Preparation method of β2-GP1-coated magnetic beads, magnetic beads prepared thereby, and applications thereof

Abstract

The present invention relates to the field of immunology, and particularly to a method for preparing β2-GP1-coated magnetic beads, the magnetic beads prepared thereby, and their applications. The present invention provides a chemiluminescent immunoassay kit for anti-β2 glycoprotein 1 magnetic microparticles. The present invention has studied the optimal conditions for the conformational change and solid-phase immobilization of β2-GP1. By studying the optimal allosteric conditions of β2-GP1, the allosteric process and the coating process are combined into one without introducing other substances, supporting each other and having an interaction. It can improve efficiency, reduce costs, and at the same time improve detection sensitivity. The native β2-GP1 antigen can be directly and stably linked to magnetic microparticles after allosteric transformation without complex processes such as dialysis, separation, and purification, which is convenient, fast, without loss, without introducing other substances, reducing interference and costs. Compared with native and recombinant antigens, the method of the present invention has stronger specificity, higher sensitivity, and better stability, and has good clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and particularly to a preparation method of β2-GP1-coated magnetic beads, the magnetic beads prepared thereby, and their applications. Background Art

[0002] Antiphospholipid syndrome (APS) is an autoimmune disease mainly manifested by recurrent arterial and venous thrombosis, morbid pregnancy (usually multiple unexplained miscarriages), thrombocytopenia, etc. The main clinical manifestation of APS is the formation of arterial and venous thrombosis, which is also common in many other diseases. Therefore, the diagnosis of APS depends on the detection of antiphospholipid antibodies (aPL). Anti-beta2 glycoprotein 1 antibody (aβ2-GP1) is one of the most important markers. However, due to its extreme heterogeneity and the detection differences of commercial kits from different manufacturers, it affects the judgment of clinicians and brings certain troubles to clinical diagnosis.

[0003] β2 glycoprotein 1 (β2-GP1), also known as apolipoprotein H (APOH), is encoded by the APOH gene in humans and is located on chromosome 17q23-24. It is a glycoprotein synthesized by hepatocytes, with a molecular weight of about 50 kD, existing in a free form in the blood circulation, and a concentration of about 150-300 μg / mL. β2-GP1 is composed of 5 domains (Domain 1-5), each domain being a short repeat sequence fragment (short consensus repeat, SCR), conforming to the structural model of complement control protein (CCP), and being a conserved CCP sequence. It is linked by Cys-Pro residues, and its Cys residues form disulfide bonds between cys1-3 and cys2-4. Domain Ⅴ additionally contains a disulfide bond and C-terminal residues, and its positively charged amino acid sequence (Lys282-Asn-Lys-Glu-Lys-Lys287) is the main site for binding to anionic phospholipids.

[0004] In normal human bodies (under physiological conditions), Domain 1 of β2-GP1 is connected to Domain 5, presenting a circular closed state (O type). In the bodies of APS patients (under pathological conditions), the positively charged Domain 5 of β2-GP1 binds to negatively charged phospholipids, presenting an open state (J type). At this time, the antigenic epitope is exposed and can bind to anti-β2-GP1 antibodies. The allosteric transformation of β2-GP1 from the closed state to the open state is a reversible process, and under different conditions, the degree of allosteric transformation is also inconsistent. If the conformation of β2-GP1 is not fully opened, it will affect the binding to some antibodies, resulting in missed detection.

[0005] Currently, the main detection methods for anti-β2-GP1 antibodies include enzyme-linked immunosorbent assay (ELISA), magnetic particle chemiluminescence immunoassay (CLIA), fluorescence enzyme immunoassay (FEIA), and multiplex bead immunoassay, etc. The detection principle of these methods is indirect detection of antibodies, that is, the solid-phase β2-GP1 antigen specifically binds to anti-β2-GP1 antibodies in the sample, and then horseradish peroxidase (HRP) / alkaline phosphatase (AP) or acridinium ester-labeled animal-derived anti-human IgG / IgM / IgA antibodies (labeled secondary antibodies) are connected.

[0006] The washing solution is used again to wash away free substances, and the substrate is added. The substrate is catalyzed or directly emits light, and the amount of the product is directly related to the amount of the substance to be detected in the specimen. Therefore, qualitative or quantitative analysis can be carried out according to the absorbance or luminescence value.

[0007] The most important factor affecting the detection of anti-β2-GP1 antibodies is the solid-phase immobilization of β2-GP1 antigen.

[0008] Currently, there are two coating methods: First, directly immobilize natural or recombinant β2-GP1 antigen; Second, pretreat (after allosteric transformation) the natural β2-GP1 antigen and then carry out solid-phase immobilization.

[0009] The first method: Passively adsorb natural or recombinant β2-GP1 antigen onto a 96-well microplate or chemically link it to magnetic particles.

[0010] Related researchers carried out differential research on 5 components of the ELISA experiment: coating buffer, microplate labeling solution, blocking buffer, dilution buffer, and enzyme-labeled secondary antibody. It was found that there are significant differences between different brands of microplates. There are also certain differences in the detection of anti-β2-GP1 antibodies between ELISA and CLISA of the same manufacturer. The main reasons for the differences are that the allosteric effects of β2-GP1 antigen under different solid-phase immobilization conditions are different, and secondly, there are also differences in the spatial structures of recombinant antigens and natural antigens.

[0011] The second method: After pre-treating (allosterically changing) the natural β2-GP1 antigen, solid-phase immobilization is carried out. In related research, by mixing natural β2-GP1 with bacterial lipopolysaccharide A and then adjusting the pH and NaCl concentration, the conformation of β2-GP1 is opened. First, dialyze natural β2-GP1 in 10 mM - 40 mM HEPES buffer (sodium ion concentration is 1.15 M, pH is 11.5) for 36 h - 60 h, and then dialyze it in 20 mM HEPES buffer (sodium ion concentration is 0.15 M, pH is 7.4) for 8 h. The dialyzed β2-GP1 is mixed with bacterial lipopolysaccharide A (mass ratio is 100 - 750:1), separated and purified using a HiTrap chelating column, and then stored. Subsequently, it is coated onto carboxyl magnetic beads under the condition of pH = 5.0.

[0012] In a normal human body (physiological state), Domain 1 of β2-GP1 is connected to Domain 5, presenting a circular closed state (O type). In the body of APS patients (pathological state), the positively charged Domain 5 of β2-GP1 binds to negatively charged phospholipids, presenting an open state (J type). At this time, the antigenic epitope is exposed and can bind to anti-β2-GP1 antibodies. The allosteric change of β2-GP1 from the closed state to the open state is a reversible process, and under different conditions, the degree of allosteric change is also inconsistent. If the conformation of β2-GP1 is not fully opened, it will affect the binding to some antibodies, resulting in missed detection.

[0013] For the first case, there are the following disadvantages:

[0014] 1. Coating with natural antigen: Direct coating of the natural β2-GP1 antigen, the closed conformation is not opened, or the conformation is not fully opened, resulting in some hidden sites not being fully exposed, which will cause missed detection.

[0015] 2. Coating with recombinant antigen: The antibody-binding sites of β2-GP1 belong to both linear and conformational sites, while the recombinant antigen cannot construct a complete spatial conformation, which will cause missed detection.

[0016] For the second case, there are the following disadvantages:

[0017] By adjusting the pH and salt concentration, it can indeed cause allosteric change of β2-GP1. However, this invention has the following disadvantages:

[0018] 1. Dialysis and separation and purification are required, the process is complex, and it takes a long time;

[0019] 2. The dialysis solution A used is 20 mM HEPES buffer with pH = 11.5, which exceeds its buffering range of 6.8 - 8.2;

[0020] 3. The introduction of additional substances (bacterial lipopolysaccharide A) may potentially affect the function of β2-GP1 and interfere with the detection results;

[0021] 4. The dialysis and separation and purification processes will cause losses of raw materials, increase costs, and there are batch-to-batch differences;

[0022] 5. The allosteric change of β2-GP1 is reversible. Under acidic conditions, β2-GP1 can be restored from the open state to the closed-loop state. Therefore, coating β2-GP1 onto carboxyl magnetic beads under the condition of pH = 5.0 will cause the structure of β2-GP1 to convert to the cyclic closed state, reducing the detection sensitivity. Summary of the Invention

[0023] In view of this, the technical problem to be solved by the present invention is to provide a preparation method of β2-GP1-coated magnetic beads, the magnetic beads prepared thereby, and their applications.

[0024] The present invention provides a preparation method of β2-GP1 magnetic beads, which includes: after activating carboxyl magnetic beads, mixing the magnetic beads with a β2-GP1 solution, making up the volume with an alkaline buffer solution having a concentration of 0.05 - 1.0 M and a pH value of 9 - 11.5, oscillating and reacting for coating for 30 - 120 min, and separating the magnetic beads;

[0025] The method of the present invention can achieve the allosteric change of β2-GP1 protein during coating. Not only are the reaction conditions simple, but also the obtained magnetic beads have good specificity, high sensitivity, and high stability. Among the parameters of this preparation method, such as the selection of the alkaline buffer solution, the concentration of the alkaline buffer solution, the pH value of the alkaline buffer solution, and the oscillating reaction coating time, each experimental condition supports each other, there is an interaction, and jointly a good effect of coating magnetic beads is achieved.

[0026] The magnetic beads described in the present invention are selected from carboxyl magnetic beads, amino magnetic beads, or tosyl magnetic beads. In the embodiments of the present invention, carboxyl magnetic beads are preferably used.

[0027] In some embodiments, the pH value of the alkaline buffer solution is 9 - 11.5; in some specific embodiments, the pH value of the alkaline buffer solution is 9; in some other specific embodiments, the pH value of the alkaline buffer solution is 9.6; in some specific embodiments, the pH value of the alkaline buffer solution is 10.5; in some other specific embodiments, the pH value of the alkaline buffer solution is 11; in some other specific embodiments, the pH value of the alkaline buffer solution is 11.5. Experiments show that when the pH value is 9.6, it can better cooperate with other parameters, the obtained magnetic beads have the strongest experimental reactivity, and the background value is relatively low, and the correlation coefficient r with known samples is the highest.

[0028] The alkaline buffer solution is selected from: carbonate buffer solution, glycine buffer solution, boric acid buffer solution, disodium hydrogen phosphate buffer solution or HEPES buffer solution. Experiments show that, compared with other buffer solutions, using carbonate buffer solution can better cooperate with other parameters, and the magnetic beads prepared have the strongest experimental reactivity and the best correlation with samples of known concentration.

[0029] Furthermore, experiments have proved that carbonate buffer solutions with a concentration of 0.05 - 0.25 M and a pH of 9 - 11.5 can all be used to prepare magnetic beads with good performance. However, when the concentration of the carbonate buffer solution is 0.05 M and the pH value is 9.6, it can better cooperate with other parameters, and the prepared magnetic beads have higher reactivity, the lowest background value, and the highest correlation with known samples. Specifically, the carbonate buffer solution includes water, sodium carbonate and sodium bicarbonate. In the carbonate buffer solution with a concentration of 0.05 M, the concentration of sodium carbonate is 0.05 M, and the concentration of sodium bicarbonate is 0.05 M.

[0030] The method of the present invention simultaneously performs allosteric transformation and coating of the antigen, and adopts an alkaline environment during the coating process, which avoids the transformation of β2-GP1 protein into a cyclic closed state, and does not require dialysis for separation and purification. The process is simple, time-consuming is less, raw material loss is reduced, and cost is lowered. And during the preparation process, no extra impurities are introduced, there is no non-specific adsorption affecting the function of β2-GP1, and the detection result will not be interfered. Therefore, the magnetic beads prepared by the method of the present invention have stronger specificity, higher sensitivity and better stability.

[0031] In the method of the present invention, no NaCl is added to the involved buffer solutions. Experiments show that, compared with the condition of adding NaCl, under the condition of not adding NaCl, the experimental reactivity is stronger, there is no non-specific adsorption, and the prepared magnetic beads have stronger specificity, high sensitivity and better stability.

[0032] The present invention optimizes the temperature and time of the coating reaction. It can be known from experiments that the coating reaction can be completed at 4 - 37°C. However, when coating magnetic beads at 4°C, the overall experimental reactivity is relatively low, and the correlation coefficient r of samples of known concentration is 0.9894, which is relatively the lowest. However, at room temperature (20 - 25°C) and 37°C, it can better cooperate with parameters such as buffer solution, and the overall reactivity and correlation coefficient r are basically the same and relatively high. Considering that coating magnetic beads at 37°C is not conducive to subsequent large-scale production, it is therefore preferred to perform the coating at room temperature (20 - 25°C).

[0033] In the method of the present invention, the time for coating the oscillating reaction is 30 to 120 min. In some embodiments, the time is 30 to 60 min; in other embodiments, the time is 60 to 120 min; it can be known from experiments that when coating for 30 min to 60 min, the reactivity gradually increases, and the reactivity basically no longer changes at 60 to 120 min. Therefore, it is considered that the experimental reactivity is the strongest at 60 min, and the correlation coefficient r is higher. Therefore, 60 min is preferably combined with other parameters to obtain a good magnetic bead coating effect.

[0034] In the method of the present invention, the β2-GP1 solution includes: β2-GP1, HEPES buffer solution and glycerol, wherein the concentration of β2-GP1 is 0.1 to 0.5 mg / mL, and the volume fraction of glycerol is 20 vol%. And when the concentration of β2-GP1 is 0.3 mg / mL, it can better cooperate with other experimental parameters to obtain a good magnetic bead coating effect.

[0035] In the method of the present invention, before magnetic bead coating, it further includes a step of activating the magnetic beads. After the coating, it further includes a step of making up the volume with magnetic particle buffer solution. The activation refers to activating the carboxyl groups on the surface of the magnetic beads, which includes two steps. Step 1: Wash, oscillate and magnetically separate the carboxyl magnetic beads; Step 2: Add 5 to 20 μg / ml EDC and 5 to 20 μg / ml NHS to the magnetic beads, and oscillate and react at room temperature for 30 to 120 min.

[0036] In the experiment, the magnetic beads coated according to the method of the present invention were stored at 2 to 8 °C and 37 °C for 14 days respectively, and then the variation range of the luminescence value was compared. The experimental results show that for the magnetic beads coated by the method of the present invention, the decrease rate after heating at 37 °C for 14 days is <5% compared with that at 2 to 8 °C. It is estimated that it can be placed at 2 to 8 °C for 24 months, indicating that it has better stability.

[0037] The present invention provides the application of the magnetic beads in the preparation of a detection reagent for β2-GP1 antibody.

[0038] The present invention provides a detection reagent for β2-GP1 antibody, which includes a magnetic bead suspension and an enzyme-labeled antibody solution;

[0039] The magnetic bead suspension includes the magnetic beads prepared by the preparation method and a magnetic particle buffer solution; the enzyme-labeled antibody solution includes an HRP-labeled anti-human antibody. The detection reagent provided by the present invention has strong specificity, high sensitivity and good stability.

[0040] In the detection reagent of the present invention, the density of the magnetic beads in the magnetic bead suspension is 1 mg / mL; the magnetic particle buffer solution includes: water, Tris, NaCl, HCl, bovine serum albumin, Proclin300 and glycerol. In some embodiments, the magnetic bead suspension includes water and the following concentrations: 6.05 g / L of Tris, 8.20 g / L of NaCl, 1 wt% - 3 wt% of bovine serum albumin, 0.05 wt% - 0.2 wt% of Proclin300, and 1 vol% - 10 vol% of glycerol. In some embodiments, in the magnetic bead suspension, the concentration of the magnetic beads is 1 mg / mL.

[0041] In the detection reagent of the present invention, it further includes a sample diluent and / or a calibrator;

[0042] The sample diluent includes water, Tris, NaCl, HCl, bovine serum albumin and Proclin300;

[0043] The calibrator includes a negative serum-diluted anti-β2-GP1 antibody positive sample, and six calibrators with concentrations of 0, 10, 30, 90, 180, and 360 RU / mL are prepared.

[0044] In the detection reagent of the present invention, the detection reagent includes a kit. In the kit, it further includes a magnetic particle buffer solution, a magnetic particle suspension, and an HRP-labeled anti-human antibody. The anti-human antibody is a mouse anti-human antibody, a rabbit anti-human antibody, and a goat anti-human antibody.

[0045] The present invention also provides a method for detecting β2-glycoprotein 1, which includes using the detection reagent to detect a test substance.

[0046] The present invention provides a method for preparing β2-GP1 magnetic beads. By providing the optimal allosteric conditions of β2-GP1, the allosteric process and the coating process are combined into one without introducing other substances. The steps support each other and there are interactions. It can improve efficiency, reduce costs, reduce batch-to-batch differences introduced by raw material processing, and at the same time improve detection sensitivity. The natural β2-GP1 antigen can be directly and stably linked to the magnetic particles after allosteric transformation, without the need for complex processes such as dialysis and separation and purification, which is convenient and fast without loss. At the same time, no other substances are introduced, reducing interference and costs. Compared with natural and recombinant antigens, the method of the present invention has stronger specificity, higher sensitivity, and better stability, and has good clinical application prospects. Detailed implementation mode

[0047] The present invention provides a method for preparing β2-GP1-coated magnetic beads, the magnetic beads prepared thereby, and their applications. Those skilled in the art can draw on the content herein and appropriately modify process parameters to achieve the same. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can clearly make changes or appropriate alterations and combinations to the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0048] In the present invention, the β2-GP1 used is natural β2-GP1, which is a commercially available antigen purified naturally by procurement, and its amino acid sequence is:

[0049]

[0050]

[0051] In the present invention,

[0052] The carbonate buffer solution includes water, sodium carbonate, and sodium bicarbonate.

[0053] The glycine buffer solution includes water, glycine, and sodium chloride.

[0054] The boric acid buffer solution includes water, boric acid, sodium tetraborate, and sodium chloride.

[0055] The disodium hydrogen phosphate buffer solution includes water, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

[0056] The HEPES buffer solution includes water, HEPES, and NaOH.

[0057] The following will describe the implementation schemes of the present invention in detail with reference to the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0058] Example 1

[0059] Preparation of the magnetic particle suspension according to the present invention:

[0060] I. Preparation process of the magnetic particle buffer solution, taking 1 L as an example:

[0061] (1) Measure 900 ml of purified water into a container, weigh 6.05 g of tris(hydroxymethyl)aminomethane (Tris) and 8.20 g of NaCl and add them to the container, and stir well until completely dissolved.

[0062] (2) Adjust the pH with concentrated hydrochloric acid and control it at 7.4 ± 0.05;

[0063] (3) Weigh 1% - 3% bovine serum albumin (BSA), 0.05% - 0.2% Proclin 300, measure 1% - 10% glycerol, add them into a container, and stir well until completely dissolved;

[0064] (4) Store at 2°C - 8°C.

[0065] II. Preparation process of magnetic particle suspension, taking 3 ml as an example:

[0066] (1) The magnetic beads contain carboxyl (COOH) active groups. The carboxyl content per gram (dry weight) of the magnetic beads is about 30 μg. They are uniform particles with a particle diameter of 1.5 μm - 3 μm.

[0067] (2) 2 - Morpholinoethanesulfonic acid (MES), N - (3 - Dimethylaminopropyl) - N' - ethylcarbodiimide hydrochloride (EDC), Tris(hydroxymethyl)aminomethane (Tris), N - Hydroxysuccinimide (NHS), Sodium carbonate (Na2CO3), Sodium bicarbonate (NaHCO3), Sodium chloride (NaCl) and other reagents should reach chemical purity.

[0068] (3) Take 30 μl of the magnetic bead stock solution, add 300 μl of MES buffer with pH = 5.0 ± 0.05 for washing, shake for 5 min, perform magnetic separation, discard the supernatant, and repeat the washing 5 times;

[0069] (4) Weigh EDC and NHS, and respectively prepare solutions with a concentration of 5 μg / ml - 20 μg / ml (preferably 10 μg / ml) using MES buffer with pH = 5.0 ± 0.05. Add 50 μl of each solution to the magnetic beads, and shake and react at room temperature for 30 min - 120 min (preferably 60 min);

[0070] (5) Perform magnetic separation, discard the supernatant, add 10 μg - 50 μg of natural β2 - GP1, use 0.05 M alkaline buffer (carbonate buffer, pH = 9.6 ± 0.1), make up the volume to 100 μl, and shake and react at room temperature for 30 min - 120 min, preferably 60 min;

[0071] (6) Perform magnetic separation, discard the supernatant, add 300 μl of magnetic particle buffer, shake and wash for 5 min, and repeat the washing 4 times;

[0072] (7) Make up the volume to 3 ml with magnetic particle buffer, with a concentration of 1 mg / mL, and complete the preparation of the magnetic particle suspension;

[0073] Preparation of enzyme conjugate reagent:

[0074] III. The preparation steps of the enzyme conjugate reagent are as follows, taking 1 L as an example:

[0075] (1) Measure 1000 ml of purified water into a container, weigh 6.05 g of Tris and 8.20 g of NaCl and add them to the container, and stir well until completely dissolved;

[0076] (2) Adjust the pH with concentrated hydrochloric acid and control it at 7.4 ± 0.05;

[0077] (3) Weigh 1% - 3% bovine serum albumin (BSA) and 0.05% - 0.2% Proclin 300, add them to the container, and stir well until completely dissolved;

[0078] (4) Take 100 μl - 1000 μl of HRP-labeled mouse / rabbit / goat anti-human IgG / IgM / IgA antibody and stir for 30 min;

[0079] (5) Store at 2°C - 8°C.

[0080] IV. Preparation of sample diluent reagent:

[0081] (1) Measure 1000 ml of purified water into a container, weigh 6.05 g of Tris and 8.20 g of NaCl and add them to the container, and stir well until completely dissolved;

[0082] (2) Adjust the pH with concentrated hydrochloric acid and control it at 7.4 ± 0.05;

[0083] (3) Weigh 1% - 3% bovine serum albumin (BSA) and 0.05% - 0.2% Proclin 300, add them to the container, and stir well until completely dissolved;

[0084] (4) Store at 2°C - 8°C.

[0085] V. Preparation of calibrators:

[0086] Dilute the anti-β2-GP1 antibody positive sample with negative serum to prepare 6 calibrators with concentrations of 0, 10, 30, 90, 180, 360 RU / mL;

[0087] The working principle and reaction steps of the anti-β2 glycoprotein 1 antibody quantitative determination kit are as follows:

[0088] The sample is first mixed with the sample diluent and the magnetic particle suspension, incubated at 37 °C for 15 min. The anti-β2-GP1 antibody in the sample binds to the allosteric β2-GP1 antigen coated on the magnetic beads. After incubation, the reaction solution is washed and separated 5 times with the washing solution. Then the enzyme conjugate is added and incubated at 37 °C for 15 min. A complex of β2-GP1 antigen - anti-β2-GP1 antibody - mouse / rabbit / goat anti-human IgG / IgM / IgA antibody - HRP is formed. After incubation, the reaction solution is washed and separated 5 times with the washing solution. The luminescent substrate is added. The reaction solution is mixed evenly, and the luminescence intensity is detected within 1 - 5 minutes. HRP will catalyze the luminescent substrate to emit photons, and the luminescence intensity is proportional to the content of anti-β2-GP1 antibody.

[0089] The sample addition mode and reaction steps for calibrator calibration are the same as those of the sample. The validity period of the calibration curve is 28 days, and repeated calibration is not required.

[0090] Example 2

[0091] Parameter improvement is carried out for item (6) in step two of Example 1:

[0092] 1. For different pH comparisons, 0.05M carbonate buffer solution is used. Detect 10 samples calibrated with other commercial kits and blank samples.

[0093]

[0094]

[0095] As can be seen from the above table, within the range of pH 9 - 11.5, the reactivity first increases and then decreases (the detection signal value of sample 10 significantly decreases within the range of pH 11 - 12). When pH = 9.6, the reactivity is the strongest, and the background value is relatively low (the detection limit is lower), and the correlation coefficient r with the known samples is the best. The preferred pH is 9.6.

[0096] 2. For different buffer comparisons, the pH is adjusted to 9.6, and the buffer concentration is 0.05M. Detect 10 samples calibrated with other commercial kits and blank samples.

[0097]

[0098] As can be seen from the above table, when pH = 9.6, the reaction trends of different buffers are the same (except for boric acid buffer). Among them, the carbonate buffer has the strongest reactivity and the best correlation with the samples of known concentration. The preferred buffer is the carbonate buffer.

[0099] 3. For different carbonate concentration buffers, the pH is adjusted to 9.6. Detect 10 samples calibrated with other commercial kits and blank samples.

[0100]

[0101] As can be seen from the above table, as the buffer concentration increases, the reactivity and the correlation coefficient r with the samples of known concentration gradually weaken. A buffer solution of 0.05 M is preferred.

[0102] 4. Different NaCl concentrations, 0.05 M carbonate buffer, pH = 9.6. Detect 10 samples calibrated with other commercial kits and blank samples.

[0103]

[0104]

[0105] As can be seen from the above table, there is no obvious pattern in the range of 0 - 0.3 M of NaCl concentration. The reactivity is the strongest at 0.3 M, but there is non-specific adsorption at the same time (blank luminescence value > 20,000 RLU). After the NaCl concentration is greater than 0.3 M, the reactivity gradually weakens. Therefore, it is not recommended to add NaCl.

[0106] In summary, a carbonate buffer solution of 0.05 M (pH = 9.6) is preferred for the solid-phase coating of β2-GP1.

[0107] 5. Coating assessment under different temperature conditions. Detect 10 samples calibrated with other commercial kits and blank samples.

[0108]

[0109]

[0110] As can be seen from the above table, when coating at 4°C, the overall reactivity is relatively low, and the correlation coefficient r with the samples of known concentration is 0.9894, which is the relatively lowest.

[0111] Compared with 25°C and 37°C, the overall reactivity and the correlation coefficient r are basically the same. Considering that the coating environment at 37°C is relatively unfavorable for subsequent large-scale production coating, it is therefore preferred to coat at 25°C (under room temperature conditions).

[0112] 6. Coating assessment for different coating times. Detect 10 samples calibrated with other commercial kits and blank samples.

[0113]

[0114] As can be seen from the above table, when the coating time is 30 min - 1 h, the reactivity is still increasing, and the reactivity basically does not change from 1 h to 2 h. Therefore, 1 h is preferred as the coating time.

[0115] 7. The present invention (allosterically modifying and coating the antigen simultaneously) and the comparative method (first allosterically modifying the antigen and then coating it); 10 samples calibrated with other commercial kits and blank samples were detected simultaneously.

[0116] The comparative method (first allosterically modifying the antigen and then coating it) uses the method described in Patent CN 111793122A: Dialyze natural β2-GP1 in 20 mM HEPES buffer (sodium ion concentration is 1.15 M, pH is 11.5) for 48 h, and then dialyze it in 20 mM HEPES buffer (sodium ion concentration is 0.15 M, pH is 7.4) for 8 h. The dialyzed β2-GP1 is mixed with bacterial lipopolysaccharide A (mass ratio is 500:1), separated and purified using a HiTrap chelating column, and stored. Subsequently, it is coated onto carboxyl magnetic beads under the condition of pH = 5.0.

[0117] Comparative Example 1: Dialyze natural β2-GP1 in 20 mM HEPES buffer (sodium ion concentration is 1.15 M, pH is 11.5) for 48 h, and then dialyze it in 20 mM HEPES buffer (sodium ion concentration is 0.15 M, pH is 7.4) for 8 h. The dialyzed β2-GP1 is mixed with bacterial lipopolysaccharide A (mass ratio is 500:1), separated and purified using a HiTrap chelating column. Then, solid-phase coating of β2-GP1 is carried out using 0.05 M carbonate buffer (pH = 9.6).

[0118] Comparative Example 2: Dialyze natural β2-GP1 in 20 mM HEPES buffer (sodium ion concentration is 1.15 M, pH is 11.5) for 48 h, and then dialyze it in 20 mM HEPES buffer (sodium ion concentration is 0.15 M, pH is 7.4) for 8 h. The dialyzed β2-GP1 is mixed with bacterial lipopolysaccharide A (mass ratio is 500:1), separated and purified using a HiTrap chelating column. Then, solid-phase coating of β2-GP1 is carried out using 0.1 M MES buffer (pH = 5.0).

[0119] Comparative Example 3: Dialyze natural β2-GP1 in 20 mM HEPES buffer (sodium ion concentration is 1.15 M, pH is 11.5) for 48 h, and then dialyze it in 20 mM HEPES buffer (sodium ion concentration is 0.15 M, pH is 7.4) for 8 h. Then, solid-phase coating of β2-GP1 is carried out using 0.05 M carbonate buffer (pH = 9.6).

[0120]

[0121]

[0122] As can be seen from the above table,

[0123] ① As can be seen from Comparative Example 1 and Comparative Example 2, samples 4 and 6 in Comparative Example 2 were detected as negative, resulting in missed detections. The reason was that conventional acidic coating was used, which caused reverse allosteric change of the antigen and reduced the detection rate.

[0124] ② As can be seen from Comparative Example 1 and Comparative Example 3, the background value and negative samples of Comparative Example 1 were relatively high. The reason might be non-specific adsorption caused by the introduction of bacterial lipopolysaccharide A (the purification efficiency could not reach 100% to completely separate bacterial lipopolysaccharide A).

[0125] ③ As can be seen from the present invention and Comparative Example 3, the reactivity of samples 1-10 in Comparative Example 3 was relatively low, and the correlation coefficient r with the known concentration samples also decreased. The reason might be the loss of antigen amount during the dialysis and separation purification processes, resulting in a lower amount of antigen coated on the magnetic particles.

[0126] In summary, compared with the present invention, the method described in Patent CN 111793122A has the following disadvantages:

[0127] ① Dialysis and separation purification are required, the process is complex, and it takes a long time.

[0128] ② The introduction of additional other substances (bacterial lipopolysaccharide A) may potentially affect the function of β2-GP1 and interfere with the detection results.

[0129] ③ The dialysis and separation purification processes will cause loss of raw materials and increase costs.

[0130] ④ The allosteric change of β2-GP1 is reversible. Under acidic conditions, β2-GP1 can be restored from the open state to the closed-loop state. Therefore, coating β2-GP1 onto carboxyl magnetic beads under the condition of pH = 5.0 will cause the structure of β2-GP1 to convert to the cyclic closed state, reducing the detection sensitivity.

[0131] 8. Clinical coincidence rate.

[0132] The diagnosis of APS must meet at least 1 clinical symptom (thrombosis-related symptom or morbid pregnancy) and 1 laboratory positive index (persistent positivity of any anti-phospholipid antibody). In this experiment, 188 patient samples with APS-related clinical symptoms and 100 samples of apparently healthy people were selected, and Comparative Example 1 (using a coating environment with pH = 5.0), Comparative Example 2 (using recombinant antigen), and a commercial ELISA kit were compared in parallel with Example 1 (the optimal method described in the present invention). The results are as follows:

[0133]

[0134] As can be seen from the above table, a total of 100 samples from healthy individuals were detected, and all four detection methods showed negative results. For the detection of 188 samples with APS-related symptoms, the sensitivity of Example 1 was the highest, at 63.30% (119 cases), the sensitivity of the commercial kit was 48.40% (91 cases), the sensitivity of Comparative Example 1 was 28.72% (54 cases), and the sensitivity of Comparative Example 2 was 42.02% (79 cases). The sensitivity of Example 1 was the best, and the specificities were all the same.

[0135] 9. Stability

[0136] The magnetic beads coated according to the description of the present invention were stored at 2°C to 8°C and 37°C for 14 days respectively, and then the variation range of the luminescence value was compared.

[0137]

[0138]

[0139] As can be seen from the above table, for the magnetic beads coated according to the description of the present invention, the reduction rates at 37°C for 14 days and at 2°C to 8°C were both <5%. It is estimated that they can be stored at 2°C to 8°C for 24 months. Therefore, they have better stability.

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Preparation method of β2-GP1-coated magnetic beads, characterized in that, Comprising: After carboxyl magnetic beads are activated, the magnetic beads are mixed with β2-GP1 solution, made up to volume with carbonate buffer solution with a concentration of 0.05 M and a pH value of 9.6, shaken and reacted for 30 - 120 min, and the magnetic beads are separated; The carbonate buffer solution comprises sodium carbonate and sodium bicarbonate, wherein the concentration of sodium carbonate is 0.05 M and the concentration of sodium bicarbonate is 0.05 M.

2. The method according to claim 1, wherein The temperature of the shaking reaction is 25 - 37 °C and the time is 1 h.

3. The method according to claim 1 or 2, characterized in that, The β2-GP1 solution comprises: β2-GP1, HEPES buffer solution, glycerol, wherein the concentration of β2-GP1 is 0.3 mg / mL.

4. Detection reagent for β2-GP1 antibody, which comprises magnetic bead suspension and enzyme-labeled antibody solution; The magnetic bead suspension includes magnetic beads prepared by the preparation method according to any one of claims 1 to 3 and a magnetic particle buffer solution; the enzyme-labeled antibody solution includes an HRP-labeled anti-human antibody; the density of the magnetic beads in the magnetic bead suspension is 1 mg / mL; the magnetic particle buffer solution includes: Water, Tris, NaCl, HCl, bovine serum albumin, Proclin300 and glycerol.

5. The detection reagent according to claim 4, wherein It further comprises sample diluent and / or calibrator; The sample diluent comprises water, Tris, NaCl, HCl, bovine serum albumin and Proclin300; The calibrator comprises negative serum diluted anti-β2-GP1 antibody positive samples, and 6 calibrators with concentrations of 0, 10, 30, 90, 180, 360 RU / mL are prepared.

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