kit: Contains SCT screening reagent (low phospholipids), SCT confirmatory reagent (high phospholipids), and 25mM Ca2+. 2+ .

[0114] 3. Detection methods and procedures

[0115] 3.1 dRVVT testing steps

[0116] The dRVVT test was performed based on the ISO 17593:2022 standard procedure and strictly followed the kit instructions.

[0117] Detection principle:

[0118] Viper venom (RVV) directly activates coagulation factor X, which, along with coagulation factor V, phospholipids, and calcium ions, promotes the conversion of prothrombin to thrombin. LA antibodies interfere with the formation of this complex by binding to the phospholipid surface, thereby prolonging coagulation time. The presence of LA is determined by comparing the difference in coagulation time under low-phospholipid (screening) and high-phospholipid (confirmatory) conditions.

[0119] Detailed steps:

[0120] (1) Preheating: Place the dRVVT screening reagent, confirmation reagent, and reconstituted quality control / plasma sample in a 37°C water bath or instrument preheating position for at least 15 minutes.

[0121] (2) Sample addition: Add 80 μL of preheated quality control sample (or NPP) to the test cup or colorimetric cup, and then add 80 μL of preheated dRVVT screening reagent.

[0122] (3) Incubation: Incubate the mixture precisely at 37°C for 120 seconds.

[0123] (4) Recording results: The instrument or observer records the number of seconds required for plasma coagulation, which is the screening time (Screening Time, Ts).

[0124] (5) Confirmation test: Repeat steps 2-4, but replace the dRVVT screening reagent with the dRVVT confirmation reagent (high phospholipid), and record the solidification time, which is the confirmation time (Tc).

[0125] 3.2 SCT Detection Procedure

[0126] The SCT test is based on the CLSI H60-A guidelines and is performed strictly in accordance with the kit instructions.

[0127] Detection principle:

[0128] Activators (such as silica and kaolin) activate the intrinsic coagulation pathway in the presence of phospholipids and calcium ions. LA antibodies prolong coagulation time by binding to phospholipids and delaying the formation of prothrombinase complexes. The presence of LA is determined by comparing the difference in coagulation time under low-phospholipid (screening) and high-phospholipid (confirmatory) conditions.

[0129] Detailed steps:

[0130] (1) Place the SCT screening reagent, confirmation reagent, reconstituted quality control / plasma sample and CaCl2 solution in a 37°C water bath or instrument preheating position for at least 15 minutes.

[0131] (2) Sample addition and incubation:

[0132] Add 50 μL of preheated quality control (or NPP) to a test cup or cuvette, then add 50 μL of preheated SCT screening reagent, and incubate the mixture precisely at 37°C for 180 seconds.

[0133] (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.

[0134] (4) Record the results: Record the number of seconds required for plasma coagulation, which is the screening time (Ts-sct).

[0135] (5) Confirmation test: Repeat steps 2-4, but replace the SCT screening reagent with the SCT confirmation reagent (high phospholipid), and record the solidification time, which is the confirmation time (Tc-sct).

[0136] The formula for calculating the dRVVT-SCT standardized ratio is:

[0137] Screening ratio = Screening result of lupus anticoagulant to be tested (in seconds) / Average value of normal screening range (in seconds);

[0138] Confirmation ratio = Confirmation result of the lupus anticoagulant to be tested (in seconds) / Average value of the confirmed normal range (in seconds);

[0139] Standardized ratio = Screening ratio / Confirmation ratio.

[0140] 4. Result Judgment and Conclusion

[0141] Positive test criteria (according to the kit instructions):

[0142] Siemens dRVVT reagent reference range:

[0143] If the ratio of standardized ratio (screening ratio / confirmation ratio) is between 1.2 and 1.5, a weak positive LA exists;

[0144] If the ratio of standardized ratio (screening ratio / confirmation ratio) is between 1.5 and 2.0, a moderately positive LA is present.

[0145] If the ratio of the standardized ratio (screening ratio / confirmation ratio) is greater than 2.0, a strong positive LA exists.

[0146] Table 5 Reference Range for Werfen SCT Reagent

[0147]

[0148] Example 5: Detection of positive quality control results for lupus anticoagulant

[0149] Table 6 shows the blood coagulation time results of lyophilized positive control samples prepared using different buffer systems and tested with SIEMENS dRVVT assay reagent after 0.5 hours of reconstitution.

[0150] Table 6. Results of lyophilization in different buffer systems detected by SIEMENS dRVVT reagent.

[0151]

[0152] Table 5 shows that the standardized ratio of Siemens dRVVT reagent in detecting mixed plasma samples from 20 normal individuals was 1.00. The detection time for the positive control screening reagent was 65.4 s, with a standardized ratio of 1.66. The Siemens reagent meets the requirements and can be used for reagent testing. When the lyophilization system is Tris-HCl pH 5.0, the highest detection time for the positive control is 79.3 s, with a standardized ratio of 1.90. The detection time and standardized ratio of the screening reagent are higher than those of the other three buffer systems. Therefore, lyophilization scheme one is preferred for the preparation of the positive control.

[0153] This product was tested using dRVVT and SCT. The screening time (Ts), confirmation time (Tc), and the final calculated dRVVT ratio to SCT ratio were used to determine its LA positive characteristic. The results of testing the positive control sample of this invention with Siemens dRVVT reagent at 0.5h, 4h, and 8h after reconstitution are shown in Table 7 below.

[0154] Table 7 Results of positive control samples detected by SIEMENS dRVVT reagent

[0155]

[0156] Table 7 shows that, for concentration-dependent positive reactions, at low antibody concentrations (0.5-1 μg / mL), the standardized ratios were all <1.2 (negative), close to the blank control (ratio 1.00), and there was no significant prolongation of hemagglutination time for screening reagent (LA1) and confirmatory reagent (LA2). At an antibody concentration of 4 μg / mL, the standardized ratio increased to 1.14 (close to the weak positive threshold of 1.2), and the screening time began to prolong. When the antibody concentration reached 8 μg / mL and above, the standardized ratio gradient increased (1.44→1.87→2.04) with increasing concentration (8 μg / mL→20 μg / mL→30 μg / mL), reaching the standards for weak positive (8 μg / mL), moderate positive (20 μg / mL), and strong positive (30 μg / mL), respectively. Furthermore, the screening time (LA1) was significantly prolonged, while the confirmatory time (LA2) showed little change due to phospholipid neutralization, exhibiting a clear dose-dependent relationship. This conclusively confirms its LA-positive characteristic.

[0157] After reconstitution of the quality control samples, the standardized ratios of positive quality control samples at concentrations of 8 μg / mL and above were consistently >1.2 (positive) within 0.5 hours, 4 hours, and 8 hours. Specifically, the ratios at concentrations of 20 μg / mL and 30 μg / mL were stable at 1.80-1.87 and 2.04-2.16, respectively. The standardized ratio of the blank control remained stable at 1.00 throughout, indicating that the product can maintain stable positive activity within 8 hours after reconstitution.

[0158] Since SCT is an endogenous detection method, the sample needs to be tested in a timely manner. The following are the results of the detection of the positive control sample of this invention by the Wofen SCT reagent 0.5h after reconstitution, as shown in Table 8.

[0159] Table 8. Reconstitution effect of positive control samples

[0160]

[0161] Table 8 shows that the Werfen SCT reagent can detect positive control samples with a concentration of 8 μg / mL or higher. When the antibody concentration is 8 μg / mL, the SCT normalized ratio is 1.24, indicating a weak positive result. As the antibody concentration increases, the second value gradient increases by 10-20 seconds, and the normalized ratio changes from weak positive to moderate positive and then to strong positive, showing a dose-dependent relationship. This is consistent with the dRVVT screening results, further confirming that it has functional LA activity.

[0162] 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. An antigen-binding protein that specifically binds to the β2-glycoprotein I-cardiolipin complex, characterized in that, The antigen-binding protein contains a heavy chain variable region and a light chain variable region. The heavy chain variable region includes complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO.1-3, respectively, and the light chain variable region includes complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO.5-7, respectively. The β2-glycoprotein I-cardiolipin complex is a product of mixing β2-glycoprotein I with cardiolipin.

2. The antigen-binding protein according to claim 1, characterized in that, The heavy chain variable region contains a frame region VH-FR, wherein the frame region VH-FR includes the sequence shown in SEQ ID NO.4 or a sequence with at least 90% homology to it.

3. The antigen-binding protein according to claim 1, characterized in that, The light chain variable region includes a frame region VL-FR, wherein the frame region VH-FR includes the sequence shown in SEQ ID NO.8 or a sequence with at least 90% homology to it.

4. A nucleic acid molecule encoding the antigen-binding protein according to any one of claims 1-3.

5. An expression vector containing the nucleic acid molecule of claim 4.

6. A host cell containing the antigen-binding protein of any one of claims 1-3, the nucleic acid molecule of claim 4, or the expression vector of claim 5.

7. The use of the antigen-binding protein of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the host cell of claim 6 in the preparation of a β2-glycoprotein I-cardiolipin complex conjugate product.

8. The use of the antigen-binding protein according to any one of claims 1-3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, or the host cell according to claim 6 in the preparation of positive control products for lupus anticoagulants.

9. A positive control product for lupus anticoagulant, characterized in that, The lupus anticoagulant control product includes the antigen-binding protein as described in any one of claims 1-3, plasma, buffer system, preservative and excipient.

10. The lupus anticoagulant positive control product according to claim 9, characterized in that, The buffer system is selected from one or more of Tris-HCl, imidazole, Hepes buffer, and phosphate buffer.

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