D-dimer chemiluminescence assay kit

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a D-dimer chemiluminescence assay kit. The present application provides a D-dimer chemiluminescence assay kit, which comprises a magnetic particle suspension (immunomagnetic beads) coated with D-dimer antibody 1, alkaline phosphatase-labeled D-dimer antibody 2 (enzyme-labeled antibody) and a calibrant. The immunomagnetic beads and the enzyme-labeled antibody are prepared by chemical coupling and stored in a buffer containing a soluble high molecular material, which can effectively improve the detection sensitivity and repeatability. The kit can be used in combination with a substrate solution containing a chromogenic substrate such as adamantane and its derivative AMPPD or APS-5 for a full-automatic immune test system, and the content of D-dimer in plasma or whole blood can be determined by a chemiluminescence instrument. The kit has high detection sensitivity and good repeatability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a kit for the determination of D-dimer chemiluminescence. Background Technology

[0002] The fibrinolytic system is an important anticoagulant system in the human body. D-Dimer mainly originates from cross-linked fibrin clots dissolved by plasmin in this system. During fibrinolysis, thrombin hydrolyzes plasminogen, releasing fibrin peptide A and fibrin peptide B. The remaining soluble fibrin monomers, under the influence of factor XII Ia, form stable cross-linked fibrin. During the degradation of cross-linked fibrin by plasmin, the released fragments further degrade into the smallest fragment, D-Dimer. Under physiological conditions, the body maintains a dynamic balance between coagulation and fibrinolysis to ensure timely fibrin formation and clearance. Under pathological conditions, this dynamic balance is disrupted, increasing the tendency to coagulate and thus increasing fibrin degradation products, leading to elevated D-Dimer levels. Therefore, D-Dimer can serve as a molecular marker for hypercoagulable states and hyperfibrinolysis in vivo. In clinical examinations, D-Dimer is a key indicator of deep vein thrombosis (DVT), pulmonary embolism (PE), and disseminated intravascular coagulation (DIC). It also has important clinical significance for the early diagnosis, disease monitoring, and monitoring of thrombolytic drug treatment for thrombotic diseases.

[0003] Currently, the conventional detection methods for D-Dimer include four types: enzyme-linked immunosorbent assay (ELISA), latex agglutination, colloidal gold method, and latex-enhanced immunoturbidimetric assay. Among the various methods, ELISA, primarily employing a double-antibody sandwich method, is recognized as a highly accurate and specific detection method. However, it is complex to operate, time-consuming, and influenced by numerous factors, making it unsuitable for rapid clinical diagnostic testing. Latex agglutination is a qualitative or semi-quantitative method for determining D-Dimer content. While simple to operate and rapid, the results rely heavily on visual observation, leading to significant subjective variations and making it unsuitable for large-scale testing. Colloidal gold immunopermeability is a simple method suitable for quantitative determination of single or batch samples, but its sensitivity and negative predictive value (excluding venous thromboembolism) have limitations in clinical applications. Latex-enhanced immunoturbidimetry is currently the most widely used detection method. It primarily measures the turbidity change after antigen-antibody binding, reflecting the D-Dimer content in plasma. Its advantages include rapid detection, high sensitivity, and stability. However, due to the limitations of the latex immunoturbidimetric detection principle, large-particle immunoturbidimetric latex exhibits high sensitivity but a narrow linear range, while small-particle immunoturbidimetric latex has low sensitivity but a wide linear range, making it impossible to simultaneously meet both sensitivity and linear range requirements.

[0004] In recent years, with the development of nanomaterials science, magnetic nanoparticles have received widespread attention, especially in the medical field, where some researchers have already applied them to clinical diagnosis. Magnetic nanoparticles are characterized by uniform fixation, rapid separation, and good reproducibility. The magnetic nanoparticle chemiluminescence immunoassay, combining magnetic nanoparticles with ELISA (double antibody sandwich and enzyme-catalyzed chemiluminescence technology), is currently the fastest-growing and most advanced immunoassay technique. Its sensitivity is higher than that of latex immunoturbidimetry, while also exhibiting a wider linear range. Furthermore, this method combines the high specificity of immunoassay, the stability of chemiluminescence measurements, and the advantages of simple and highly automated magnetic separation.

[0005] Chemiluminescence assay kits primarily rely on magnetic separation technology, double-antibody sandwich method, and enzyme-catalyzed chemiluminescence principles to achieve the quantitative determination of specific antigens (see details). Figure 1 In this type of kit, the antigen first binds to the antibody coated on the magnetic bead surface, forming a stable antigen-antibody complex. Then, the enzyme-labeled antibody binds to the exposed (opposite) antigenic determinant, ultimately forming a double-antibody sandwich complex. After magnetic separation, the complex is mixed with a luminescent substrate solution. The terminal alkaline phosphatase catalyzes the hydrolysis of luminescent substrates such as AMPPD and APS-5, emitting a high-intensity light signal. This signal value is positively correlated with the alkaline phosphatase concentration and can be used to quantify the concentration of alkaline phosphatase (double-antibody sandwich complex). Throughout the process, the formation of the double-antibody sandwich complex is fundamental for quantitative assay, and the antigen-antibody reaction is crucial for its formation. Ideally, the antigen and antibody react rapidly in the mixed reaction solution, forming a double-antibody sandwich complex within a short time after contact. However, many factors can interfere with the immune reaction, including antigen-antibody affinity, reaction concentration, environmental temperature, pH, and electrolyte conditions, all of which affect the antigen-antibody binding effect.

[0006] To address the aforementioned issues, biotin-avidin amplification systems are often used in reagent kit research to enhance sensitivity and improve measurement stability during magnetic bead antibody conjugation. However, reagent kits relying on this system are susceptible to interference from excessive (endogenous / exogenous) biotin. Summary of the Invention

[0007] In view of this, the present invention provides a chemiluminescence assay kit for D-dimer. The present invention provides the application of soluble polymeric materials in the preparation of products with enhanced D-dimer detection sensitivity; the soluble polymeric materials include one or more of polyethylene glycol, povidone, dextran, polyethyleneimine, cellulose, or amine polysaccharides. The present invention has experimentally discovered that the immunomagnetic beads and enzyme-labeled antibodies prepared by chemical coupling and stored in a buffer solution containing soluble polymeric materials can effectively improve detection sensitivity and repeatability. The kit of the present invention can be used in conjunction with substrate solutions for fully automated immunoassay systems containing chromogenic substrates such as adamantane and its derivatives AMPPD or APS-5, to determine the content of D-dimer in plasma or whole blood using a chemiluminescence analyzer. The kit of the present invention has high detection sensitivity and good repeatability.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides the application of soluble polymer materials in the preparation of products with enhanced detection sensitivity and repeatability of D-dimer;

[0010] The soluble polymeric material includes one or more of polyethylene glycol, povidone, dextran, polyethyleneimine, cellulose, or aminopolysaccharides.

[0011] The present invention also provides a storage buffer, the storage buffer comprising one or more of a sensitizer, a buffer system, an ion concentration regulator, a stabilizer, or a preservative;

[0012] The sensitizer includes soluble polymeric materials;

[0013] The soluble polymeric material includes one or more of polyethylene glycol, povidone, dextran, polyethyleneimine, cellulose, or aminopolysaccharides.

[0014] In some specific embodiments of the present invention, the povidone includes povidone K30.

[0015] In some specific embodiments of the present invention, the polyethylene glycol molecules include 200 to 20,000; and / or

[0016] The cellulose compounds include sodium carboxymethyl cellulose and / or ethyl cellulose; and / or

[0017] The amino polysaccharides include chondroitin sulfate and / or chitosan.

[0018] In some specific embodiments of the present invention, the polyethylene glycol includes PEG8000.

[0019] In some specific embodiments of the present invention, the concentration of the polyethylene glycol is 0.5–6.0% (w / v); and / or

[0020] The concentration of the povidone is 0.5–5.0% (w / v); and / or

[0021] The concentration of the dextran is 1.0–6.0% (w / v); and / or

[0022] The concentration of the polyethyleneimine is 0.5–3.0% (w / v); and / or

[0023] The concentration of the cellulose is 0.1–0.4% (w / v); and / or

[0024] The concentration of the amino polysaccharides is 0.25–2.0% (w / v).

[0025] In some specific embodiments of the present invention, the buffer system of the storage buffer includes MOPS buffer, Tris-HCl buffer, PBS buffer, HEPES buffer or glycine buffer;

[0026] Tris-HCl buffer is preferred.

[0027] In some specific embodiments of the present invention, the ion concentration regulator of the storage buffer includes salts;

[0028] The salts include one or more of NaCl, CaCl2, Al2(SO4)3, KCl, ZnCl2, CuCl2, or MgCl2.

[0029] In some specific embodiments of the present invention, the stabilizer of the storage buffer includes one or more of bovine serum albumin (BSA), human serum albumin, rabbit serum albumin, ovalbumin, or myoglobin.

[0030] In some specific embodiments of the present invention, the preservative of the storage buffer includes one or more of NaN3, Proclin 300, or Proclin 600.

[0031] In some specific embodiments of the present invention, the pH of the storage buffer solution includes 6.0 to 9.0.

[0032] In some specific embodiments of the present invention, the pH of the storage buffer is preferably 8.0.

[0033] The present invention also provides a magnetic microparticle suspension comprising the storage buffer solution and immunomagnetic beads.

[0034] In some specific embodiments of the present invention, the method for preparing the immunomagnetic beads includes:

[0035] a) Disperse the magnetic microparticles in a cross-linking buffer solution;

[0036] b) Dissolve / dilute the activator using cross-linking buffer, add it to the magnetic microparticle suspension, mix well, and incubate at room temperature (10-30℃) for 10 min to 1 h to obtain activated magnetic beads;

[0037] c) Remove residual activator by magnetic separation, and redisperse magnetic particles by adding cross-linking buffer;

[0038] d) After dissolving / diluting D-dimer antibody 1 with cross-linking buffer, add it to the activated magnetic microparticle suspension, mix well, and incubate at 4–30°C for 0.5–12 h to obtain immunomagnetic bead suspension;

[0039] e) Dissolve the blocking agent in cross-linking buffer, add it to the immunomagnetic bead suspension after the reaction is complete, and incubate at 4–30°C for 0.5–3 h;

[0040] f) After magnetic separation, disperse the immunomagnetic beads using storage buffer and store for later use.

[0041] In some specific embodiments of the present invention, the activator includes one or more of EDC, EDC / NHS, or EDC / sulfo-NHS.

[0042] In some specific embodiments of the present invention, the feeding ratio of the magnetic beads to the antibody is 10:1 to 200:1 (w / w), preferably 50:1.

[0043] In some specific embodiments of the present invention, the feeding ratio of the magnetic beads to the activator is 0.1:1 to 40:1 (w / w), preferably 10:1.

[0044] In some specific embodiments of the present invention, the blocking agent comprises free amino groups.

[0045] In some specific embodiments of the present invention, the blocking agent includes one or more of the following substances containing free amino groups: ethanolamine, bovine serum albumin, casein, amino acids, etc.

[0046] In some specific embodiments of the present invention, the crosslinking buffer does not contain amino and carboxyl groups.

[0047] In some specific embodiments of the present invention, the crosslinking buffer includes one of MES buffer, borate buffer or PBS buffer, preferably MES buffer.

[0048] In some specific embodiments of the present invention, the pH range of the crosslinking buffer is 4.5 to 7.0, preferably 5.5.

[0049] In some specific embodiments of the present invention, the magnetic microparticles exhibit superparamagnetism;

[0050] The magnetic microparticle core material includes one or more of iron oxide, zinc ferrite, or ferrous oxide.

[0051] The surface of the magnetic microparticles is modified with a uniform and sufficient amount of carboxyl groups.

[0052] In some specific embodiments of the present invention, the magnetic microparticles may have a particle size ranging from 0.5 μm to 5 μm, preferably 1.5 μm.

[0053] In some specific embodiments of the present invention, the D-dimer antibody 1 is a monoclonal antibody, a modified antibody fragment with Fab activity, or an antibody or antibody fragment multimer;

[0054] The D-dimer antibody 1 can specifically bind to the human D-dimer surface antigenic determinant;

[0055] The D-dimer antibody 1 was derived from mice, rabbits, sheep, or dogs;

[0056] In some specific embodiments of the present invention, the D-dimer antibody 1 is preferably a murine monoclonal antibody.

[0057] The present invention also provides a reagent combination comprising any combination of the aforementioned magnetic microparticle suspension, enzyme-labeled antibody, or calibrator.

[0058] In some specific embodiments of the present invention, the enzyme-labeled antibody can be prepared by the glutaraldehyde method, the sodium periodate method, or the heterobifunctional reagent cross-linking method (SMCC / sulfo-SMCC), with the heterobifunctional reagent cross-linking method being preferred.

[0059] In some specific embodiments of the present invention, the method for preparing the enzyme-labeled antibody includes:

[0060] a) Dissolve / dilute alkaline phosphatase using enzyme-labeled buffer, add a certain amount of sulfo-SMCC / SMCC, and gently incubate at 4–30°C for 0.5–3 hours. Desalt the product and set it aside for later use.

[0061] b) Dissolve / dilute D-dimer antibody 2 with enzyme-labeled buffer, add a certain amount of Traut's / DTT / 2-MEA reagent, incubate gently at 4-30℃ for 0.5-3h, then quench with 0.5%-2.5% (w / v) glycine solution for 5min, and desalt the product for later use.

[0062] c) After mixing the activated alkaline phosphatase with the thiolized antibody, gently stir and incubate at 4–30°C for 2–30 h, then add 0.5–2.5% (w / v) cysteine ​​solution to block for 0.5–2 h;

[0063] d) After desalting, the product is stored in a storage buffer for later use.

[0064] In some specific embodiments of the present invention, the D-dimer antibody 2 and D-dimer antibody 1 are validated paired antibodies.

[0065] In some specific embodiments of the present invention, the alkaline phosphatase / antibody to sulfo-SMCC feed ratio is 2 to 30:1 (w / w), preferably 10:1.

[0066] In some specific embodiments of the present invention, the ratio of alkaline phosphatase / antibody to Traut's / DTT / 2-MEA is 20 to 250:1 (w / w), preferably 100:1.

[0067] In some specific embodiments of the present invention, the ratio of alkaline phosphatase to antibody is 0.5 to 10:1 (w / w), preferably 2:1.

[0068] In some specific embodiments of the present invention, the enzyme-labeled buffer does not contain amino and carboxyl groups.

[0069] In some specific embodiments of the present invention, the enzyme-labeled buffer includes one of MES buffer, borate buffer or PBS buffer, preferably PBS buffer.

[0070] In some specific embodiments of the present invention, the pH of the enzyme-labeled buffer is 7.0 to 8.0, preferably 7.2.

[0071] In some specific embodiments of the present invention, the D-dimer calibrator is prepared by dissolving the D-dimer antigen in physiological saline to form D-dimer calibrators of different concentrations.

[0072] This invention also provides the application of any of the following in the preparation of a D-dimer chemiluminescence assay kit:

[0073] (I) the storage buffer solution; and / or

[0074] (II) the magnetic particle suspension; and / or

[0075] (III) The reagent combination.

[0076] This invention also provides a D-dimer chemiluminescence assay kit, which includes any of the following:

[0077] (I) the storage buffer solution; and / or

[0078] (II) the magnetic particle suspension; and / or

[0079] (III) The reagent combination.

[0080] In some specific embodiments of the present invention, the D-dimer chemiluminescence assay kit can be applied to a chemiluminescence analyzer with an enzyme-catalyzed glow discharge chemiluminescence detection system.

[0081] In some specific embodiments of the present invention, the D-dimer chemiluminescence assay kit has an immunomagnetic bead concentration of 0.1–1 mg / mL (based on the magnetic bead concentration), preferably 0.2 mg / mL.

[0082] In some specific embodiments of the present invention, the concentration of enzyme-labeled antibody in the D-dimer chemiluminescence assay kit is 0.1–1 μg / mL (based on antibody concentration), preferably 0.5 μg / mL.

[0083] In some specific embodiments of the present invention, the linear range of the kit is 10 to 20000 ng / mL (FEU).

[0084] This invention also provides a method for determining the D-dimer content, wherein the D-dimer content is determined using any of the following methods:

[0085] (I) the storage buffer solution; and / or

[0086] (II) the magnetic particle suspension; and / or

[0087] (III) The reagent combination; and / or

[0088] (IV) The D-dimer chemiluminescence assay kit.

[0089] This invention includes, but is not limited to, the following beneficial effects:

[0090] This invention provides a chemiluminescence assay kit for D-dimer, comprising a magnetic microparticle suspension (immunomagnetic beads) coated with D-dimer antibody 1, alkaline phosphatase-labeled D-dimer antibody 2 (enzyme-labeled antibody), and calibrators. The immunomagnetic beads and enzyme-labeled antibody described in this invention are prepared by chemical coupling and stored in a buffer solution containing soluble polymeric material, which effectively improves detection sensitivity and measurement stability. This kit can be used in conjunction with substrate solutions from fully automated immunoassay systems containing chromogenic substrates such as adamantane and its derivatives AMPPD or APS-5 to determine the D-dimer content in plasma or whole blood using a chemiluminescence analyzer. The kit described in this invention is stable, has high detection sensitivity, and good repeatability. Attached Figure Description

[0091] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0092] Figure 1 This demonstrates the principle of magnetic particle chemiluminescence method;

[0093] Figure 2 Calibration curves for different storage buffer solutions (four-parameter Logistic fitting curves);

[0094] Figure 3 Consistency analysis of storage buffer group 1 and samples from commercially available ELISA kits;

[0095] Figure 4 Consistency analysis of two groups of storage buffer solutions with samples from commercially available ELISA kits;

[0096] Figure 5 Consistency analysis of three groups of storage buffer samples with those from commercially available ELISA kits;

[0097] Figure 6 The consistency analysis of four groups of storage buffer samples with those of commercially available ELISA kits was presented.

[0098] Figure 7 Consistency analysis of five groups of storage buffer samples with those from commercially available ELISA kits;

[0099] Figure 8 Consistency analysis of six groups of storage buffer samples with those from commercially available ELISA kits;

[0100] Figure 9 Consistency analysis of 7 groups of storage buffer samples with those of commercially available ELISA kits;

[0101] Figure 10Consistency analysis of 8 groups of storage buffer samples with commercially available ELISA kits;

[0102] Figure 11 Consistency analysis of nine groups of storage buffer samples with those from commercially available ELISA kits;

[0103] Figure 12 Consistency analysis of 10 groups of storage buffer samples with those from commercially available ELISA kits;

[0104] Figure 13 Consistency analysis of 11 groups of storage buffer samples with those of commercially available ELISA kits;

[0105] Figure 14 Consistency analysis of 12 groups of storage buffer samples with those from commercially available ELISA kits;

[0106] Figure 15 Consistency analysis of 13 groups of storage buffer samples with those from commercially available ELISA kits;

[0107] Figure 16 Consistency analysis of 14 groups of storage buffer samples with commercially available ELISA kits;

[0108] Figure 17 Consistency analysis of 15 groups of storage buffer samples with those of commercially available ELISA kits;

[0109] Figure 18 Consistency analysis of 16 groups of storage buffer samples with commercially available ELISA kits;

[0110] Figure 19 Consistency analysis of 17 groups of storage buffer samples with those of commercially available ELISA kits;

[0111] Figure 20 Consistency analysis of 18 groups of storage buffer samples with those from commercially available ELISA kits;

[0112] Figure 21 The consistency analysis of 19 groups of storage buffer samples with those of commercially available ELISA kits was shown. Detailed Implementation

[0113] This invention discloses a D-dimer chemiluminescence assay kit. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0114] This invention provides a D-Dimer chemiluminescence assay kit, which uses a double-antibody sandwich method and an enzyme-catalyzed chemiluminescence method for detection. The kit includes a magnetic microparticle suspension (immunomagnetic beads) coated with D-Dimer antibody 1, D-Dimer antibody 2 labeled with alkaline phosphatase (enzyme-labeled antibody), and calibrators.

[0115] The magnetic microparticles should be superparamagnetic, and the core material can be one or more of the following: iron oxide, zinc ferrite, or ferrous oxide. The surface should be modified with a uniform and sufficient amount of carboxyl groups.

[0116] The magnetic microparticles may have a particle size ranging from 0.5 μm to 5 μm, preferably 1.5 μm.

[0117] The D-Dimer antibody 1 can be a monoclonal antibody, a modified antibody fragment with Fab activity, an antibody or antibody fragment polymer, etc., which can specifically bind to the human D-Dimer surface antigenic determinant cluster, and can be derived from animals such as mice, rabbits, sheep, and dogs, preferably mouse monoclonal antibodies.

[0118] The immunomagnetic beads can be coupled using activators such as EDC, EDC / NHS, and EDC / sulfo-NHS. The coupling steps are as follows:

[0119] a) Disperse the magnetic microparticles in a cross-linking buffer solution;

[0120] b) Dissolve / dilute the activator using cross-linking buffer, add it to the magnetic microparticle suspension, mix well, and incubate at room temperature (10-30℃) for 10 min to 1 h to obtain activated magnetic beads;

[0121] c) Remove residual activator by magnetic separation, and redisperse magnetic particles by adding cross-linking buffer;

[0122] d) After dissolving / diluting D-Dimer antibody 1 with cross-linking buffer, add it to the activated magnetic microparticle suspension, mix well, and incubate at 4–30°C for 0.5–12 h to obtain the immunomagnetic bead suspension;

[0123] e) Dissolve the blocking agent in cross-linking buffer, add it to the immunomagnetic bead suspension after the reaction is complete, and incubate at 4–30°C for 0.5–3 h;

[0124] f) After magnetic separation, disperse the immunomagnetic beads using storage buffer and store for later use.

[0125] The ratio of magnetic beads to antibodies is 10:1 to 200:1 (w / w), preferably 50:1.

[0126] The feeding ratio of the magnetic beads to the activator is 0.1:1 to 40:1 (w / w), preferably 10:1.

[0127] The blocking agent may be one or more of the following substances containing free amino groups: ethanolamine, bovine serum albumin, casein, amino acids, etc.

[0128] The cross-linking buffer can be one of the buffers that do not contain amino and carboxyl groups, such as MES buffer, borate buffer, and PBS buffer, with MES buffer being preferred; the pH range of the cross-linking buffer is 4.5 to 7.0, preferably 5.5.

[0129] The storage buffer solution mainly consists of a buffer system, sensitizer, ion concentration regulator, stabilizer and preservative.

[0130] The storage buffer system can be one of MOPS buffer, Tris-HCl buffer, PBS buffer, HEPES buffer, glycine buffer, etc., preferably Tris-HCl buffer.

[0131] The sensitizer of the storage buffer can be one or more of the following soluble polymer materials: (0.5-6.0% (w / v)) polyethylene glycol (molecular weight 200-20000), (0.5-5.0% (w / v)) povidone, (1.0-6.0% (w / v)) dextran, (0.5-3.0% (w / v)) polyethyleneimine, (0.1-0.4% (w / v)) cellulose (sodium carboxymethyl cellulose, ethyl cellulose, etc.), and (0.25-2.0% (w / v)) amine polysaccharides (chondroitin sulfate, chitosan, etc.).

[0132] The ion concentration adjuster of the storage buffer can be one or more of commonly used salts such as NaCl, CaCl2, Al2(SO4)3, KCl, ZnCl2, CuCl2, and MgCl2.

[0133] The storage buffer stabilizer can be one or more of bovine serum albumin (BSA), human serum albumin, rabbit serum albumin, ovalbumin, myoglobin, etc.

[0134] The preservative in the storage buffer solution can be one or more of NaN3, Proclin 300, and Proclin 600.

[0135] The pH range of the storage buffer solution is 6.0 to 9.0, preferably 8.0.

[0136] The enzyme-labeled antibody can be prepared by methods such as the glutaraldehyde method, the sodium periodate method, or the heterobifunctional reagent cross-linking method (SMCC / sulfo-SMCC), with the heterobifunctional reagent cross-linking method being preferred. The preparation process can involve coupling SMCC / sulfo-SMCC activated alkaline phosphatase with 2-iminothiophene (Traut's) / dithiothreitol (DTT) / 2-mercaptoethylamine (2-MEA) thiolated D-Dimer antibody 2, or coupling SMCC / sulfo-SMCC activated D-Dimer antibody 2 with Traut's / DTT / 2-MEA reagent thiolated alkaline phosphatase.

[0137] The D-Dimer antibody 2 and D-Dimer antibody 1 are verified paired antibodies.

[0138] The preparation steps are as follows:

[0139] a) Dissolve / dilute alkaline phosphatase with enzyme-labeled buffer, add a certain amount of sulfo-SMCC / SMCC, and gently incubate at 4–30°C for 0.5–3 hours. Desalt the product and set it aside for later use.

[0140] b) Dissolve / dilute D-Dimer antibody 2 using enzyme-labeled buffer, and add a certain amount of Traut'

[0141] After incubating the s / DTT / 2-MEA reagent at 4–30°C for 0.5–3 h, quench the product with 0.5%–2.5% (w / v) glycine solution for 5 min. Desalt the product and set it aside for later use.

[0142] c) After mixing the activated alkaline phosphatase with the thiolized antibody, gently stir and incubate at 4–30°C for 2–30 h, then add 0.5–2.5% (w / v) cysteine ​​solution to block for 0.5–2 h;

[0143] d) After desalting, the product is stored in a storage buffer for later use.

[0144] The alkaline phosphatase / antibody to sulfo-SMCC feed ratio is 2 to 30:1 (w / w), preferably 10:1.

[0145] The ratio of alkaline phosphatase / antibody to Traut's / DTT / 2-MEA is 20-250:1 (w / w), preferably 100:1.

[0146] The ratio of alkaline phosphatase to antibody is 0.5 to 10:1 (w / w), preferably 2:1.

[0147] The enzyme-labeled buffer can be one of the buffers that do not contain amino and carboxyl groups, such as MES buffer, borate buffer, or PBS buffer, with PBS buffer being preferred.

[0148] The pH of the enzyme-labeled buffer solution is 7.0 to 8.0, preferably 7.2.

[0149] The D-Dimer calibrators are mainly prepared by dissolving D-Dimer antigen in physiological saline to form D-Dimer calibrators of different concentrations.

[0150] The D-Dimer chemiluminescence assay kit can be used in chemiluminescence analyzers with an enzyme-catalyzed glow discharge chemiluminescence detection system.

[0151] The D-Dimer chemiluminescence assay kit uses an immunomagnetic bead concentration of 0.1–1 mg / mL (based on bead concentration), preferably 0.2 mg / mL.

[0152] The D-Dimer chemiluminescence assay kit uses an enzyme-labeled antibody concentration of 0.1–1 μg / mL (based on antibody concentration), preferably 0.5 μg / mL.

[0153] Furthermore, the linear range of the kit is 10–20000 ng / mL (FEU).

[0154] This invention, based on the conventional immunomagnetic bead preparation method (direct antibody labeling of magnetic microparticles), introduces a soluble polymer material (sensitizer) in the later stages of immunomagnetic bead and enzyme-labeled antibody preparation. This component will improve the test results in the following three aspects:

[0155] 1) In terms of preparation, polymer materials can further block the prepared immunomagnetic beads and enzyme-labeled antibodies, reduce non-specific adsorption during testing, and improve detection sensitivity;

[0156] 2) In terms of storage, the addition of polymer materials can improve the suspension stability of enzyme-labeled antibodies and immunomagnetic beads, inhibit the aggregation, degradation and denaturation of bioactive components, and improve the detection sensitivity and repeatability of the kit.

[0157] 3) In terms of detection, polymer materials can improve the binding efficiency of immunomagnetic beads and enzyme-labeled antibodies to antigens (especially low-concentration antigens) through volume exclusion, thereby further improving detection sensitivity.

[0158] Advantages of the technical solution of this invention:

[0159] The kit described in this invention utilizes a double-antibody sandwich and enzyme-catalyzed chemiluminescence detection principle, offering higher detection sensitivity and a wider linear range compared to the latex immunoturbidimetric kits widely used in clinical practice. Furthermore, based on the conventional immunomagnetic bead preparation method (direct antibody labeling of magnetic microparticles), this invention introduces soluble polymeric materials, avoiding interference from excessive biotin while further improving detection sensitivity and repeatability.

[0160] Unless otherwise specified, all raw materials and reagents used in the D-dimer chemiluminescence assay kit provided by this invention are commercially available.

[0161] The present invention will be further illustrated below with reference to the embodiments:

[0162] Example 1: Reagent Kit Preparation

[0163] 1.1 Preparation of Immunomagnetic Beads

[0164] Use a vortex mixer to thoroughly mix the carboxyl magnetic beads (Magnosphere). TM For MS160 / Carboxyl), take 10 mg of magnetic bead solution and add cross-linking buffer (0.1 M MES, pH 6.0) to a final volume of 1 mL. Mix well and then magnetically separate for 1 min. Discard the supernatant. Add another 1 mL of cross-linking buffer, mix well, and then magnetically separate for 1 min. Discard the supernatant, add another 1 mL of cross-linking buffer, and mix well before use.

[0165] Add 100 μL of activator (10 mg / mL EDC & 10 mg / mL sulfo-NHS, freshly prepared) and mix well. Incubate at 25±5℃ by rotation for 30 min. After magnetic separation, remove the supernatant.

[0166] Take 200 μg of D-Dimer antibody 1, add cross-linking buffer to make up to 1 mL, then add it to the activated magnetic beads, mix well, and incubate at 25±5℃ for 4 h by rotation mixing. After magnetic separation for 1 min, remove the supernatant.

[0167] Use 1% (w / v) BSA solution (dissolved in crosslinking buffer) to rotate and block for 1 h at 25±5℃, then magnetically separate for 1 min and remove the supernatant.

[0168] The conjugated immunomagnetic beads were dispersed using storage buffer (0.1M Tris, 0.15M NaCl, 0.5% (w / v) BSA, 0.2% (w / v) NaN3, 3% (w / v) dextran 20000, pH 8.0) and stored at 2–8°C. Before testing, the beads were vortexed and diluted with storage buffer to 0.2 mg / mL (based on bead concentration).

[0169] 1.2 Preparation of enzyme-labeled antibodies

[0170] Dissolve / dilute 10 mg of alkaline phosphatase to a 10 mg / mL alkaline phosphatase solution using 1 mL of enzyme-labeled buffer (0.1 M PBS, 0.15 M NaCl, pH 7.2), add 200 μL of sulfo-SMCC (5 mg / mL), and gently incubate at 25 ± 5 °C for 1 h. After desalting the product, disperse it in enzyme-labeled buffer for later use.

[0171] Dissolve / dilute 10 mg D-Dimer antibody 2 to a 10 mg / mL antibody solution using 1 mL of enzyme-labeled buffer, add 10 μL of Traut's (10 mg / mL), and incubate gently at 25 ± 5 °C for 1 h. Add 10 μL of 1% (w / v) glycine solution, mix well, and continue incubating for 5 min. After desalting the product, disperse it with enzyme-labeled buffer for later use.

[0172] After thoroughly mixing the activated alkaline phosphatase and thiolized D-Dimer antibody 2, incubate gently with stirring at 4°C for 24 h or at 25±5°C for 6 h. Then, add 50 μL of 1% (w / v) cysteine, mix well, and continue incubating with stirring for 30 min. After desalting the product, dilute and disperse it with enzyme-labeled storage buffer (0.1 M Tris, 0.15 M NaCl, 0.5% (w / v) BSA, 0.2% (w / v) NaN3, 3% (w / v) dextran 20000, pH 8.0). Before testing, dilute to 0.5 μg / mL (based on antibody concentration) with enzyme-labeled storage buffer.

[0173] 1.3 Calibration Sample Preparation

[0174] The linear range of this kit is 10–20000 ng / mL. To ensure measurement accuracy, commercially available D-Dimer was prepared with physiological saline to the concentrations shown in Table 1.

[0175] Table 1. Calibration concentration distribution

[0176] Calibrator Name S0 S1 S2 S3 S4 S5 S6 S7 S8 Concentration, ng / mL 0 200 500 1200 1500 3000 5000 10000 16000

[0177] Example 2: Calibration Curve and Sample Concentration Detection Method

[0178] Place the immunomagnetic beads, enzyme-labeled antibodies, and calibrators / samples in the designated positions on the chemiluminescence analyzer. The testing procedure and process are as follows:

[0179] a) Mix 50 μL of immunomagnetic beads with 20 μL of sample / calibrator and incubate at 37°C for 10 min;

[0180] b) After magnetic separation, the magnetic bead composite 1 was washed four times;

[0181] c) Add 50 μL of enzyme-labeled antibody to the complex and incubate at 37°C for 5 min;

[0182] d) After magnetic separation, the magnetic bead composite 2 was washed four times;

[0183] e) Add 200 μL of substrate solution for fully automated immunoassay system (containing 0.02% (w / v) AMPPD and 0.005% (w / v) sodium fluorescein) to the cleaned magnetic bead complex 2, and incubate at 37°C in the dark for 5 min.

[0184] f) Use a photoelectrorea to measure the photon quantity of the substrate solution after the reaction;

[0185] g) Fit the calibration curve using a weighted four-parameter Logistic regression with the calibrator concentration as the x-axis and the photon quantity as the y-axis.

[0186] h) Substitute the sample photon measurement value into the calibration curve to calculate the sample concentration.

[0187] Example 3: Study on the effects of adding different types and amounts of polymeric materials

[0188] 3.1 Composition of storage buffer solutions with different formulations

[0189] Table 2 Different storage buffer formulations

[0190]

[0191]

[0192] 3.2 Calibration Tests of Storage Buffers with Different Formulations

[0193] Immunomagnetic beads and enzyme-labeled antibodies with different storage buffers were tested according to the procedure specified in Example 2 on the master curve calibrators S0-S8 described in Example 1. The obtained photon quantity results were fitted with a four-parameter logistic regression using Graphpad. The calibration curve results are shown in Table 3 and... Figure 2 As shown in Table 3, the addition of polymeric sensitizers to the storage buffer significantly improves photon measurement values ​​(see Table 3 for details; the analysis of variance shows that different types of polymers listed in this invention exhibit different abilities to improve measurement values). The increased difference in photon measurement values ​​between calibration points can effectively improve test sensitivity.

[0194] Table 3. Calibration test results for different storage buffers

[0195]

[0196]

[0197]

[0198] Note: 1. The gray box in the table represents the average photon quantity at each calibration point (n=3);

[0199] 2. The asterisks (*) after the data in the table indicate the results of the ANOVA between each group and the data in storage buffer 1: a) When the ANOVA P > 0.05, there is no significant difference; b) "*" indicates that the ANOVA P < 0.05, there is a statistically significant difference; c) "**" indicates that the ANOVA P < 0.001, there is a significant difference; d) "***" indicates that the ANOVA P < 0.001, there is an extremely significant difference.

[0200] Example 4 Repeatability Test

[0201] For different storage buffer groups, low-value control plasma with a target value of 640 ng / mL and high-value control plasma with a target value of 1600 ng / mL were selected and tested repeatedly 10 times. The photon quantity measurement value and sample measurement value were counted and the coefficient of variation (CV) was calculated. The results are shown in Table 4. Compared with the control group (storage buffer 1), the experimental group had a lower CV in the repeatability test and better repeatability.

[0202] Table 4. Repeatability test results for different experimental groups

[0203]

[0204]

[0205] Example 5: Blank Limit (Detection Sensitivity) Test

[0206] For different storage buffer groups, zero-concentration calibrators (S0) were used as samples for testing. The measurements were repeated 20 times, and the RLU values ​​of the 20 measurements were obtained. The average value was then calculated. and standard deviation SD, where The blank limit is the photon measurement value corresponding to the blank limit. Substituting the photon measurement value corresponding to the blank limit into the calibration curve formula will yield the blank limit measurement value. The results are shown in Table 5. As can be seen from the table, compared with the control group (storage buffer 1), the blank limits of the experimental group with added polymer sensitizer are all lower than that of the control group (57.83 ng / mL), which improves the detection sensitivity.

[0207] Table 5. Blank limit test results for different experimental groups

[0208]

[0209]

[0210] Example 6: Consistency Study of Reagent Kit Samples

[0211] (Correlation between the D-Dimer Assay Kit of this invention (chemiluminescence method) and commercially available D-Dimer Assay Kits (ELISA))

[0212] One hundred blood samples were randomly collected from inpatients and outpatients at Longhua Hospital affiliated with Shanghai University of Traditional Chinese Medicine. The blood was mixed with 0.109 mol / L sodium citrate anticoagulant at a ratio of 9:1 and centrifuged at 2500 rpm for 15 minutes to separate plasma. The samples were measured using the kit of this invention (containing storage buffers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 as described in Example 3) and a commercially available ELISA kit. The measured values ​​were subjected to linear regression, and the correlation coefficient between the two was calculated. The results are as follows: Figures 3-21 As shown, except for storage buffer 1 (control group), the detection data of the kit of the present invention and the commercially available kits show good consistency. Furthermore, compared to the method described in this invention, commercially available ELISA kits are more complex to operate, take longer, and require trained professionals to operate.

[0213] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of a reagent combination in the preparation of a D-dimer chemiluminescence assay kit: The reagent combination is a magnetic particle suspension, an enzyme-labeled antibody, and a calibrator; The magnetic particle suspension is composed of a storage buffer and immunomagnetic beads; The storage buffer is composed of a sensitizer, a buffer system, an ion concentration regulator, a stabilizer, and a preservative; The sensitizer is a soluble high molecular material; the soluble high molecular material is one or more of povidone, dextran, polyethyleneimine, cellulose, or amine polysaccharide; The povidone is povidone K30; The dextran is dextran 20000; The cellulose is sodium carboxymethyl cellulose; The amine polysaccharide is chondroitin sulfate; The concentration of povidone is 0.5-5.0% (w / v); The concentration of dextran is 1.0-6.0% (w / v); The concentration of polyethyleneimine is 0.5-3.0% (w / v); The concentration of cellulose is 0.1-0.4% (w / v); The concentration of amine polysaccharide is 0.25-2.0% (w / v); The pH of the storage buffer is 6.0-9.0; The buffer system is Tris-HCl buffer, the ion concentration regulator is sodium chloride, and the stabilizer is BSA.

2. A D-dimer chemiluminescence assay kit characterized in that, The reagent combination is composed of: The reagent combination is a magnetic particle suspension, an enzyme-labeled antibody, and a calibrator; The magnetic particle suspension is composed of a storage buffer and immunomagnetic beads; The storage buffer is composed of a sensitizer, a buffer system, an ion concentration regulator, a stabilizer, and a preservative; The sensitizer is a soluble high molecular material; The soluble high molecular material is one or more of povidone, dextran, polyethyleneimine, cellulose, or amine polysaccharide; The povidone is povidone K30; The dextran is dextran 20000; The cellulose is sodium carboxymethyl cellulose; The amine polysaccharide is chondroitin sulfate; The concentration of povidone is 0.5-5.0% (w / v); The concentration of dextran is 1.0-6.0% (w / v); The concentration of polyethyleneimine is 0.5-3.0% (w / v); The concentration of cellulose is 0.1-0.4% (w / v); The concentration of amine polysaccharide is 0.25-2.0% (w / v); The pH of the storage buffer is 6.0-9.0; The buffer system is Tris-HCl buffer, the ion concentration regulator is sodium chloride, and the stabilizer is BSA.

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