A myocardial triple time-resolved fluorescent immunochromatographic test strip, kit, preparation method and application thereof

Abstract

The present application relates to a myocardial triple time-resolved fluorescence immunoassay test strip, kit and its preparation method and application, belong to the field of in vitro diagnosis technology, the present application provides a myocardial triple time-resolved fluorescence immunoassay test strip, the present application adopts Fab fragment antibody, eliminates the non-specific binding mediated by Fc section and the interference of heterophilic antibody;Adopt cTnI-Eu 3+ , CK-MB-Sm 3+ , MYO-Tb 3+ Fixed wavelength matching and scribe sequence, realize spectrum without crosstalk, signal accurate split, guarantee multi-index without cross, high accuracy, strong anti-interference, solve the pain point of traditional triple detection reagent spectrum color, item and matrix interference, false positive / false negative frequent, suitable for rapid and accurate quantitative detection of emergency whole blood, serum, plasma sample.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, and particularly relates to a myocardial triple time-resolved fluorescence immunochromatographic test strip, a reagent kit, its preparation method and application. Background Technology

[0002] Cardiac troponin I (cTnI) is a specific biomarker for myocardial injury and the gold standard for diagnosing acute myocardial infarction (AMI), exhibiting high specificity and a long window period. Creatine kinase isoenzyme MB (CK-MB) is an auxiliary diagnostic indicator of myocardial injury, reflecting the degree of myocardial necrosis and used for infarct area assessment. Myoglobin (MYO) is an early marker of elevated levels in myocardial injury, possessing high sensitivity and early onset, and is used for rapid early screening of AMI. Therefore, current rapid and accurate early detection of AMI relies on the combined detection of cTnI, CK-MB, and MYO. However, existing clinical products for myocardial combined testing generally suffer from the following drawbacks: (1) parallel testing of multiple indicators is prone to spectral cross-coloration and inter-item cross-interference; (2) they are easily bound by rheumatoid factor (RF), HAMA, and heterophile antibodies, leading to a high false positive rate; (3) they suffer from severe matrix interference from whole blood, hemolysis, lipemia, jaundice, etc., resulting in a poor signal-to-noise ratio; (4) high concentrations of MYO / CK-MB are prone to interference with cTnI detection, causing low-value missed detection or high-value false low detection; (5) ordinary fluorescence cannot distinguish between specific signals and autofluorescence, resulting in insufficient sensitivity and stability. It is difficult to simultaneously achieve detection without inter-item cross-interference, strong resistance to sample interference, and high accuracy. Therefore, providing a myocardial triple detection reagent with good anti-interference ability and high accuracy is of great significance in this field. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a myocardial triple time-resolved fluorescence immunochromatographic test strip, a reagent kit, and the preparation method and application thereof, to improve the anti-interference ability and accuracy of detecting cTnI, CK-MB, and MYO, so as to be more accurately used for early detection of acute myocardial infarction.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a myocardial triple time-resolved fluorescence immunochromatographic test strip, comprising a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the nitrocellulose membrane has a first detection line T1, a second detection line T2, a third detection line T3, and a control line C arranged sequentially along the chromatography direction; T1 is coated with cTnI capture antibody, T2 with CK-MB capture antibody, T3 with MYO capture antibody, and C with goat anti-mouse IgG; the conjugate pad is coated with Eu... 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3+ Microsphere-labeled CK-MB for detecting Fab fragments and Tb 3+ Microsphere-labeled MYO detection of Fab fragments.

[0005] Preferably, the Eu 3+ The microspheres have a particle size of 180~200nm, and the Sm 3+ The microspheres have a particle size of 190~210 nm, and the Tb 3+ The microspheres have a particle size of 210~230nm.

[0006] Preferably, the distance between two adjacent test lines or between an adjacent test line and a quality control line is ≥4mm; a hydrophobic isolation strip is provided between T1 and T2.

[0007] The present invention also provides a method for preparing the test strip, comprising the following steps: The Fc fragments of the cTnI detection antibody, CK-MB detection antibody, and MYO detection antibody were removed respectively to obtain the cTnI detection Fab fragment, CK-MB detection Fab fragment, and MYO detection Fab fragment; Activated Eu 3+ Microspheres, Sm 3+ Microspheres and Tb 3+ Microspheres were coupled to cTnI detection Fab fragments, CK-MB detection Fab fragments, and MYO detection Fab fragments, respectively, and then sealed to obtain Eu. 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3+ Microsphere-labeled CK-MB for detecting Fab fragments and Tb 3+ Microsphere-labeled MYO detection of Fab fragments; Eu 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3+ Microsphere-labeled CK-MB for detecting Fab fragments and Tb 3+ Microspheres labeled with MYO were used to detect the mixing of Fab fragments, resulting in a mixture that was sprayed onto the binding pad and dried to obtain the sprayed binding pad. Fluorine-containing hydrophobic ink was coated and cured between blank nitrocellulose membranes T1 and T2 to form a hydrophobic isolation zone; cTnI capture antibody was coated on T1 of the nitrocellulose membrane, CK-MB capture antibody was coated on T2; MYO capture antibody was coated on T3; and goat anti-mouse IgG was coated on C. The membranes were dried to obtain the coated nitrocellulose membranes. The sample pad, the sprayed bonding pad, the coated nitrocellulose membrane, and the absorbent pad are overlapped on the base plate and cut into test strips.

[0008] Preferably, in the mixture, the mass ratio of cTnI detection Fab fragment, CK-MB detection Fab fragment and MYO detection Fab fragment is 1:(0.8~1.2):(0.8~1.2), the concentration of cTnI detection Fab fragment is 0.2~0.5 mg / mL, and the spraying amount of the mixture is 3~6 μL / cm.

[0009] Preferably, the streaking concentrations of the antibodies on T1, T2, T3 and C are 0.8~1.2 mg / mL, and the streaking volumes are 0.8~1.2 μL / cm, respectively.

[0010] This invention provides a myocardial triple time-resolved fluorescence immunochromatographic assay kit, the kit comprising the aforementioned test strip and sample diluent, the sample diluent comprising: a heterophile antibody blocking agent, a rheumatoid factor blocking agent, irrelevant xenobiotic IgG, EDTA, PEG, and a surfactant.

[0011] Preferably, the sample diluent comprises: 10-50 mM PBS pH 7.00-7.50, heterophile antibody blocking agent 0.5%-1.0%, rheumatoid factor blocking agent 0.1%-0.5%, Tween-20 0.05%-0.5%, PEG6000 0.05%-0.2%, EDTA-Na2 0.5-2 mM, irrelevant heterologous IgG 0.05-0.1 mg / mL, and ProClin300 0.05%-0.5%.

[0012] This invention also provides the application of the test strips described herein, the test strips prepared by the described method, or the kit described herein in the preparation of the following products a and / or b: a) Simultaneous detection of cTnI, CK-MB and MYO concentrations; b) Early detection of acute myocardial infarction.

[0013] The present invention also provides a method for simultaneous detection of cTnI, CK-MB and MYO for non-diagnostic purposes, comprising the following steps: mixing the sample to be tested with the sample diluent in the kit at a volume ratio of 1:(2~4) to obtain a diluted sample, adding it to the sample pad of the test strip in the kit, and detecting it using a fluorescence immunochromatographic analyzer; The parameters detected by the fluorescence immunochromatographic analyzer are: excitation wavelength 350~365nm, delay time 100~200μs, integration time 200~400μs, and three-channel time-division acquisition and spectral demixing correction are used.

[0014] The beneficial effects of this invention are: This invention provides a time-resolved fluorescence immunochromatographic test strip for myocardial triplet assays. It employs a dual-antibody sandwich method for directional binding, time-resolved fluorescence immunochromatography technology, and a multi-dimensional anti-interference design, effectively solving the core problems of traditional myocardial triplet assay reagents, such as spectral crosstalk, inter-term crossover, significant matrix interference, and frequent false positives / false negatives. Compared to conventional fluorescence immunochromatographic test strips, this test strip eliminates Fc-mediated non-specific binding and utilizes tricolor rare-earth fluorescence (Eu)... 3+ 、Sm 3+ 、Tb 3+ Fixed wavelength matching and time-division acquisition enable simultaneous, accurate, and color-cross-color quantification of three indicators; simultaneously, this is combined with the fixed-order capture antibody arrangement of cTnI(Eu) 3+ )-CK-MB(Sm 3+ )-MYO(Tb 3+ This technology ensures natural separation of the detection spectral wavelengths, eliminating crosstalk even in adjacent detections, significantly improving detection accuracy and anti-interference capabilities. It enables rapid detection of various sample types, including whole blood, serum, and plasma. With simple sample pretreatment and fast detection speed, it is suitable for precise screening of myocardial biomarkers in demanding scenarios such as emergency departments and clinical laboratories.

[0015] The present invention also provides a kit comprising the test strip and sample diluent. The present invention relies on a multiple blocking system of sample diluent, especially the addition of heterophile antibody blocker (HBR), rheumatoid factor (RF) blocker and irrelevant xenobiotic IgG, to enhance the ability of the kit to detect endogenous interferences such as hemolysis, lipemia, jaundice, rheumatoid factor, and human anti-mouse antibody, thereby improving the anti-interference ability and accuracy of myocardial triple detection.

[0016] The kit of this invention can simultaneously meet the following requirements: relative deviation <10% under interference from hemolyzed Hb ≤5g / L, lipid-lowering TG ≤10g / L, jaundice Bil ≤34μmol / L, RF ≤4000IU / mL, and human anti-mouse antibody HAMA positive samples; when simultaneously detecting cTnI, CK-MB, and MYO, the cross-interference rate among the three indicators is <5%; intra-assay / inter-assay CV <8%; and the spiked recovery rate is 95%~105%. This indicates that the kit of this invention has strong anti-interference ability, high accuracy, good specificity, excellent repeatability, and stable and reliable detection results, meeting the needs of rapid and accurate clinical testing. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the test strip structure in some embodiments of the present invention; wherein, 1-base plate, 2-sample pad, 3-conjugation pad, 4-nitrocellulose membrane, 5-absorbent pad, T1-T1 detection line, T2-T2 detection line, T3-T3 detection line, C-control line. Detailed Implementation

[0018] This invention provides a myocardial triple time-resolved fluorescence immunochromatographic test strip, comprising a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad; the nitrocellulose membrane has a first detection line T1, a second detection line T2, a third detection line T3, and a control line C sequentially arranged along the chromatography direction; T1 is coated with cTnI capture antibody, T2 with CK-MB capture antibody, T3 with MYO capture antibody, and C with goat anti-mouse IgG; the conjugate pad is coated with Eu... 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3+ Microsphere-labeled CK-MB for detecting Fab fragments and Tb 3+ Microsphere-labeled MYO detection of Fab fragments.

[0019] In this invention, the cTnI detection Fab fragment, CK-MB detection Fab fragment, and MYO detection Fab fragment are preferably obtained by removing the Fc fragment from the cTnI detection antibody, CK-MB detection antibody, and MYO detection antibody, respectively. The detection antibody for the same protein and its corresponding capture antibody are preferably paired with non-overlapping epitopes. Their binding is sterically unhindered, exhibiting high affinity and high specificity, and can specifically recognize different antigenic epitopes of the corresponding antigen, ensuring detection specificity and accuracy. The cTnI detection Fab fragment, CK-MB detection Fab fragment, and MYO detection Fab fragment are preferably derived from different preparation systems with different genetic backgrounds, which can eliminate cross-reactivity and avoid mutual interference between different proteins. This invention has found that full-length IgG antibodies containing the Fc fragment are easily bound by RF, HAMA, and heterophile antibodies, leading to a high false positive rate. By removing the Fc fragment, Fc-mediated non-specific binding and heterophile antibody interference can be eliminated, improving detection accuracy.

[0020] This invention does not specifically limit the source of the cTnI detection antibody, CK-MB detection antibody, and MYO detection antibody; conventional commercially available products in the field can be used. In some embodiments, the cTnI detection antibody can be a cTnI monoclonal antibody (Phipeng Biotechnology Co., Ltd. CTNI-McAB-24#), the CK-MB detection antibody can be a CK-MB monoclonal antibody (Phipeng Biotechnology Co., Ltd. CK-MB-03), and the MYO detection antibody can be a MYO monoclonal antibody (Phipeng Biotechnology Co., Ltd. MYO-Ab2#).

[0021] In this invention, the Eu 3+ The particle size of the microspheres is preferably 180~200nm, specifically 180nm, 190nm or 200nm, for cTnI ultrasensitive detection; the Sm 3+ The particle size of the microspheres is preferably 190~210nm, specifically 190nm, 200nm or 210nm; the Tb 3+ The particle size of the microspheres is preferably 210~230nm, specifically 210nm, 220nm, or 230nm, for wide linearity and anti-hooking effect of MYO. Terbium (Tb) is used in this process. 3+ The average particle size of the microspheres is preferably greater than that of europium (Eu). 3+ Microspheres and samarium (Sm) 3+ The average particle size of the microspheres. This invention optimizes the particle size and particle size differentiation by selecting suitable particle sizes, combined with tricolor rare earth fluorescence (Eu). 3+ 、Sm 3+ 、Tb 3+ Fixed wavelength matching and time-division acquisition can achieve simultaneous, accurate, and color-cross-color quantification of three indicators, reduce detection interference, and improve detection accuracy.

[0022] The microspheres described in this invention are preferably carboxylated time-resolved fluorescent microspheres, which encapsulate lanthanide ions (Eu). 3 + 、Sm 3+ 、Tb 3+The carboxylated time-resolved fluorescent microspheres exhibit strong fluorescence signals and large Stokes shifts. A single microsphere can carry a large number of fluorescent groups, which, after covalent coupling with the detection antibody, form a significant signal amplification effect, greatly improving the sensitivity and linear range of the detection system. The carboxylated time-resolved fluorescent microspheres possess excellent physicochemical stability. During storage, transportation, and immunoreaction at room temperature, they are not easily affected by environmental factors such as pH, temperature, and ionic strength, preventing fluorescence quenching, aggregation, or sedimentation, thus ensuring a continuous and stable output of the fluorescence signal. The carboxyl groups on the microsphere surface easily achieve efficient covalent coupling with antibodies through activation, resulting in high coupling efficiency and strong binding, effectively preventing antibody detachment from the microsphere surface during storage or chromatography, ensuring the uniformity and batch-to-batch stability of the labeled antibody activity. Simultaneously, the antibodies labeled with the microspheres bind to the target antigen quickly and with high affinity, shortening the chromatography reaction time and meeting the needs of rapid emergency testing. This invention does not specifically limit the source of the carboxylated time-resolved fluorescent microspheres; conventional commercially available products in the art can be used. In some embodiments, Eu... 3+ Time-resolved fluorescent microspheres (MD016, Nanjing Microtest Biotechnology Co., Ltd.), Sm 3+ Time-resolved fluorescent microspheres (MD057 from Nanjing Microtest Biotechnology Co., Ltd.) and Tb 3+ Time-resolved fluorescent microspheres (R-PS60-G, Xi'an Ruixi Biotechnology Co., Ltd.)

[0023] In this invention, by sequentially arranging T1-T3 coated with specific capture antibodies along the chromatography direction, a fixed sequence of capture antibody arrangement cTnI(Eu) is formed. 3+ )-CK-MB(Sm 3+ )-MYO(Tb 3+ The distance between two adjacent detection lines or between an adjacent detection line and a control line is preferably ≥4mm, specifically 4mm, 5mm, or 5.5mm; a hydrophobic isolation band is provided between T1 and T2, preferably a UV-cured hydrophobic ink band containing fluorine. By arranging the capture antibodies in a fixed order and providing a hydrophobic isolation band between T1 and T2, lateral diffusion crosstalk can be blocked, thereby blocking interference between detection indicators. This invention does not specifically limit the source of the capture antibodies and goat anti-mouse IgG; conventional commercially available products in the field can be used. In some embodiments, the capture antibodies can be selected from cTnI monoclonal antibody (cTnI104 from Hangzhou Boyue Biotechnology Co., Ltd.), CK-MB monoclonal antibody (CKMB102 from Hangzhou Boyue Biotechnology Co., Ltd.), MYO monoclonal antibody (MYO102 from Hangzhou Boyue Biotechnology Co., Ltd.), and goat anti-mouse IgG antibody (goat anti-mouse IgG from Feipeng Biotechnology Co., Ltd.).

[0024] The present invention also provides a method for preparing the test strip, comprising the following steps: The Fc fragments of the cTnI detection antibody, CK-MB detection antibody, and MYO detection antibody were removed respectively to obtain the cTnI detection Fab fragment, CK-MB detection Fab fragment, and MYO detection Fab fragment; In this invention, the preferred method for removing the Fc fragment is papain enzymatic digestion. This invention does not specifically limit the details of the digestion steps; conventional steps in the art are acceptable. In some embodiments, the enzyme and antibody can be mixed at a mass ratio of 1:10 and digested at 37°C for 4 hours. Prior to the digestion, it is preferred to include dialysis of the detection antibody and activation of the enzyme. This invention does not specifically limit the dialysis of the detection antibody and activation of the enzyme; conventional steps in the art are acceptable. After digestion, the resulting solution is preferably ultrafiltered at 10 kDa and then loaded onto a Protein A / G affinity chromatography column. The unbound flow-through is collected; then concentrated using a 10 kDa ultrafiltration tube and replaced with the same buffer solution to obtain a high-purity Fab fragment with a purity ≥95%.

[0025] Activated Eu 3+ Microspheres, Sm 3+ Microspheres and Tb 3+ Microspheres were coupled to cTnI detection Fab fragments, CK-MB detection Fab fragments, and MYO detection Fab fragments, respectively, and then sealed to obtain Eu. 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3+ Microsphere-labeled CK-MB for detecting Fab fragments and Tb 3+ Microsphere-labeled MYO detection of Fab fragments; The present invention does not specifically limit the activation, coupling, and blocking steps, and conventional steps in the art can be used. In some embodiments, the activation step includes: adding 50 mM MES buffer (pH 5.0~6.0, such as 5.0, 5.5, or 6) to the microspheres and mixing well; adding EDC and NHS activation solution sequentially to make the final concentration of EDC 0.8~1.2 mM (such as 0.8, 0.94, 1, or 1.2 mM) and the final concentration of NHS 1.8~2.2 mM (such as 1.8, 1.89, 2, or 2.2 mM); and reacting by shaking or rotating at 35~37℃ (such as 35, 36, or 37℃) for 20~30 min (20 min, 25 min, or 30 min) to complete the carboxyl activation of the microspheres and obtain the activated microspheres.

[0026] After activation, centrifugation and washing are performed to discard unreacted reagents before coupling. In some embodiments, the coupling step includes: adding Fab fragments to the corresponding activated microspheres, coupling at room temperature (35~37℃, such as 35, 36 or 37℃), in the dark, and by shaking or rotation for 1.5~3h (1.5h, 2h, 2.5h or 3h). The preferred mass ratio of microspheres to Fab fragments is 1:(0.08~0.2), specifically 1:0.08, 1:0.1, 1:0.15 or 1:0.2.

[0027] After coupling, blocking is performed. In some embodiments, the blocking step includes: adding BSA blocking solution to the coupled microsphere system and blocking by rotation at room temperature (35~37℃, such as 35, 36 or 37℃) in the dark for 30~60 min (such as 30 min, 40 min, 50 min or 60 min) to block unreacted activated sites on the surface of the microspheres; the mass fraction of the BSA blocking solution is preferably 8%~12% (w / v), specifically 8%, 10% or 12%, and the amount added is preferably 10~50 μL per 100 μL of microsphere system, specifically 10 μL, 30 μL, 40 μL or 50 μL per 100 μL of microsphere system.

[0028] The preferred method after sealing also includes centrifugation to discard the supernatant, yielding Eu. 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3 + Microsphere-labeled CK-MB for detecting Fab fragments and Tb 3+ Microsphere-labeled MYO-detecting Fab fragments were stored in preservation solutions at 2–8°C. Preferred preservation solutions included: 10–50 mM PBS, pH 7.00–7.50, Tween-20 0.05%–0.5% (w / v), BSA 0.5–2% (w / v), ProClin300 0.05%–0.5% (v / v), and sucrose 5%–10% (w / v) or trehalose 5%–10% (w / v); more preferred solutions included: 10, 20, 30, 40, or 50 mM PBS, pH 7.00, 7.40, or 7.50, Tween-20 0.05%, 0.1%, 0.3%, or 0.5% (w / v), BSA 0.5%, 1%, or 2% (w / v), and ProClin300. 0.05%, 0.1%, 0.3% or 0.5% (v / v), sucrose 5%, 8% or 10% (w / v) or trehalose 5%, 8% or 10% (w / v).

[0029] In this invention, the centrifugation is preferably performed at 14,000 to 20,000 rpm (e.g., 14,000, 15,000, 18,000 or 20,000 rpm) and at 4°C for 8 to 15 minutes (e.g., 8 minutes, 10 minutes or 15 minutes).

[0030] Eu 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3+ Microsphere-labeled CK-MB for detecting Fab fragments and Tb 3+ Microspheres labeled with MYO detection Fab fragments are mixed to obtain a mixture. The preferred mass ratio of the cTnI detection Fab fragment, CK-MB detection Fab fragment, and MYO detection Fab fragment in the mixture is 1:(0.8~1.2):(0.8~1.2), specifically 1:0.8:0.8, 1:1:0.8, 1:1:1, 1:1.2:0.8, 1:0.8:1, 1:0.8:1.2, or 1:1.2:1.2. The preferred concentration of the cTnI detection Fab fragment in the mixture is 0.2~0.5 mg / mL, specifically 0.2, 0.33, or 0.5 mg / mL. The preferred spraying amount of the mixture is 3~6 μL / cm, specifically 3, 4, 5, or 6 μL / cm. The mixture is sprayed onto the conjugate pad and dried to obtain the sprayed conjugate pad. A fluorinated hydrophobic ink is coated and cured between blank nitrocellulose membranes T1 and T2 to form a hydrophobic barrier. The coating thickness is preferably 10-15 μm, specifically 10 μm, 13 μm, or 15 μm. After coating, it is preferably cured online under 365nm LED ultraviolet light, with the light intensity preferably being 500-1000 mW / cm². 2 Specifically, the power output can be selected as 500, 800, or 1000 mW / cm. 2 The exposure time is preferably 0.5~1.0s, specifically 0.5, 0.8 or 1.0s, to form a hydrophobic isolation band that runs through the width of the film. The width of the hydrophobic isolation band is preferably 1~1.5mm, specifically 1, 1.3 or 1.5mm.

[0031] A nitrocellulose membrane is streaked on T1 and coated with cTnI capture antibody, streaked on T2 and coated with CK-MB capture antibody, streaked on T3 and coated with MYO capture antibody, and streaked on C and coated with goat anti-mouse IgG. After drying, the coated nitrocellulose membrane is obtained. The streaking concentrations of the antibodies on T1, T2, T3 and C are preferably 0.8~1.2 mg / mL (the antibody concentration is preferably obtained by dilution with 10 mM PBS buffer), specifically 0.8, 1.0 or 1.2 mg / mL, and the streaking volume is preferably 0.8~1.2 μL / cm, specifically 0.8, 1.0 or 1.2 μL / cm.

[0032] In this invention, the drying temperature during the bonding pad spraying and nitrocellulose membrane coating processes is preferably 35°C to 37°C, specifically 35°C, 36°C or 37°C, and the drying time is preferably 12h to 28h, specifically 12h, 24h or 28h.

[0033] The sample pad, the sprayed binding pad, the coated nitrocellulose membrane, and the absorbent pad are overlapped on the base plate and cut into test strips. The preferred cut width is 3.8 mm. The overlap is preferably performed by attaching the binding pad and sample pad to the NC membrane near the T1 end, and attaching the absorbent pad to the other end of the NC membrane, ensuring a tight overlap without gaps. The structural diagram of the test strip is shown below. Figure 1 As shown, the chromatography test strip includes a base plate 1, and a sample pad 2, a conjugate pad 3, a nitrocellulose membrane 4 (NC membrane), and an absorbent pad 5, which are sequentially arranged on the base plate 1 and overlap each other. The nitrocellulose membrane 4 has a first detection line T1, a second detection line T2, a third detection line T3, and a control line C sequentially arranged along the chromatography direction; the first detection line T1 is closer to the conjugate pad 3, and the control line C is closer to the absorbent pad 5. The base plate 1 is preferably made of PVC (Polyvinyl chloride); the sample pad 2 and the conjugate pad 3 are preferably made of glass fiber; and the absorbent pad 5 is preferably made of highly absorbent paper.

[0034] This invention provides a myocardial triple time-resolved fluorescence immunochromatographic assay kit. The kit includes the test strip and sample diluent. The sample diluent preferably includes: a heterophile antibody blocker, a rheumatoid factor blocker, irrelevant xenobiotic IgG, EDTA, PEG, and a surfactant; more preferably, it includes 10-50 mM PBS pH 7.00-7.50, 0.5%-1.0% heterophile antibody blocker, 0.1%-0.5% rheumatoid factor blocker, 0.05%-0.5% Tween-20, 0.05%-0.2% PEG6000, 0.5%-2 mM EDTA-Na2, 0.05-0.1 mg / mL irrelevant xenobiotic IgG, and 0.05%-0.5% ProClin300; and even more preferably, it includes 10, 20, 30, 40, or 50 mM. PBS, pH 7.00, 7.20, 7.40, or 7.50; heterophile antibody blocking agent 0.5%, 0.8%, or 1.0% (w / v); rheumatoid factor blocking agent 0.1%, 0.3%, or 0.5% (w / v); Tween-20 0.05%, 0.08%, 0.1%, 0.3%, or 0.5% (w / v); PEG6000 0.05%, 0.1%, 0.15%, or 0.2% (w / v); EDTA-Na2 0.5%, 1%, 1.5%, or 2 mM; irrelevant xenobiotic IgG 0.05%, 0.08%, or 0.1 mg / mL; and ProClin300 0.05%, 0.08%, 0.1%, 0.3%, or 0.5% (v / v). This invention does not specifically limit the source of each substance in the sample diluent; conventional commercially available products or conventional methods in the art can be used. This invention relies on a multiple blocking system of sample diluents, especially the addition of heterophile antibody blockers, rheumatoid factor blockers, and irrelevant xenobiotic IgG, to enhance the ability of the kit to detect endogenous interferences such as hemolysis, lipemia, jaundice, rheumatoid factor, and human anti-mouse antibodies, thereby improving the anti-interference ability and accuracy of myocardial triple detection.

[0035] In this invention, the preferred method for preparing the reagent kit includes: preparing a sample diluent according to the concentration of each component; after preparation, preferably filtering and sterilizing the sample; and then assembling the sample kit with the test strips. The assembly process preferably involves inserting the test strips into a cartridge.

[0036] This invention also provides the application of the test strips described herein, the test strips prepared by the described method, or the kit described herein in the preparation of the following products a and / or b: a) Simultaneous detection of cTnI, CK-MB and MYO concentrations; b) Early detection of acute myocardial infarction.

[0037] The kit prepared in this invention can simultaneously detect the concentrations of cTnI, CK-MB, and MYO, effectively solving the core problems of traditional myocardial triple detection kits, such as spectral crosstalk, inter-item crossover, large matrix interference, and frequent false positives / false negatives. Compared with conventional fluorescence immunochromatographic kits, it significantly improves detection accuracy and anti-interference ability, and can be used more accurately and reliably for the early detection of acute myocardial infarction. It is suitable for rapid and accurate quantitative detection of whole blood, serum, and plasma samples in the emergency department.

[0038] The present invention also provides a method for simultaneous detection of cTnI, CK-MB and MYO for non-diagnostic purposes, comprising the following steps: mixing the sample to be tested with the sample diluent in the kit at a volume ratio of 1:(2~4) (specifically 1:2, 1:3 or 1:4) to obtain a diluted sample, adding it to the sample pad of the test strip in the kit, and detecting it using a fluorescence immunochromatographic analyzer; The parameters detected by the fluorescence immunochromatographic analyzer are: excitation wavelength 350~365nm (e.g. 350nm, 355nm, 360nm or 365nm), delay time 100~200μs (e.g. 100μs, 150μs or 200μs), integration time 200~400μs (e.g. 200μs, 300μs or 400μs), and three-channel time-division acquisition and spectral demixing correction are used.

[0039] In this invention, the test sample includes whole blood, serum, or plasma.

[0040] This invention eliminates matrix autofluorescence by using time-resolved delayed acquisition, time-division reading, and spectral demixing, achieving ultra-high signal-to-noise ratio and accurate quantification. It ensures simultaneous quantification of the three indicators without crosstalk, thus improving detection accuracy.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Unless otherwise specified, the following embodiments are all conventional methods.

[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0044] Example 1 The preparation of a myocardial triple time-resolved fluorescence immunochromatographic assay kit (cTnI / CK-MB / MYO triple time-resolved fluorescence immunochromatographic assay kit) includes the following steps: (1) First carboxylation time-resolved fluorescent microsphere labeling cTnI detection antibody (Eu) 3+ Preparation of microsphere-labeled cTnI detection Fab fragments a. Dialyze the in vitro expressed cTnI monoclonal antibody (CTNI-McAB-24# from Phytobio Biotechnology Co., Ltd.) in 0.01 mol / L, pH 7.4 phosphate buffer and adjust the concentration to 4 mg / mL to obtain an antibody solution. Activate papain in 0.1 mol / L, pH 7.4 phosphate buffer containing 0.01 mol / L cysteine ​​and 0.2 mol / L EDTA at 37°C for 20 minutes to prepare an activation enzyme solution. Add the activation enzyme solution to the antibody solution at an enzyme-to-antibody mass ratio of 1:10, and digest at 37°C for 4 hours (monitored by SDS-PAGE until complete digestion). Terminate the reaction by adding iodoacetamide to a final concentration of 0.1 mol / L. Centrifuge the digested product in a 10 kDa ultrafiltration tube to replace the 0.01 mol / L, pH 7.4 phosphate buffer. Load the resulting solution into Protein. An A / G affinity chromatography column was used to collect the unbound flow-through. The flow-through was then concentrated using a 10 kDa ultrafiltration tube and replaced with the same buffer solution to obtain the Fab fragment (cTnI was used to detect the Fab fragment). SDS-PAGE analysis showed that the non-reducing molecular weight was 50 kDa, and the anti-Fc specific antibody test was negative.

[0045] b. Activation: Take 100 μL (1% w / v) Eu 3+ Time-resolved fluorescent microspheres (MD016, Nanjing Microtest Biotechnology Co., Ltd.), with an average particle size of 190 nm, were gently mixed after adding 0.5 mL of 50 mM pH 5.50 MES solution. Then, 2 μL of 50 mM MEDC solution was added, followed by 4 μL of 50 mM Sulfo-NHS solution. The mixture was incubated at 37 °C for 30 min by rotation to complete activation. Subsequently, the mixture was centrifuged at 15000 rpm at 4 °C for 15 min, and the supernatant was discarded.

[0046] c. Washing: Redissolve the product from step b. with 1 mL of 20 mM BB buffer, and sonicate (30% power, sonicate for 30 s, with 30 s intervals, sonicate 3 times). Centrifuge at 15000 rpm and 4℃ for 10 min, discard the supernatant, and repeat once.

[0047] d. Coupling: Add 1 mL of 20 mM BB buffer to reconstitute the product from step c., sonicate to disperse, add 0.15 mg of cTnI to detect the Fab fragment, and spin-couple at 37 °C for 2 h to complete the labeling.

[0048] e. Blocking: Add 40µL of 10% BSA to every 100µL of the coupled microsphere system and mix gently. Incubate at 37℃ for 1 hour by rotation. Centrifuge at 15000rpm and 4℃ for 10 minutes, discard the supernatant, and obtain Eu. 3+ Microsphere-labeled cTnI detection of Fab fragments.

[0049] f. Save: Save the above Eu3+ Microsphere-labeled cTnI-detected Fab fragments were resuspended in preservation solution by sonication (30% power, 30s sonication, 30s interval, 2 sonications), and stored at 2–8°C. The preservation solution consisted of 20mM PBS pH 7.40, Tween-20 0.1% (w / v), BSA 0.5% (w / v), ProClin300 0.05% (v / v), and trehalose 5% (w / v).

[0050] (2) Second carboxylation time-resolved fluorescent microsphere labeling of CK-MB detection antibody (Sm 3+ Preparation of microsphere-labeled CK-MB for detecting Fab fragments a. The CK-MB monoclonal antibody (CK-MB-03 from Fipeng Biotechnology Co., Ltd.) expressed in vitro was used to prepare the Fab fragment (CK-MB detection Fab fragment) according to step a in step (1) of this embodiment. The molecular weight was 50kDa under non-reducing conditions as identified by SDS-PAGE, and the anti-Fc specific antibody test was negative. The antigen binding activity was verified by ELISA and retained.

[0051] b. Activation ~ c. Washing: Take 100 μL of Sm 3+ Time-resolved fluorescent microspheres (MD057 from Nanjing Microtest Biotechnology Co., Ltd.) (1% w / v) with an average particle size of 200 nm were activated and washed according to steps b and c in step (1) of this embodiment.

[0052] d. Coupling: Add 1 mL of 20 mM BB buffer to reconstitute the product from step c., sonicate to disperse, add 0.08 mg of CK-MB to detect the Fab fragment, and spin-couple at 37°C for 2 h to complete the labeling.

[0053] e. Blocking: Add 40µL of 10% BSA to every 100µL of the coupled microsphere system and mix gently. Incubate at 37℃ for 1 hour by rotation. Centrifuge at 15000rpm and 4℃ for 10 minutes, discard the supernatant, and obtain Sm... 3+ Microsphere-labeled CK-MB for detecting Fab fragments.

[0054] f. Save: Save the above Sm 3+ Microsphere-labeled CK-MB was used to detect Fab fragments. The fragments were resuspended by sonication (same as step (1)) in preservation solution (30% power, sonication for 30s, interval of 30s, sonication twice), and stored at 2~8℃.

[0055] (3) Third carboxylation time-resolved fluorescent microsphere labeling of MYO detection antibody (Tb 3+ Preparation of microsphere-labeled MYO detection Fab fragments a. Prepare the Fab fragment (MYO detection Fab fragment) by using MYO monoclonal antibody (MYO-Ab2# from Fipeng Biotechnology Co., Ltd.) derived from mouse ascites fluid according to step a. in step (1) of this embodiment; the molecular weight is 50kDa under non-reducing conditions as identified by SDS-PAGE, and the detection of anti-mouse IgG-Fc specific antibody is negative. The antigen binding activity is retained as verified by ELISA.

[0056] b. Activation ~ c. Washing: Take 100 μL of Tb 3+ Time-resolved fluorescent microspheres (R-PS60-G, Xi'an Ruixi Biotechnology Co., Ltd.) (1% w / v), with an average particle size of 220 nm, were activated and washed according to steps b and c in step (1) of this embodiment.

[0057] d. Coupling: Add 1 mL of 20 mM BB buffer to reconstitute the product from step c., sonicate to disperse, add 0.10 mg of MYO to detect the Fab fragment, and spin-couple at 37°C for 2 h to complete the labeling.

[0058] e. Blocking: Add 40µL of 10% BSA to every 100µL of the coupled microsphere system and mix gently. Incubate at 37℃ for 1 hour by rotation. Centrifuge at 15000rpm and 4℃ for 10 minutes, discard the supernatant, and obtain Tb. 3+ Microsphere-labeled MYO detection of Fab fragments.

[0059] f. Save: Save the above Tb 3+ Microsphere-labeled MYO-detected Fab fragments were resuspended by sonication in preservation solution (same as step (1)) (30% power, sonication for 30s, interval of 30s, sonication twice), and stored at 2~8℃.

[0060] (4) Preparation of liquid reagents The three types of labeled microspheres obtained in steps (1), (2), and (3) were diluted with preservation solution, namely Eu 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3+ Microsphere-labeled CK-MB detection of Fab fragments and Tb 3+ Microspheres were labeled with MYO to detect Fab fragments. The calculated concentration of the antibody coupled to the surface of the microspheres in each labeled microsphere suspension was adjusted to 1.0 mg / mL. The three diluted labeled microsphere suspensions were mixed in equal volume ratios to prepare the conjugate pad spray mixture.

[0061] The mixture was evenly applied to the glass fiber bonding pad at a spraying rate of 5 μL / cm, and then dried in an oven at 37°C for 24 hours.

[0062] (5) Coating of NC membrane a. Preparation of NC film: A blank NC plate was equilibrated under constant temperature and humidity. UV-curable fluorinated hydrophobic ink was then applied via screen printing at pre-defined gaps between the T1 and T2 detection lines, resulting in a wet film thickness of 13 μm. After coating, the film was cured online under 365 nm LED ultraviolet light at an intensity of 800 mW / cm². 2 The exposure time is 0.8s, forming a hydrophobic isolation band that runs through the film width, blocking the lateral diffusion of liquid; the spacing between T1 and T2 lines is 5mm, and the width of the hydrophobic isolation band is 1.5mm.

[0063] b. Coating the NC membrane: Goat anti-mouse IgG antibody (Feipeng Biotechnology Co., Ltd. goat anti-mouse IgG) was diluted to 0.8 mg / mL with 10 mM PBS buffer and used as the C line at a volume of 1 μL / cm. cTnI mouse monoclonal antibody capture antibody (Hangzhou Boyue Biotechnology Co., Ltd. cTnI104) was diluted to 1.2 mg / mL with 10 mM PBS buffer and used as the first T line (T1). CK-MB mouse monoclonal antibody capture antibody (Hangzhou Boyue Biotechnology Co., Ltd. CKMB102) was diluted to 1 mg / mL with 10 mM PBS buffer and used as the second T line (T2). MYO mouse monoclonal antibody capture antibody (Hangzhou Boyue Biotechnology Co., Ltd. MYO102) was diluted to 1 mg / mL with 10 mM PBS buffer and used as the third T line (T3). These were sequentially streaked onto the NC membrane for coating. The spacing between adjacent test lines and between the test line and the control line was 5 mm. Then place it in a 37℃ oven to dry for 24 hours.

[0064] (6) Assembly of test strips Using an overlapping method on an adhesive-backed PVC base plate, the bonding pad and sample pad are overlapped and pasted at the T1 end of the NC membrane, and the absorbent pad is overlapped and pasted at the other end of the NC membrane. All components are tightly overlapped without gaps to obtain a large test strip plate. This plate is then cut into 3.8mm wide test strips using a coded strip cutter. These strips are then packed into a three-part card plastic casing and pressed firmly. The structure is as follows: Figure 1 As shown.

[0065] (7) Sample diluents: 20mM PBS pH7.40, heterophile antibody blocking agent (HBR blocking agent from Hangzhou Boyue Biotechnology Co., Ltd.) 0.8% (w / v), rheumatoid factor blocking agent (C04002 blocking agent from Jiangsu Dongkang Biopharmaceutical Co., Ltd.) 0.3% (w / v), Tween-20 0.08% (w / v), PEG6000 0.15% (w / v), EDTA-Na2 0.037% (w / v) (1mM), irrelevant xenogeneic IgG (anti-human IgG monoclonal antibody from Jiangsu Dongkang Biopharmaceutical Co., Ltd., catalog number: C01206) 0.008% (w / v), ProClin300 0.08% (v / v) (8) Detection method: Mix the sample to be tested (whole blood, serum or plasma) with the sample diluent at a volume ratio of 1:3. Take an appropriate amount of diluted sample and drop it onto the sample pad. The sample migrates towards the conjugate pad under capillary chromatography. If the sample contains cTnI, CK-MB, or MYO antigens, they can be reacted with the corresponding Eu antigens coated on the conjugate pad. 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3 + Microsphere-labeled CK-MB detection of Fab fragments and Tb 3+ Microspheres specifically bind to the Fab fragment, forming an "antigen-fluorescently labeled antibody" complex. This complex flows to a nitrocellulose membrane via chromatography and sequentially binds specifically to the cTnI capture antibody (T1), the CK-MB capture antibody (T2), and the MYO capture antibody (T3), forming a "capture antibody-antigen-fluorescently labeled antibody" double-antibody sandwich complex. Unbound fluorescently labeled antibodies continue chromatography to the control line, where they are captured by goat anti-mouse IgG antibody on the control line. Detection is then performed using a fluorescence immunochromatographic analyzer. Fluorescence signal intensities at each detection line and control line are read using 365nm excitation and time-resolved delayed acquisition. The time-resolved detection parameters are: excitation wavelength 365nm, delay time 150μs, integration time 300μs, three-channel independent time-sharing acquisition with spectral demixing correction. Through three-channel time-sharing acquisition and spectral demixing correction, the instrument automatically calculates and outputs the precise concentrations of cTnI, CK-MB, and MYO in the sample based on the built-in standard curve. The entire detection cycle is 600s.

[0066] Examples 2-3 Examples 2-3 all describe the preparation of a myocardial triple time-resolved fluorescence immunochromatographic assay kit. The only difference between these kits and Example 1 is the content in the table below. All other aspects are the same as in Example 1.

[0067] Table 1. Differences between Examples 2-3 and Example 1

[0068] Experimental Example 1 Methodological validation of the reagent kit prepared in Example 1 The detection gradient settings were as follows: cTnI: low 0.3 ng / mL, medium 2.0 ng / mL, high 12.0 ng / mL; CK-MB: low 3.0 ng / mL, medium 18.0 ng / mL, high 90.0 ng / mL; MYO: low 50 ng / mL, medium 200 ng / mL, high 800 ng / mL.

[0069] Anti-interference performance determination: Interference matrix samples containing hemolysis (Hb 5g / L), lipemia (TG 10g / L), jaundice (Bil 34μmol / L), rheumatoid factor (4000 IU / mL), and human anti-mouse antibody HAMA positive were prepared. Low, medium, and high concentrations of cTnI, CK-MB, and MYO antigen were added respectively. The kit prepared in Example 1 was used for detection. The concentrations of each analyte in the non-interference matrix sample and the interference matrix sample were obtained, and the relative deviation of the detection results was calculated.

[0070] Accuracy (spiking recovery) determination: Negative serum matrix was selected. The negative serum background concentrations were: cTnI T / C < 0.005, CK-MB T / C < 0.008, and MYO T / C < 0.010, which were negligible. Low, medium, and high concentrations of cTnI, CK-MB, and MYO standards were added separately, and spiked recovery tests were performed to calculate the recovery rates.

[0071] Inter-item cross-interference test: Single high-concentration samples of cTnI, CK-MB, and MYO were prepared: samples containing only high-concentration cTnI (20 ng / mL), samples containing only high-concentration CK-MB (120 ng / mL), and samples containing only high-concentration MYO (1000 ng / mL). The kits in Example 1 were used for detection. Fluorescence T / C signals of each detection channel were collected, and the cross-interference rate between the indicators was calculated. The signal responses of the other two indicators were observed, and the cross-interference rate was calculated.

[0072] Precision determination: Quality control samples at three concentration levels (low, medium, and high) were selected, and the samples were repeatedly measured 10 times within each batch and 3 batches were measured consecutively between batches. The coefficient of variation (CV) was then calculated.

[0073] The results showed that the relative deviation of the kit of the present invention was <10% under conditions of hemolysis, lipemia, jaundice, RF, and HAMA interference; the spiked recovery rate was 95%~105%; the cross-interference rate among the three indicators was <5%; the intra-assay CV was <8% and the inter-assay relative range was <8%. This indicates that the kit of the present invention has strong anti-interference ability, high accuracy, good specificity, excellent repeatability, and stable and reliable detection results, meeting the clinical needs for rapid and accurate detection. Performance test data are shown in Tables 2, 3, 4, 5 (5-1, 5-2, 5-3), and 6.

[0074] Table 2 Results of Anti-interference Performance Measurement

[0075] Table 3. Accuracy (Recovery Rate) Measurement Results

[0076] Table 4 Results of Inter-item Cross-Interference Test

[0077] Table 5-1 Results of intra-batch repeatability assays for cTnI

[0078] Table 5-2 Results of CK-MB Intra-assay Repeatability Test

[0079] Table 5-3 Results of MYO intra-batch repeatability determination

[0080] Table 6 Results of inter-batch coefficient of variation determination

[0081] Comparative Example 1 (1) The kit of this comparative example differs from that of Example 1 only in that the sample diluent used in step (7) does not contain heterophile antibody blocking agent, rheumatoid factor blocking agent and irrelevant heterologous IgG, while the rest are the same as in Example 1, which is used to verify the effect of the multiple blocking system on the detection performance of the kit.

[0082] The comparative sample dilutions were: 20 mM PBS pH 7.40, Tween 20 0.08% (w / v), PEG6000 0.15% (w / v), EDTA Na2 0.037% (w / v), and ProClin300 0.08% (v / v).

[0083] (2) The comparison example focuses on the impact of the absence of the blocking agent in the sample diluent on the core detection performance of the kit. The blocking agent is sensitive and the anti-interference performance, detection accuracy and low concentration precision are specifically selected for verification. The detection methods and detection gradient settings for anti-interference performance measurement, accuracy (spiking recovery rate) measurement, and precision measurement (only low concentration level quality control samples are measured. Medium and high concentration samples are only slightly affected by matrix interference and are not used as the comparison evaluation indicators) are the same as those in Example 1.

[0084] Results: In the absence of an inhibitor, the core detection performance of the kit showed significant degradation. Under conditions of multiple interferences including hemolysis, lipemia, jaundice, high RF, and HAMA, the relative deviations for each concentration ranged from 12.80% to 22.50%, generally exceeding 10%. The spiked recoveries fluctuated between 89.50% and 113.20%, exceeding the acceptable range of 95% to 105%. The precision degradation was most pronounced in low-concentration samples, with intra-assay CVs of 12.48% to 15.89% and inter-assay CVs of 13.25% to 17.92%, significantly exceeding the 8% threshold. The precision fluctuations for medium and high concentrations were smaller, with minimal impact from the absence of an inhibitor.

[0085] Comparing the results of Example 1 and Comparative Example 1, it can be seen that the multiple blocking system configured in this invention can effectively resist interference from complex clinical matrices, stabilize the immunoreaction of trace antigens, and significantly improve the anti-interference ability, detection accuracy, and repeatability of low-concentration detection of the test strip. This blocking system specifically addresses the core defects of low-concentration myocardial marker detection, such as large deviation, poor anti-interference, and insufficient precision, under non-blocking formulations, and plays a key role in improving the clinical reliability of the triplet test kit. Performance test data are shown in Tables 7, 8, and 9.

[0086] Table 7 Results of Anti-interference Performance (System Without Blocking Agent)

[0087] Table 8. Accuracy (Blocker-Free System) Test Results

[0088] Table 9. Precision Measurement Results for Low Concentration Systems (Without Blockers)

[0089] Comparative Example 2 (1) The only difference between the kit of this comparative example and Example 1 is that the arrangement order of the three detection lines on the nitrocellulose membrane in step (5) is adjusted. The original chromatography flow direction arrangement in Example 1 was: T1(cTnI) → UV hydrophobic isolation band → T2(CK-MB) → T3(MYO); the arrangement in this comparative example is: T1(MYO) → T2(cTnI) → T3(CK-MB), and the UV-cured hydrophobic isolation band between T1 and T2 is removed. The rest is the same as in Example 1. This kit is used to verify the effect of the detection line arrangement order and the absence of lateral hydrophobic isolation on the performance of simultaneous detection of multiple indicators.

[0090] (2) This comparative study examines the three performance aspects most significantly affected by the arrangement of the detection lines and lateral diffusion: anti-interference, accuracy, and low-concentration precision. The detection methods and detection gradient settings for anti-interference performance determination, accuracy (spiking recovery rate) determination, and precision determination (only low-concentration level quality control samples) are the same as those for Experiment 1.

[0091] Results: Compared to Example 1 with its standardized arrangement and T1 / T2 hydrophobic isolation structure, this comparative example, with its altered detection line order and lack of a UV hydrophobic isolation band, exhibited severe lateral diffusion of the labeled complex along the short side of the test strip. Under multiple interfering matrices, the relative deviations of various indicators ranged from 13.50% to 24.16%. The spiked recovery rate ranged from 87.33% to 114.17%, exceeding the acceptable range of 95% to 105%. The intra-batch CV at low concentrations was 13.22% to 14.64%, and the inter-batch CV was 14.49% to 16.30%, both exceeding the 8% acceptance standard. Among these, the original MYO high-signal Tb...3+ The line is positioned before T1, and the lateral diffusion extends to the adjacent T2 (cTnI), causing a significant increase in the deviation of the full concentration gradient of cTnI. This verifies that the invention's "prepositioning of cTnI, adding a UV hydrophobic isolation band between T1 / T2, and fixing the arrangement of T1 (cTnI)-T2 (CK-MB)-T3 (MYO)" can significantly suppress lateral diffusion and reduce detection errors. Performance test data are shown in Tables 10, 11, and 12.

[0092] Table 10 Results of anti-interference performance test (test line order changed, no UV hydrophobic isolation)

[0093] Table 11 Accuracy Measurement Results (with altered test line order and no UV hydrophobic isolation)

[0094] Table 12 Results of Low Concentration Precision Measurements (Detection Line Order Changed, No UV Hydrophobic Isolation)

[0095] Comparative Example 3 (1) The only difference between the kit of this comparative example and Example 1 is that the three rare earth time-resolved fluorescent microspheres used in steps (1) to (3) are all antibody-conjugated with larger particle size microspheres, and the rest are the same as in Example 1.

[0096] Example 1: Three rare earth time-resolved fluorescent microspheres with different particle sizes: Eu 3+ Time-resolved fluorescent microspheres at 190 nm, Sm 3+ Time-resolved fluorescent microspheres at 200 nm, Tb 3+ Time-resolved fluorescent microspheres at 220 nm; This comparative example uses microspheres replaced by Eu. 3+ Time-resolved fluorescent microspheres at 320 nm, Sm 3+ Time-resolved fluorescent microspheres at 350 nm, Tb 3+ Time-resolved fluorescent microspheres at 380 nm.

[0097] (2) This comparative example only examines the changes in fluid migration and immune binding efficiency caused by the increase in microsphere particle size. Three core performance indicators, namely anti-interference, accuracy, and low-concentration precision, are selected for verification. The detection methods and detection gradient settings for anti-interference performance determination, accuracy (spiking recovery rate) determination, and precision determination (only low-concentration quality control samples are measured) are the same as those in Example 1.

[0098] Results: Compared to the standard particle size microspheres in Example 1, the larger particle size microspheres exhibited increased fluid migration resistance, slower chromatographic ascent speed, and incomplete immunobinding. Furthermore, the larger particles were prone to non-specific adsorption. Under multiple interfering matrices, the relative deviations of each indicator ranged from 13.49% to 25.51%; the spiked recoveries ranged from 86.17% to 115.10%, exceeding the acceptable range of 95% to 105%; the intra-batch CV for low concentrations was 11.70% to 12.78%, and the inter-batch CV was 13.87% to 14.75%, all exceeding the 8% acceptance standard. Low-concentration samples were most severely affected by particle migration hindrance, exhibiting a higher degree of deviation degradation than Comparative Example 1 (without blocking agent) and Comparative Example 2 (without an isolation band after changing the detection line order), but a lower degree of repeatability degradation than Comparative Example 1 and Comparative Example 2. This verifies that the use of 190 / 200 / 220 nm gradient small particle size microspheres in this invention can ensure chromatographic smoothness, reduce non-specific adsorption, and significantly improve detection stability. The performance test data are shown in Tables 13, 14, and 15.

[0099] Table 13 Results of anti-interference performance testing (large particle size fluorescent microsphere system)

[0100] Table 14 Accuracy Measurement Results (Large Particle Size Fluorescent Microsphere System)

[0101] Table 15 Results of Precision Measurement for Low Concentration (Large Particle Size Fluorescent Microsphere System)

[0102] Comparative Example 4 (1) The only difference between the kit of this comparative example and Example 1 is that the labeling antibody used in steps (1) to (3) was not digested with papain to remove the Fc fragment, and the complete monoclonal antibody IgG whole antibody in the antibody solution in step a. was directly used to couple fluorescent microspheres in step d. The rest are the same as in Example 1.

[0103] In Example 1, the labeling system used an in vitro expressed cTnI monoclonal antibody, an in vitro expressed CKMB monoclonal antibody, and a Fab fragment (without the Fc region) purified by enzyme digestion of a mouse ascites-derived MYO monoclonal antibody. In this comparative example, the papain digestion and Protein A / G purification steps were omitted. The in vitro expressed cTnI monoclonal antibody, the in vitro expressed CKMB monoclonal antibody, and the mouse ascites-derived MYO monoclonal antibody were directly dialyzed and coupled to the activated microspheres. The rest was exactly the same as in Example 1.

[0104] (2) This comparative example only examines the effect of heterophilic nonspecific adsorption caused by the Fc segment of intact IgG on the detection performance. Three sensitive indicators, namely anti-interference, accuracy, and low concentration precision, are selected for verification. The detection methods and detection gradient settings for anti-interference performance determination, accuracy (spiking recovery rate) and precision determination (only low concentration level quality control samples are measured, and medium and high concentration samples are only slightly affected by matrix interference and are not used as the comparative evaluation indicators) are the same as those in Experiment 1.

[0105] Detection Results: Compared to Example 1, which used Fc-free Fab-labeled antibodies, intact IgG carries the Fc fragment, which readily binds non-specifically to RF, HAMA, and lipoproteins in the sample. Even with the addition of multiple blocking agents in the diluent, Fc-mediated conjugation could not be completely eliminated. The relative deviations of each indicator under multiple interfering matrices ranged from 14.32% to 27.68%; the spiked recovery rate ranged from 85.18% to 116.23%, exceeding the 95% to 105% acceptable range; the intra-assay CV at low concentrations was 12.47% to 14.55%, and the inter-assay CV was 11.51% to 14.37%, all exceeding the 8% acceptance standard. Low-concentration trace samples were most severely affected by Fc non-specific interference, with an overall degradation level higher than Comparative Example 1 (no blocking agent), Comparative Example 3 (large-particle microspheres), and Comparative Example 2 (reversed line sequence without UV isolation). This verifies that the present invention, using Fc-free Fab fragments as labeling antibodies, can significantly avoid Fc-mediated endogenous matrix interference, which is a key technical point for ensuring accurate detection of low-concentration samples. The performance test data are shown in Tables 16, 17, and 18.

[0106] Table 16 Results of anti-interference performance assay (intact IgG antibody, Fc fragment not removed)

[0107] Table 17 Accuracy Measurement Results (Intact IgG Antibody, Fc Fragment Not Removed)

[0108] Table 18 Results of Low-Concentration Precision Assay (Intact IgG Antibody, Fc Fragment Not Removed)

[0109] 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. A myocardial triple time-resolved fluorescence immunochromatographic test strip, characterized in that, The test strip includes a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane has a first detection line T1, a second detection line T2, a third detection line T3, and a control line C arranged sequentially along the chromatography direction. T1 is coated with cTnI capture antibody, T2 with CK-MB capture antibody, T3 with MYO capture antibody, and C with goat anti-mouse IgG. The conjugate pad is coated with Eu. 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3+ Microsphere-labeled CK-MB for detecting Fab fragments and Tb 3+ Microsphere-labeled MYO detection of Fab fragments.

2. The test strip according to claim 1, characterized in that, The Eu 3+ The microspheres have a particle size of 180~200nm, and the Sm 3+ The microspheres have a particle size of 190~210 nm, and the Tb 3+ The microspheres have a particle size of 210~230nm.

3. The test strip according to claim 1, characterized in that, The distance between two adjacent test lines or between an adjacent test line and a quality control line is ≥4mm; a hydrophobic isolation strip is provided between T1 and T2.

4. The method for preparing the test strip according to any one of claims 1 to 3, characterized in that, Includes the following steps: The Fc fragments of the cTnI detection antibody, CK-MB detection antibody, and MYO detection antibody were removed respectively to obtain the cTnI detection Fab fragment, CK-MB detection Fab fragment, and MYO detection Fab fragment; Activated Eu 3+ Microspheres, Sm 3+ Microspheres and Tb 3+ Microspheres were coupled to cTnI detection Fab fragments, CK-MB detection Fab fragments, and MYO detection Fab fragments, respectively, and then sealed to obtain Eu. 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3+ Microsphere-labeled CK-MB for detecting Fab fragments and Tb 3+ Microsphere-labeled MYO detection of Fab fragments; Eu 3+ Microsphere-labeled cTnI detection of Fab fragments and Sm 3+ Microsphere-labeled CK-MB for detecting Fab fragments and Tb 3+ Microspheres labeled with MYO were used to detect the mixing of Fab fragments, resulting in a mixture that was sprayed onto the binding pad and dried to obtain the sprayed binding pad. Fluorine-containing hydrophobic ink was coated and cured between blank nitrocellulose membranes T1 and T2 to form a hydrophobic isolation zone; cTnI capture antibody was coated on T1 of the nitrocellulose membrane, CK-MB capture antibody was coated on T2; MYO capture antibody was coated on T3; and goat anti-mouse IgG was coated on C. The membranes were dried to obtain the coated nitrocellulose membranes. The sample pad, the sprayed bonding pad, the coated nitrocellulose membrane, and the absorbent pad are overlapped on the base plate and cut into test strips.

5. The preparation method according to claim 4, characterized in that, In the mixture, the mass ratio of cTnI detection Fab fragment, CK-MB detection Fab fragment and MYO detection Fab fragment is 1:(0.8~1.2):(0.8~1.2), the concentration of cTnI detection Fab fragment is 0.2~0.5 mg / mL, and the spraying amount of the mixture is 3~6 μL / cm.

6. The preparation method according to claim 4, characterized in that, The antibody concentrations for T1, T2, T3, and C were 0.8–1.2 mg / mL, and the streaking volumes were 0.8–1.2 μL / cm, respectively.

7. A myocardial triple time-resolved fluorescence immunochromatographic assay kit, characterized in that, The kit comprises the test strip as described in any one of claims 1 to 3 and a sample diluent, wherein the sample diluent comprises: a heterophile antibody blocking agent, a rheumatoid factor blocking agent, irrelevant heterologous IgG, EDTA, PEG and a surfactant.

8. The reagent kit according to claim 7, characterized in that, The sample diluents included: 10-50 mM PBS pH 7.00-7.50, heterophile antibody blocking agent 0.5%-1.0%, rheumatoid factor blocking agent 0.1%-0.5%, Tween-20 0.05%-0.5%, PEG6000 0.05%-0.2%, EDTA-Na2 0.5-2 mM, irrelevant xenobiotic IgG 0.05-0.1 mg / mL, and ProClin300 0.05%-0.5%.

9. The use of the test strip according to any one of claims 1 to 3, the test strip prepared by the preparation method according to any one of claims 4 to 6, or the kit according to any one of claims 7 to 8 in the preparation of the following products a and / or b: a) Simultaneous detection of cTnI, CK-MB and MYO concentrations; b) Early detection of acute myocardial infarction.

10. A method for simultaneously detecting cTnI, CK-MB, and MYO for non-diagnostic purposes, characterized in that, Includes the following steps: The sample to be tested is mixed with the sample diluent in the kit of claim 7 or 8 at a volume ratio of 1:(2~4) to obtain a diluted sample, which is then dropped onto the sample pad of the test strip in the kit and detected by a fluorescence immunochromatographic analyzer. The parameters detected by the fluorescence immunochromatographic analyzer are: excitation wavelength 350~365nm, delay time 100~200μs, integration time 200~400μs, and three-channel time-division acquisition and spectral demixing correction are used.

Images