D-dimer detection kit and preparation method thereof

Abstract

The invention provides a D-dimer detection kit and a preparation method thereof. A chromatographic test strip of the kit comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad. And the combination pad is coated with a D-dimer detection antibody marked by carboxylated time-resolved fluorescent microspheres. A detection line of the nitrocellulose membrane is coated with a D-dimer capture antibody, and a quality control line of the nitrocellulose membrane is coated with a goat anti-mouse IgG antibody. The D-dimer detection antibody and the D-dimer capture antibody are a pair of monoclonal antibodies for specifically recognizing different epitopes of the D-dimer. According to the kit, the convenience of immunochromatography and the high sensitivity characteristic of the time-resolved fluorescent microspheres are fused, accurate and quantitative detection of the D-dimer is achieved through the synergistic effect of the detection antibody and the capture antibody which specifically recognize different epitopes, and the kit is high in sensitivity, good in specificity, easy and convenient to operate, high in anti-interference capacity and high in sensitivity. The clinical high-precision quantitative monitoring requirement can be met, and the diagnosis and treatment application value and the market prospect are achieved.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic testing technology, and in particular to a D-dimer detection kit based on fluorescence immunochromatography and its preparation method. Background Technology

[0002] D-dimer is a specific degradation product of cross-linked fibrin produced by plasmin. As a key molecular marker of thrombosis and related fibrinolytic activation, it plays an important role in the clinical diagnosis and exclusion of thrombotic diseases such as deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation. High concentrations of D-dimer indicate the presence of secondary hyperfibrinolysis in vivo, providing direct experimental evidence for thrombosis and its subsequent fibrinolytic process.

[0003] Currently, commonly used clinical methods for D-dimer detection include enzyme-linked immunosorbent assay (ELISA), latex-enhanced immunoturbidimetry, and colloidal gold immunochromatography. Among these, ELISA is considered the "gold standard" due to its high sensitivity and specificity; however, its complex and time-consuming procedure makes it unsuitable for emergency situations requiring rapid results. Latex-enhanced immunoturbidimetry, with its automated analysis capabilities, has become the mainstream choice for laboratories in large and medium-sized hospitals. However, this method relies on expensive fully automated biochemical analysis equipment, and the results are easily affected by interference factors such as lipemia and hemolysis in the sample. Colloidal gold immunochromatography is characterized by its ease of operation and rapid detection, but it typically only provides qualitative or semi-quantitative analysis, with limitations in sensitivity and precision, making it difficult to meet the clinical needs for high-precision quantitative monitoring of D-dimer.

[0004] Fluorescent immunochromatography is a rapidly developing novel detection technique in recent years. It combines the convenience and efficiency of immunochromatography with the high sensitivity of fluorescent labeling, achieving precise measurement of target analytes by detecting the intensity of fluorescence signals. Compared to colloidal gold immunochromatography, its sensitivity can be improved by 1-2 orders of magnitude, providing excellent quantitative accuracy. Therefore, developing a detection method that is simple to operate, rapid, highly sensitive, and highly accurate, and capable of precise quantification of D-dimer, is of significant importance for improving clinical diagnosis and treatment, and holds great promise for market applications. Summary of the Invention

[0005] One objective of this invention is to address the shortcomings of existing technologies by providing a D-dimer detection kit that is highly sensitive, specific, easy to operate, and capable of accurate quantitative detection, thereby meeting clinical needs for D-dimer detection.

[0006] A second objective of this invention is to provide a method for preparing D-dimer test strips that is simple, efficient, and feasible, and can stably prepare D-dimer detection kits with high sensitivity, good specificity, simple operation, and accurate quantitative detection, thus providing reliable technical support for large-scale production.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: According to a first aspect of the present invention, a D-dimer detection kit is provided, the D-dimer detection kit comprising at least a chromatographic test strip, the chromatographic test strip comprising a base plate and a sample pad, a conjugation pad, a nitrocellulose membrane and an absorbent pad sequentially disposed on the base plate and overlapping each other.

[0008] The binding pad is coated with D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres. The D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres is prepared by covalent coupling, blocking, and purification of carboxylated time-resolved fluorescent microspheres and D-dimer detection antibody.

[0009] The nitrocellulose membrane is provided with a detection line and a control line at intervals. The detection line is coated with D-dimer capture antibody, and the control line is coated with goat anti-mouse IgG antibody.

[0010] The D-dimer detection antibody and the D-dimer capture antibody are a pair of monoclonal antibodies that specifically recognize different antigenic epitopes of D-dimers.

[0011] Preferably, the carboxylated time-resolved fluorescent microspheres have an excitation wavelength of 355–375 nm, an emission wavelength of 600–620 nm, and an average particle size of 100–300 nm.

[0012] Preferably, the concentration of the D-dimer capture antibody on the detection line is 0.9–1.1 mg / mL, and the spray volume is 0.9–1.1 μL / cm.

[0013] Preferably, the concentration of goat anti-mouse IgG antibody on the quality control line is 0.9–1.1 mg / mL, and the spray volume is 0.9–1.1 μL / cm.

[0014] Preferably, the D-dimer detection kit further includes a detection card, sample diluent, calibrators, and quality control materials, with the chromatographic test strip disposed in the detection card.

[0015] According to a second aspect of the present invention, a method for preparing a D-dimer detection kit as described above is provided, the method comprising: A pair of monoclonal antibodies that specifically recognize different antigenic epitopes of D-dimer were selected as D-dimer detection antibodies and D-dimer capture antibodies, respectively.

[0016] Preparation of D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres: The fluorescent microspheres were resuspended, and EDC and NHS were added in a set ratio to activate the carboxyl groups. After centrifugation, excess cross-linking agent was removed to obtain carboxylated time-resolved fluorescent microspheres. The carboxylated time-resolved fluorescent microspheres were covalently coupled with the D-dimer detection antibody, blocked, and purified to obtain D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres, which was then stored in a preservation solution.

[0017] Preparation of chromatographic test strips: Spray the D-dimer detection antibody solution labeled with carboxylated time-resolved fluorescent microspheres onto the conjugate pad and vacuum dry; spray the D-dimer capture antibody onto the detection line of the nitrocellulose membrane and the goat anti-mouse IgG antibody onto the control line of the nitrocellulose membrane and dry; sequentially overlap and paste the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad onto the base plate, and cut into chromatographic test strips of the required width.

[0018] Assembly: Insert the chromatography strip into the test card, and assemble the test card, sample diluent, calibrator and quality control to form the D-dimer detection kit.

[0019] Preferably, the steps for preparing D-dimer detection antibodies labeled with carboxylated time-resolved fluorescent microspheres include: S1-1: Take 150-250 μL of fluorescent microspheres with a solid content of 0.8-1.1% and resuspend them in 0.4-0.6 mL of MES buffer; S1-2: Add 90-110 μg EDC and 90-110 μg NHS to activate the carboxyl groups, and rotate the reaction for 10-20 min; after the rotation reaction is complete, centrifuge to remove excess crosslinking agent to obtain carboxylated time-resolved fluorescent microspheres. S1-3: Resuspend the carboxylated time-resolved fluorescent microspheres in MES buffer, add 0.05-0.20 mg of D-dimer detection antibody for covalent coupling, and react with shaking at room temperature in the dark for 1.5-3.0 hours; S1-4: Add 40-60 μL of BSA solution with a solid content of 8-12% to block the remaining active sites; after blocking, centrifuge and purify to obtain D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres; S1-5: Resuspend the D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres in the preservation solution and store at 4°C.

[0020] Preferably, in step S1-1, the excitation wavelength of the fluorescent microspheres is 365 nm, the emission wavelength is 610 nm, and the average particle size is 200 nm.

[0021] Preferably, the pH value of the MES buffer in step S1-1 is 5.0-6.0, and the concentration is 90-110 mM.

[0022] Preferably, the preservation solution in steps S1-5 is a Tris buffer containing 0.8-1.2% BSA.

[0023] Preferably, the steps for preparing the chromatographic test strip include: S2-1: The D-dimer detection antibody solution labeled with carboxylated time-resolved fluorescent microspheres obtained in step S1-5 is sprayed onto the conjugate pad at a spraying rate of 4-6 μL / cm, and the sprayed conjugate pad is vacuum dried at 37°C. S2-2: Using a spotting apparatus, D-dimer capture antibody with a concentration of 0.9–1.1 mg / mL is sprayed onto the detection line of the nitrocellulose membrane at a spraying volume of 0.9–1.1 μL / cm, and goat anti-mouse IgG antibody with a concentration of 0.9–1.1 mg / mL is sprayed onto the control line of the nitrocellulose membrane at a spraying volume of 0.9–1.1 μL / cm. The sprayed nitrocellulose membrane is then dried at 37°C. S2-3: Sequentially overlap and paste the sample pad, the binding pad prepared in step S2-1, the nitrocellulose membrane prepared in step S2-2, and the absorbent pad on the base plate to form a large test paper plate; S2-4: Cut the test strip plate into the required width to obtain the chromatography test strip.

[0024] Preferably, in step S2-3, the overlap distance between the sample pad and the conjugate pad, the overlap distance between the conjugate pad and the nitrocellulose membrane, and the overlap distance between the nitrocellulose membrane and the absorbent pad are 1.5 to 2.5 mm, respectively, and in step S2-4, the width of the chromatography strip is 3.5 to 4.0 mm.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a D-dimer detection kit that combines the convenience of immunochromatography with the high sensitivity of time-resolved fluorescent microspheres. Through the synergistic effect of detection antibodies that specifically recognize different antigenic epitopes and capture antibodies, it achieves accurate quantitative detection of D-dimers. Compared to ELISA, this kit is simpler to operate and has a shorter detection time, making it suitable for rapid testing needs in emergency settings. Compared to latex-enhanced immunoturbidimetry, it does not require expensive automated equipment and has stronger resistance to interference from lipemia and hemolysis. Compared to colloidal gold immunochromatography, it effectively improves sensitivity and significantly optimizes precision and accuracy, meeting the needs of high-precision quantitative clinical monitoring and possessing both diagnostic and therapeutic application value and market potential.

[0026] The preparation method of the D-dimer detection kit of the present invention, through steps such as EDC / NHS activation coupling, BSA blocking, and centrifugal purification, ensures stable binding and excellent specificity between carboxylated time-resolved fluorescent microspheres and D-dimer detection antibodies. The method also optimizes process parameters such as solution ratio and coating amount to ensure consistency and reproducibility in batch production of the kit. This method is simple, efficient, and feasible, and can stably prepare D-dimer detection kits with high sensitivity, good specificity, ease of operation, and accurate quantitative detection, providing reliable technical support for large-scale production. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of the structure of the chromatography strip in a D-dimer detection kit of the present invention.

[0028] Figure 2 This is a flowchart illustrating the preparation of D-dimer detection antibodies labeled with carboxylated time-resolved fluorescent microspheres in this invention.

[0029] Figure 3 This is a flowchart of the preparation of the chromatography test strip in this invention.

[0030] In the diagram, 10 is the base plate, 20 is the sample pad, 30 is the binding pad, 40 is the nitrocellulose membrane, 41 is the detection line, 42 is the quality control line, and 50 is the absorbent pad. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. It is understood that, without conflict, some technical means of the various embodiments described herein can be substituted for or combined with each other.

[0032] In the description of this invention, the terms "first," "second," etc., are used only to distinguish the described objects and have no sequential or technical meaning. Therefore, objects specified with "first," "second," etc., may explicitly or implicitly include one or more of those objects. Furthermore, the words "one" or "a" do not indicate a quantity limitation, but rather indicate the presence of at least one, while "multiple" indicates at least two.

[0033] In the description of this invention, references to "one embodiment" or "some embodiments" mean that one or more embodiments of the invention include a particular feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "one embodiment," "some embodiments," "other embodiments," "and other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized.

[0034] According to a first aspect of the present invention, a D-dimer detection kit is provided, the D-dimer detection kit comprising at least a chromatographic test strip, as shown in the reference... Figure 1 The chromatography test strip includes a base plate 10 and a sample pad 20, a conjugate pad 30, a nitrocellulose membrane 40 (NC membrane), and an absorbent pad 50, which are sequentially arranged on the base plate 10 and overlap each other. The base plate 10 is made of PVC (Polyvinyl chloride), the sample pad 20 and the conjugate pad 30 can be made of glass fiber, and the absorbent pad 50 can be made of a highly absorbent paper material.

[0035] The binding pad 30 is coated with D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres. The D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres is prepared by covalent coupling, blocking and purification of carboxylated time-resolved fluorescent microspheres and D-dimer detection antibody.

[0036] The nitrocellulose membrane 40 has a detection line 41 (T line) and a control line 42 (C line) spaced apart. The detection line 41 is close to the conjugate pad 30, and the control line 42 is close to the absorbent pad 50. The detection line 41 is coated with D-dimer capture antibody, and the control line 42 is coated with goat anti-mouse IgG antibody.

[0037] Among them, the D-dimer detection antibody and the D-dimer capture antibody are a pair of monoclonal antibodies that specifically recognize different antigenic epitopes of D-dimer, ensuring that there is no steric hindrance when they bind to D-dimer, and have high affinity and high specificity.

[0038] This invention utilizes carboxylated time-resolved fluorescent microspheres as a label, which possess high fluorescence intensity. After conjugation with D-dimer detection antibodies, they exhibit a "signal amplification effect." A single microsphere can load multiple fluorescent molecules, making the detection system more sensitive to D-dimers. The carboxylated time-resolved fluorescent microspheres themselves possess excellent physicochemical stability. During storage, transportation, and detection reactions at room temperature, they are not easily affected by pH fluctuations, temperature changes, or ionic strength, preventing fluorescence quenching or aggregation, ensuring the stability of the labeled antibody's fluorescence signal throughout the detection cycle. The carboxylated time-resolved fluorescent microspheres readily covalently conjugate with antibodies. In detection applications, the binding reaction between the labeled antibody and D-dimer is rapid and has strong affinity, shortening the overall detection time and meeting the clinical demand for rapid results. Furthermore, the strong binding bond between the microspheres and the D-dimer detection antibody through covalent conjugation effectively prevents antibody detachment from the microsphere surface during storage or reaction, ensuring the consistency and stability of the labeled antibody activity.

[0039] During the testing procedure, the sample to be tested (e.g., plasma / whole blood) is mixed with diluent and then added to sample pad 20. Under chromatographic action, the sample moves towards conjugation pad 30. When the sample contains D-dimer, the D-dimer in the sample first specifically binds to the D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres in conjugation pad 30, forming an "antigen-fluorescent antibody" complex. This complex continues chromatographically to detection line 41, where it specifically binds to the D-dimer capture antibody on detection line 41, forming a "capture antibody-antigen-fluorescent antibody" double antibody sandwich complex. It then continues to the control line 42, where it is captured by goat anti-mouse IgG antibody on the control line. Afterwards, the fluorescence signal intensity of detection line 41 and control line 42 is read using a fluorescence immunochromatographic analyzer, and the precise concentration of D-dimer in the sample can be automatically calculated based on the instrument's built-in standard curve.

[0040] This invention provides a D-dimer detection kit that combines the convenience of immunochromatography with the high sensitivity of time-resolved fluorescent microspheres. Through the synergistic effect of detection antibodies that specifically recognize different antigenic epitopes and capture antibodies, it achieves accurate quantitative detection of D-dimers. Compared to ELISA, this kit is simpler to operate and has a shorter detection time, making it suitable for rapid testing needs in emergency settings. Compared to latex-enhanced immunoturbidimetry, it does not require expensive automated equipment and has stronger resistance to interference from lipemia and hemolysis. Compared to colloidal gold immunochromatography, it effectively improves sensitivity and significantly optimizes precision and accuracy, meeting the needs of high-precision quantitative clinical monitoring and possessing both diagnostic and therapeutic application value and market potential.

[0041] In some preferred embodiments, the carboxylated time-resolved fluorescent microspheres have an excitation wavelength of 355 nm to 375 nm, an emission wavelength of 600 nm to 620 nm, and an average particle size of 100 nm to 300 nm. These carboxylated time-resolved fluorescent microspheres exhibit high brightness, which helps to improve detection sensitivity and detection linearity. Specifically, in one embodiment, the fluorescent microspheres have an excitation wavelength of 365 nm, an emission wavelength of 610 nm, and an average particle size of 200 nm.

[0042] In some preferred embodiments, the concentration of the D-dimer capture antibody on the detection line 41 is 0.9–1.1 mg / mL, and the spray volume is 0.9–1.1 μL / cm. This concentration ensures that the detection line 41 has sufficient capture sites for efficient binding of the antigen-fluorescent antibody complex, thus improving binding efficiency. The uniform spray volume ensures that the D-dimer capture antibody is evenly distributed on the detection line 41, reducing detection errors, ensuring stable intra-batch / inter-batch results, and improving detection repeatability. Further, in one embodiment, the concentration of the D-dimer capture antibody on the detection line 41 is 1.0 mg / mL, and the spray volume is 1.0 μL / cm.

[0043] In some preferred embodiments, the concentration of goat anti-mouse IgG antibody on control line 42 is 0.9–1.1 mg / mL, and the spray volume is 0.9–1.1 μL / cm. Goat anti-mouse IgG antibody can specifically bind to labeled antibodies; a suitable concentration can stably capture free labeled antibodies, forming a clear control signal and ensuring detection validity. A uniform spray volume ensures stable signal intensity on control line 42; color development on control line 42 is a prerequisite for valid detection results, guaranteeing reliability. Further, in one embodiment, the concentration of goat anti-mouse IgG antibody on control line 42 is 1.0 mg / mL, and the spray volume is 1.0 μL / cm.

[0044] In some preferred embodiments, the D-dimer detection kit further includes a test card, sample diluent, calibrators, and quality control components, with the chromatographic test strip housed within the test card. The test card, a plastic casing, provides physical protection for the chromatographic test strip and standardizes the sample application and reading areas, improving ease of operation. The sample diluent adjusts the sample concentration and removes matrix interference (such as impurities in blood), ensuring D-dimer is within the appropriate detection range. The calibrators are used for instrument calibration to ensure accurate quantitative results. The quality control components validate the performance of each batch of reagents, guaranteeing detection reliability. The synergistic effect of these components creates a complete workflow from sample processing and calibration to detection and result verification, eliminating the need for additional consumables, making it suitable for rapid clinical testing scenarios, and lowering the operational threshold.

[0045] In one embodiment, 100 clinical plasma samples were simultaneously tested using the D-dimer detection kit of the present invention and a commercially available immunoturbidimetric kit. The D-dimer detection kit of the present invention has significant advantages in terms of precision, accuracy, sensitivity, and specificity. (1) Precision: The quality control samples with high, medium and low concentrations were tested separately, and the coefficients of variation (CV) within and between batches were less than 10%.

[0046] (2) Accuracy: Correlation analysis with the results of immunoturbidimetric assay showed a correlation coefficient R² > 0.95, indicating good consistency.

[0047] (3) Sensitivity: The limit of detection (LoD) is 0.05 mg / L.

[0048] (4) Specificity: No cross-reaction with fibrinogen, fibrin monomers and other similar substances.

[0049] The test results show that the kit provided by the present invention has excellent performance, high sensitivity, good specificity, simple operation and can achieve accurate quantitative detection, which can meet the clinical needs for D-dimer detection.

[0050] The above-mentioned test kit exhibits significantly higher detection sensitivity than the colloidal gold method, with a detection limit as low as 0.05 mg / L. It also boasts a wide linear range, from 0.05 to 10 mg / L, covering the clinically required detection range. The concentration of D-dimer in the sample is automatically obtained by reading the fluorescence signal, providing objective and accurate results and achieving precise quantitative analysis of D-dimer, superior to the semi-quantitative interpretation of the colloidal gold method. The entire detection process takes only 15 minutes, is simple to operate, requires no complex equipment, and is suitable for use in outpatient clinics, emergency rooms, and primary healthcare institutions. The stability of the fluorescent microsphere label is far superior to that of enzyme-labeled labels, and the kit can be stored at room temperature for more than 2 years.

[0051] According to a second aspect of the present invention, a method for preparing a D-dimer detection kit is provided, the method comprising: A pair of monoclonal antibodies specifically recognizing different antigenic epitopes of D-dimer were selected as the D-dimer detection antibody and the D-dimer capture antibody, respectively. This antibody pair selection ensures no steric hindrance when binding to D-dimers, exhibiting high affinity and high specificity.

[0052] Preparation of D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres: The fluorescent microspheres were resuspended, and EDC and NHS were added in a set ratio to activate the carboxyl groups. After centrifugation, excess cross-linking agent was removed to obtain carboxylated time-resolved fluorescent microspheres. The carboxylated time-resolved fluorescent microspheres were covalently coupled with the D-dimer detection antibody, blocked, and purified to obtain D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres, which was then stored in a preservation solution.

[0053] Preparation of chromatographic test strips: Spray the D-dimer detection antibody solution labeled with carboxylated time-resolved fluorescent microspheres onto the conjugate pad and vacuum dry; spray the D-dimer capture antibody onto the detection line of the nitrocellulose membrane and the goat anti-mouse IgG antibody onto the control line of the nitrocellulose membrane and dry; sequentially overlap and paste the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad onto the base plate, and cut into chromatographic test strips of the required width.

[0054] Assembly: Insert the chromatography strip into the test card, and assemble the test card, sample diluent, calibrator and quality control to form the D-dimer detection kit.

[0055] The preparation method of the D-dimer detection kit of the present invention, through steps such as EDC / NHS activation coupling, BSA blocking, and centrifugal purification, ensures stable binding and excellent specificity between carboxylated time-resolved fluorescent microspheres and D-dimer detection antibodies. This method is simple, efficient, and feasible, and can stably prepare D-dimer detection kits with high sensitivity, good specificity, easy operation, and accurate quantitative detection, providing reliable technical support for large-scale production.

[0056] Reference Figure 2 In some embodiments, the steps for preparing carboxylated time-resolved fluorescent microsphere-labeled D-dimer detection antibodies include: S1-1: Take 150–250 μL of fluorescent microspheres with a solid content of 0.8–1.1% and resuspend them in 0.4–0.6 mL of MES buffer. The MES buffer provides a suitable acid-base environment for the subsequent carboxyl activation reaction. Buffer resuspension can adjust the concentration of fluorescent microspheres to a suitable range for subsequent reactions, ensuring uniform dispersion of the microspheres and reserving reasonable reaction space for the subsequent addition of activators, antibodies, and other reagents.

[0057] S1-2: Add 90-110 μg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 90-110 μg NHS (N-hydroxysuccinimide) to activate the carboxyl groups, and rotate the reaction for 10-20 min. After the rotation reaction is complete, centrifuge to remove excess crosslinking agent to obtain carboxylated time-resolved fluorescent microspheres. EDC, as a carbodiimide crosslinking agent, can specifically activate the carboxyl groups (-COOH) on the surface of the fluorescent microspheres, converting them into unstable O-acylisourea intermediates. NHS can react with this intermediate to generate stable succinimide esters (-CO-NHS). The reactivity of this ester group with the antibody amino group (-NH2) is much higher than that of the original carboxyl group, which can significantly improve the subsequent coupling efficiency.

[0058] S1-3: Resuspend the carboxylated time-resolved fluorescent microspheres in MES buffer, add 0.05–0.20 mg of D-dimer detection antibody for covalent coupling, and allow to react at room temperature in the dark with shaking for 1.5–3.0 hours. The MES buffer ensures the coupling reaction is carried out in a stable acid-base environment; the amount of D-dimer detection antibody is matched to the number of activated carboxyl sites on the surface of the carboxylated time-resolved fluorescent microspheres to achieve optimal coupling efficiency. Due to the characteristics of the time-resolved fluorescent microspheres, light-protected operation is used to prevent quenching of fluorescent molecules by light exposure, ensuring the fluorescence intensity of the final product. Slow shaking allows for sufficient contact between the microspheres and the antibody, promoting the formation of stable amide bonds between the succinimide ester groups and the antibody amino groups, ensuring the structural stability of the coupling product and guaranteeing specific binding in subsequent detection.

[0059] S1-4: Add 40–60 μL of BSA (bovine serum albumin) solution with a solid content of 8–12% to block the remaining active sites. After blocking, centrifuge and purify to obtain D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres. After the coupling reaction, activated carboxyl sites that have not bound to the antibody may still remain on the surface of the microspheres. If these sites are not blocked, they are prone to non-specific binding with impurities in the sample during subsequent detection, leading to false positive results. The amino groups in the BSA molecule can bind to the activated carboxyl groups remaining on the microspheres, efficiently blocking the active sites. At the same time, BSA is stable and will not cross-react with the D-dimer detection antibody or subsequent detection system. Centrifugation purification removes free antibody (antibody not coupled to the microspheres), excess BSA, and other impurities from the reaction system, obtaining high-purity labeled antibody. This avoids non-specific binding of free antibody to capture antibody or sample antigen during detection, further improving detection specificity, while ensuring uniform labeled antibody concentration and enhancing batch-to-batch reproducibility.

[0060] S1-5: Resuspend the D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres in storage solution and store at 4°C. The purpose of this step is to extend the shelf life of the labeled antibody and maintain its activity, reduce reagent waste, and improve the convenience of clinical use.

[0061] The preparation method of this invention, through steps such as EDC / NHS activation coupling, BSA blocking, and centrifugal purification, ensures stable binding and excellent specificity between carboxylated time-resolved fluorescent microspheres and D-dimer detection antibodies; the optimized process parameters such as solution ratios ensure the consistency and repeatability of kit mass production.

[0062] In some preferred embodiments, the excitation wavelength of the fluorescent microspheres in step S1-1 is 355–375 nm, the emission wavelength is 600–620 nm, and the average particle size is 100–300 nm. These fluorescent microspheres have advantages such as high fluorescence intensity, good stability, and ease of covalent coupling with antibodies, which helps to improve detection sensitivity and detection linearity. Further, in one embodiment, the excitation wavelength of the fluorescent microspheres in step S1-1 is 365 nm, the emission wavelength is 610 nm, and the average particle size is 200 nm.

[0063] In some preferred embodiments, the pH of the MES buffer in step S1-1 is 5.0-6.0 and the concentration is 90-110 mM, which can provide a suitable acid-base environment for the subsequent carboxyl activation reaction.

[0064] In some preferred embodiments, the preservation solution in steps S1-5 is a Tris buffer containing 0.8–1.2% BSA. Tris buffer provides a stable neutral-to-alkaline environment (typically pH 7.0–8.0), which is more compatible with the natural active environment of the antibody. It effectively inhibits conformational changes in antibody molecules due to pH fluctuations. Simultaneously, its excellent buffering capacity can withstand slight acid-base changes that may occur during storage, providing a continuously stable physicochemical environment for the labeled antibody. The 0.8–1.2% BSA not only acts as a protein stabilizer, encapsulating the surface of the antibody and fluorescent microspheres through intermolecular interactions, reducing protein aggregation and precipitation, but also further blocks any remaining unbound active sites on the microsphere surface, reducing the risk of nonspecific binding in subsequent detection.

[0065] Reference Figure 3 In some embodiments, the steps for preparing the chromatographic test strip include: S2-1: The D-dimer detection antibody solution labeled with carboxylated time-resolved fluorescent microspheres obtained in step S1-5 is sprayed onto the conjugate pad at a spray volume of 4–6 μL / cm. The sprayed conjugate pad is then vacuum-dried at 37°C. The conjugate pad can be made of glass fiber. The precise spray volume of 4–6 μL / cm is optimized to ensure that the labeled antibody loading on the conjugate pad is within an optimal range, guaranteeing sufficient binding of the labeled antibody to the D-dimer in the sample without wasting raw materials or causing antibody buildup and blockage during chromatography due to excessive antibody.

[0066] S2-2: Using a spotting apparatus, D-dimer capture antibody at a concentration of 0.9–1.1 mg / mL is sprayed onto the detection line of the nitrocellulose membrane at a spray rate of 0.9–1.1 μL / cm. This concentration ensures sufficient capture sites on detection line 41 for efficient binding of the antigen-fluorescent antibody complex, improving binding efficiency. A uniform spray rate ensures even distribution of the D-dimer capture antibody on detection line 41, reducing detection errors, ensuring stable intra-batch / inter-batch results, and improving detection repeatability. Goat anti-mouse IgG antibody at a concentration of 0.9–1.1 mg / mL is sprayed onto the control line of the nitrocellulose membrane at a spray rate of 0.9–1.1 μL / cm. The sprayed nitrocellulose membrane is then dried at 37°C. Goat anti-mouse IgG antibody can specifically bind to labeled antibody. A reasonable concentration of 0.9–1.1 mg / mL can stably capture free labeled antibody, forming a clear quality control signal and ensuring detection validity. Uniform spray volume ensures stable signal intensity of control line 42. Color development through control line 42 is a prerequisite for valid detection results, ensuring reliability.

[0067] S2-3: Sequentially overlap and paste the sample pad, the binding pad prepared in step S2-1, the nitrocellulose membrane prepared in step S2-2, and the absorbent pad onto the base plate to form a large test paper plate.

[0068] S2-4: Cut the test strip plate into the required width to obtain the chromatography test strip.

[0069] In some preferred embodiments, the overlap distances between the sample pad and the conjugate pad, the conjugate pad and the nitrocellulose membrane, and the nitrocellulose membrane and the absorbent pad in steps S2-3 are 1.5 mm to 2.5 mm, respectively, and the width of the chromatography strip in step S2-4 is 3.5 to 4.0 mm. Further, in one embodiment, the overlap distances between the components are 2.0 mm, and the width of the chromatography strip in step S2-4 is 4.0 mm.

[0070] In one embodiment, the preparation method of the D-dimer detection kit includes: A pair of monoclonal antibodies that specifically recognize different antigenic epitopes of D-dimer were selected as D-dimer detection antibodies and D-dimer capture antibodies, respectively.

[0071] Preparation of D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres: S1-1: Take 200 μL of fluorescent microspheres with a solid content of 1% and resuspend them in 0.5 mL of MES buffer with a concentration of 100 mM and a pH of 6.0; S1-2: Add 100ug EDC and 100ug NHS to activate the carboxyl groups, and rotate the reaction for 15min; after the rotation reaction is complete, centrifuge to remove excess crosslinking agent to obtain carboxylated time-resolved fluorescent microspheres; S1-3: Resuspend the carboxylated time-resolved fluorescent microspheres in MES buffer, add 0.1 mg of D-dimer detection antibody for covalent coupling, and react with shaking at room temperature in the dark for 2 hours; S1-4: Add 50 μL of BSA solution with a solid content of 10% to block the remaining active sites; after blocking, centrifuge and purify to obtain D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres; S1-5: Resuspend the D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres in the preservation solution and store at 4°C.

[0072] Preparation of chromatography test strips: S2-1: The D-dimer detection antibody solution labeled with carboxylated time-resolved fluorescent microspheres obtained in step S1-5 is sprayed onto the conjugate pad at a spraying rate of 5 μL / cm, and the sprayed conjugate pad is dried under vacuum at 37°C. S2-2: Using a dotting instrument, D-dimer capture antibody with a concentration of 1 mg / mL was sprayed onto the detection line of the nitrocellulose membrane at a spraying rate of 1 μL / cm, and goat anti-mouse IgG antibody with a concentration of 1 mg / mL was sprayed onto the quality control line of the nitrocellulose membrane at a spraying rate of 1 μL / cm. The sprayed nitrocellulose membrane was then dried at 37°C. S2-3: Sequentially overlap and paste the sample pad, the binding pad prepared in step S2-1, the nitrocellulose membrane prepared in step S2-2, and the absorbent pad on the base plate, with an overlap distance of 2.0 mm between each component to form a large test paper plate; S2-4: Cut the test strip plate into 4mm widths to obtain chromatography test strips.

[0073] The D-dimer detection kit prepared using the above method and a commercially available immunoturbidimetric kit were used to simultaneously detect 100 clinical plasma samples. The D-dimer detection kit of this invention has significant advantages in terms of precision, accuracy, sensitivity, and specificity. (1) Precision: The quality control samples with high, medium and low concentrations were tested separately, and the coefficients of variation (CV) within and between batches were less than 10%.

[0074] (2) Accuracy: Correlation analysis with the results of immunoturbidimetric assay showed a correlation coefficient R² > 0.95, indicating good consistency.

[0075] (3) Sensitivity: The limit of detection (LoD) is 0.05 mg / L.

[0076] (4) Specificity: No cross-reaction with fibrinogen, fibrin monomers and other similar substances.

[0077] The test results show that the kit provided by the present invention has excellent performance, high sensitivity, good specificity, simple operation and can achieve accurate quantitative detection, which can meet the clinical needs for D-dimer detection.

[0078] The present invention has been described in the above-described embodiments; however, these embodiments are merely examples for implementing the present invention. It must be noted that the disclosed embodiments do not limit the scope of the present invention. Conversely, any modifications and refinements made without departing from the spirit and scope of the present invention are within the scope of patent protection of the present invention.

Claims

1. A D-dimer detection kit, characterized in that: It includes at least a chromatography test strip, the chromatography test strip including a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad sequentially disposed on the base plate and overlapping each other; The binding pad is coated with D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres. The D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres is prepared by covalently coupling, blocking and purifying carboxylated time-resolved fluorescent microspheres and D-dimer detection antibody. The nitrocellulose membrane is provided with a detection line and a control line at intervals. The detection line is coated with D-dimer capture antibody, and the control line is coated with goat anti-mouse IgG antibody. The D-dimer detection antibody and the D-dimer capture antibody are a pair of monoclonal antibodies that specifically recognize different antigenic epitopes of D-dimers.

2. The D-dimer detection kit as described in claim 1, characterized in that: The carboxylated time-resolved fluorescent microspheres have an excitation wavelength of 355–375 nm, an emission wavelength of 600–620 nm, and an average particle size of 100–300 nm.

3. The D-dimer detection kit as described in claim 1, characterized in that: The concentration of the D-dimer capture antibody on the detection line is 0.9–1.1 mg / mL, and the spray volume is 0.9–1.1 μL / cm.

4. The D-dimer detection kit as described in claim 1, characterized in that: The concentration of goat anti-mouse IgG antibody on the quality control line is 0.9–1.1 mg / mL, and the spray volume is 0.9–1.1 μL / cm.

5. The D-dimer detection kit as described in claim 1, characterized in that: The D-dimer detection kit also includes a detection card, sample diluent, calibrators, and quality control materials, with the chromatographic test strip disposed in the detection card.

6. A method for preparing a D-dimer detection kit as described in any one of claims 1-5, characterized in that: The preparation method of the D-dimer detection kit includes: A pair of monoclonal antibodies that specifically recognize different antigenic epitopes of D-dimer were selected as D-dimer detection antibody and D-dimer capture antibody, respectively. Preparation of D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres: The fluorescent microspheres were resuspended, and EDC and NHS were added in a set ratio to activate the carboxyl groups. After centrifugation, excess cross-linking agent was removed to obtain carboxylated time-resolved fluorescent microspheres. The carboxylated time-resolved fluorescent microspheres were covalently coupled with the D-dimer detection antibody, blocked, and purified to obtain D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres, which was then stored in a preservation solution. Preparation of chromatographic test strips: Spray the D-dimer detection antibody solution labeled with carboxylated time-resolved fluorescent microspheres onto the conjugate pad and vacuum dry; spray the D-dimer capture antibody onto the detection line of the nitrocellulose membrane and the goat anti-mouse IgG antibody onto the control line of the nitrocellulose membrane and dry; sequentially overlap and paste the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad on the base plate, and cut into chromatographic test strips of the required width; Assembly: Insert the chromatography strip into the test card, and assemble the test card, sample diluent, calibrator and quality control to form the D-dimer detection kit.

7. The method for preparing the D-dimer detection kit as described in claim 6, characterized in that: The steps for preparing D-dimer detection antibodies labeled with carboxylated time-resolved fluorescent microspheres include: S1-1: Take 150-250 μL of fluorescent microspheres with a solid content of 0.8-1.1% and resuspend them in 0.4-0.6 mL of MES buffer; S1-2: Add 90-110 μg EDC and 90-110 μg NHS to activate the carboxyl groups, and rotate the reaction for 10-20 min; after the rotation reaction is complete, centrifuge to remove excess crosslinking agent to obtain carboxylated time-resolved fluorescent microspheres. S1-3: Resuspend the carboxylated time-resolved fluorescent microspheres in MES buffer, add 0.05-0.20 mg of D-dimer detection antibody for covalent coupling, and react with shaking at room temperature in the dark for 1.5-3.0 hours; S1-4: Add 40-60 μL of BSA solution with a solid content of 8-12% to block the remaining active sites; after blocking, centrifuge and purify to obtain D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres; S1-5: Resuspend the D-dimer detection antibody labeled with carboxylated time-resolved fluorescent microspheres in the preservation solution and store at 4°C.

8. The method for preparing the D-dimer detection kit as described in claim 7, characterized in that: In step S1-1, the pH of the MES buffer is 5.0–6.0, and the concentration is 90–110 mM.

9. The method for preparing the D-dimer detection kit as described in claim 7, characterized in that: The preservation solution in steps S1-5 is Tris buffer containing 0.8-1.2% BSA.

10. The method for preparing the D-dimer detection kit as described in claim 6, characterized in that: The steps for preparing chromatographic test strips include: S2-1: The prepared D-dimer detection antibody solution labeled with carboxylated time-resolved fluorescent microspheres was sprayed onto the conjugate pad at a spraying rate of 4-6 μL / cm, and the sprayed conjugate pad was vacuum dried at 37°C. S2-2: Using a spotting apparatus, D-dimer capture antibody with a concentration of 0.9–1.1 mg / mL is sprayed onto the detection line of the nitrocellulose membrane at a spraying volume of 0.9–1.1 μL / cm, and goat anti-mouse IgG antibody with a concentration of 0.9–1.1 mg / mL is sprayed onto the control line of the nitrocellulose membrane at a spraying volume of 0.9–1.1 μL / cm. The sprayed nitrocellulose membrane is then dried at 37°C. S2-3: Sequentially overlap and paste the sample pad, the binding pad prepared in step S2-1, the nitrocellulose membrane prepared in step S2-2, and the absorbent pad on the base plate to form a large test paper plate; S2-4: Cut the test strip plate into the required width to obtain the chromatography test strip.

Images