A dual-target sicm diagnostic prognostic joint detection kit and a preparation method thereof

Abstract

The application discloses a double-target septic cardiomyopathy (SICM) diagnosis and prognosis combined detection kit and a preparation method thereof, and belongs to the technical field of in-vitro diagnosis kits. The kit comprises a first antibody pair specifically combined with human cardiac troponin I (cTnI), a second antibody pair specifically combined with human B-type natriuretic peptide (BNP), a carboxyl-modified magnetic microsphere solid-phase carrier, an HRP enzyme label and a luminol chemiluminescence substrate, and synchronous double-target combined detection of cTnI and BNP is realized in the same reaction system. The diagnosis and prognosis stratification of SICM are effectively realized.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent kit technology, specifically relating to a dual-target combined detection kit for simultaneously detecting human cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP), its preparation method, and its application. Background Technology

[0002] Sepsis-induced cardiomyopathy (SICM) is one of the most serious complications of sepsis, characterized by myocardial depression and ventricular dysfunction. Clinical studies show that approximately 30%–50% of sepsis patients experience a decline in cardiac systolic function, with some progressing to irreversible myocardial damage. The 28-day mortality rate for SICM patients is as high as 50%–70%, significantly higher than that for sepsis patients without myocardial damage. Therefore, early diagnosis and prognostic assessment of SICM have significant clinical value.

[0003] Currently, the main biomarkers used clinically to assess myocardial injury and cardiac function include:

[0004] Cardiac troponin I (cTnI) is a specific marker of cardiomyocyte injury, released into the bloodstream during cardiomyocyte necrosis or apoptosis. cTnI exhibits high myocardial specificity (expressed only in myocardial tissue) and is the gold standard for diagnosing myocardial injury. Patients with septic cardiomyopathy often have elevated serum cTnI levels, and the degree of elevation is positively correlated with the severity of myocardial injury. However, a single cTnI test cannot assess ventricular function.

[0005] B-type natriuretic peptide (BNP) and its N-terminal pro-BNP (NT-proBNP): BNP is mainly secreted by ventricular myocytes and its expression is upregulated when ventricular wall tension increases or volume overload occurs. BNP is an important indicator for assessing ventricular function and heart failure, and its level is negatively correlated with ventricular ejection fraction (LVEF) in patients with spontaneous cardiac angina (SICM). However, BNP testing alone cannot determine whether cardiomyocyte damage has occurred.

[0006] In existing technologies, cTnI and BNP are usually detected as independent tests using different kits, which has the following drawbacks:

[0007] (1) Separate testing increases sample volume and operation time: Two independent tests require two sample volumes, the operation process is cumbersome, and it cannot meet the needs of rapid testing in emergency and ICU.

[0008] (2) Inability to achieve simultaneous joint interpretation: Individual detection of cTnI and BNP is difficult to reflect the synchronous state of myocardial injury and ventricular dysfunction, and cannot meet the clinical needs of SICM "injury + function" dual-dimensional assessment.

[0009] (3) Existing dual-marker detection kits have complex structures: Most existing cTnI+BNP combined detection kits are immunochromatographic qualitative / semi-quantitative cards, which are not sensitive enough to meet the ICU’s requirements for accurate quantification. Summary of the Invention

[0010] In response to the above situation and to overcome the shortcomings of the prior art, the present invention provides a dual-target SICM diagnostic and prognostic combined detection kit and its preparation method, which effectively solves the technical problems of existing technologies, such as the inability of separate detection of cTnI and BNP to meet the requirements of simultaneous "diagnosis + prognosis" combined assessment of SICM, and the lack of sensitivity, complex operation, and inability to quantify simultaneously in existing combined detection kits.

[0011] The technical solution adopted in this invention is as follows: This invention proposes a dual-target SICM diagnostic and prognostic combined detection kit, including...

[0012] (1) Double antibody sandwich system: The first antibody pair (capture antibody + detection antibody) specifically binds to cTnI, and the second antibody pair (capture antibody + detection antibody) specifically binds to BNP. The two antibody pairs recognize different epitopes of the target antigen respectively, avoiding mutual interference;

[0013] (2) Magnetic microsphere solid-phase support: Carboxyl-modified magnetic microspheres (particle size 1~3 μm) are used as solid-phase support. Antibodies are captured by covalent coupling through amide bonds. The solid-phase support is rapidly separated under a magnetic field to achieve high-throughput automated detection.

[0014] (3) Simultaneous detection of dual targets: Two capture antibody-conjugated microspheres are mixed in proportion and used as a solid phase, and two enzyme-labeled detection antibodies are mixed and used as enzyme-labeled reagents to achieve simultaneous sandwich reaction of cTnI and BNP and detection of chemiluminescent signals in the same reaction tube;

[0015] (4) HRP-luminol chemiluminescence system: The enzyme-labeled antibody is labeled with HRP, and the substrate is a luminol + p-iodophenol + hydrogen peroxide luminescence system. The detection signal intensity is positively correlated with the target antigen concentration, and the sensitivity is 10 to 100 times higher than that of ELISA.

[0016] Furthermore, the first capture antibody and the first detection antibody target different epitopes of cTnI, wherein the epitopes are selected from the epitopes located at positions 28-36, 56-76, and / or 80-110 of the cTnI amino acid sequence; the second capture antibody and the second detection antibody target different epitopes of BNP, wherein the epitopes are selected from the cyclic region at positions 77-108 and / or the region at position 13-32 of the C-terminus of the BNP molecule.

[0017] Furthermore, the magnetic microspheres have a particle size of 0.5~5 μm, preferably 1~3 μm, a magnetic core of Fe3O4 or γ-Fe2O3, and a surface modified with carboxyl or amino groups.

[0018] Furthermore, the first capture antibody and the second capture antibody are immobilized on the surface of the magnetic microspheres in the following ways: first, the two capture antibodies are coupled to separate magnetic microspheres, and then the two antibody-coupled microspheres are mixed in proportion; or, the two capture antibodies are pre-mixed and then coupled together to the surface of the same batch of magnetic microspheres.

[0019] Further, the enzyme marker is HRP, and the chemiluminescent substrate solution contains luminol, p-iodophenol, and hydrogen peroxide; or the enzyme marker is AP, and the chemiluminescent substrate solution is APS-5 or CDP-Star.

[0020] Furthermore, when used for detection in the kit, the volume ratio of the solid-phase reagent to the sample to be tested is 1:1, and the volume ratio of the enzyme-labeled antibody reagent to the solid-phase complex is 1:1 to 1:2; and the solid-phase reagent and the enzyme-labeled antibody reagent have a shelf life of not less than 12 months when stored at 2~8℃ and not less than 18 months when stored at -20℃.

[0021] This invention also proposes a method for preparing a dual-target SICM diagnostic prognostic combined detection kit, specifically including the following steps:

[0022] S1. Take the carboxyl-modified magnetic microspheres, wash them with MES buffer, add EDC and NHS to activate the carboxyl groups, and prepare an activated magnetic microsphere suspension;

[0023] S2. Mix the first capture antibody with the activated magnetic microspheres and react at room temperature for 0.5 to 2 hours to couple the first capture antibody to the magnetic microspheres to obtain the first antibody-coupled microspheres; similarly, couple the second capture antibody to obtain the second antibody-coupled microspheres.

[0024] S3. Mix the first antibody-conjugated microspheres and the second antibody-conjugated microspheres at a volume ratio of 1:0.5 to 1:2, and resuspend them in phosphate buffer containing BSA and surfactant to obtain a mixed solid suspension.

[0025] S4. The first detection antibody and the second detection antibody are respectively coupled with HRP to prepare enzyme-labeled first detection antibody and enzyme-labeled second detection antibody;

[0026] S5. Dispense the mixed solid-phase suspension to obtain solid-phase reagent, and dispense the enzyme-labeled antibody to obtain enzyme-labeled antibody reagent. Seal and assemble the reagent together with the chemiluminescent substrate solution, calibrator and quality control sample to obtain the final product.

[0027] Further, the concentration of the MES buffer in S1 is 10~50 mM, pH 5.0~6.5; the concentrations of EDC and NHS are 1~10 mg / mL, and the activation reaction is carried out for 5~30 minutes under pH 5.5~6.5 conditions.

[0028] Furthermore, in S2, the mass ratio of the first capturing antibody to the magnetic microspheres is 1:20, and the mass ratio of the second capturing antibody to the magnetic microspheres is 1:20. The coupling reaction is carried out at pH 7.0~7.5 for 1~1.5 hours. In step three, the volume ratio of the first antibody-coupled microspheres to the second antibody-coupled microspheres is 1:1.

[0029] Furthermore, the coupling of HRP and the detection antibody in S4 was performed using a modified sodium periodate oxidation method: HRP was dissolved in deionized water, NaIO4 solution was added, and the mixture was stirred at room temperature in the dark for 30 minutes. After the reaction was terminated, the detection antibody was added, and the mixture was reacted at room temperature for 2 hours. Then, NaBH4 was added for reduction to obtain the enzyme-labeled antibody.

[0030] This invention proposes a dual-target SICM diagnostic prognosis combined detection kit, its preparation method and its usage method, with the following beneficial effects: (1) Simultaneous dual-target detection: cTnI and BNP are detected simultaneously in the same reaction system, reducing the sample volume by 50% and shortening the detection time to within 90 minutes, meeting the rapid detection needs of ICU emergency departments;

[0031] (2) Ultra-high sensitivity: The detection limit of cTnI in CLIA chemiluminescence system is 0.01 ng / mL, which is 10 times higher than that of traditional ELISA (0.1 ng / mL); the detection limit of BNP is 10 pg / mL, which meets the requirements of accurate quantification in ICU.

[0032] (3) Dual-dimensional clinical assessment: cTnI reflects the degree of myocardial cell damage, BNP reflects the ventricular function status, and the combination of dual targets realizes the simultaneous assessment of SICM "damage + function", which has higher diagnostic accuracy than single marker detection (expected AUC≥0.92).

[0033] (4) Improved prognostic value: The 28-day mortality rate of patients with double positive results in combined testing was significantly higher than that of patients with single positive results, which effectively guided clinical stratification management and treatment decisions;

[0034] (5) The preparation process is stable and reliable: HRP-antibody conjugation is carried out by modified sodium periodate oxidation method, with a conjugation rate of ≥80%, good batch-to-batch consistency, and a shelf life of ≥12 months; Attached Figure Description

[0035] Figure 1 This is a flowchart illustrating the preparation method of a dual-target SICM diagnostic prognostic combined detection kit proposed in this invention;

[0036] Figure 2 This is a schematic diagram of the joint detection procedure for dual-target SICM.

[0037] Figure 3 This is a comparison chart showing the effects of different scales.

[0038] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] Separately coupled-mixed solid-phase dual-target kit (HRP / luminol system)

[0042] Antibody selection

[0043] The first capture antibody is a mouse anti-human cTnI monoclonal antibody (clone number CTNI-1), which specifically binds to the 28th to 36th amino acid epitopes of cTnI.

[0044] The first detection antibody is a mouse anti-human cTnI monoclonal antibody (clone number CTNI-2), which specifically binds to the 80th to 110th amino acid epitopes of cTnI.

[0045] The second capture antibody is a mouse anti-human BNP monoclonal antibody (clone number BNP-1), which specifically binds to the circular structure region of BNP at positions 77-108.

[0046] The second detection antibody is a mouse anti-human BNP monoclonal antibody (clone number BNP-2), which specifically binds to the C-terminal region 13-32 of BNP.

[0047] S1: Activation of magnetic microspheres

[0048] Take 100 mg of Fe3O4 magnetic microspheres with a particle size of 1.5 μm and surface carboxyl modification (5% solid content, 2 mL) and place them in a 15 mL centrifuge tube.

[0049] Add 10 mL of pre-cooled MES buffer (50 mM, pH 5.5), vortex to mix, magnetically separate and discard the supernatant, and repeat the washing twice.

[0050] Resuspend the microspheres in 4 mL of MES buffer (pH 5.5), then add freshly prepared EDC solution (200 μL, containing 20 mg EDC, final concentration 5 mg / mL) and NHS solution (200 μL, containing 20 mg NHS, final concentration 5 mg / mL) sequentially, and vortex to mix.

[0051] Adjust the pH of the suspension to 6.0 with 1 M HCl, place it on a rotary mixer, and activate it at room temperature in the dark for 20 minutes.

[0052] After activation, the supernatant was discarded by magnetic separation, and the mixture was quickly washed twice with 5 mL of pre-cooled PBS buffer (10 mM PB, pH 7.2) and immediately used for coupling.

[0053] S2: Preparation of antibody-conjugated microspheres

[0054] Take half of the S1 activated microspheres (about 50 mg) and resuspend them in 2.5 mL PBS (pH 7.2).

[0055] Add 2.5 mg of the first capture antibody (1 mg / mL PBS solution, 2.5 mL) to the suspension to make the antibody-to-microsphere mass ratio 1:20. Add PBS to a total volume of 6 mL and vortex to mix.

[0056] Adjust the pH to 7.2 with 1 M NaOH, place in a rotary mixer, and shake at room temperature for 1.5 hours.

[0057] After coupling, magnetic separation was performed, and the supernatant was collected for coupling rate determination. The microspheres were washed three times with 5 mL of PBS containing 0.5% BSA and 0.05% Tween-20 (pH 7.4).

[0058] Finally, the suspension was resuspended in 5 mL of blocking buffer (PBS containing 2% BSA, 0.1% Tween-20, and 0.05% ProClin 300) and blocked overnight at 4°C. The next day, after washing, the suspension was reconstituted in 5 mL of storage solution (PBS containing 1% BSA and 0.05% Tween-20) to obtain the first antibody-conjugated microsphere suspension, which was stored at 4°C.

[0059] S3: Preparation of second antibody-conjugated microspheres

[0060] Take the remaining activated microspheres (approximately 50 mg) from S1 and resuspend them in PBS using the same method.

[0061] Add 2.5 mg of the second capture antibody at a mass ratio of 1:20, and perform conjugation, washing, and blocking under the same conditions as S2 to obtain a suspension of the second antibody-conjugated microspheres (5 mL).

[0062] S4: Preparation of mixed solid-phase reagents

[0063] The solid content of the two coupled microsphere suspensions was determined and adjusted to 5 mg / mL with preservation solution.

[0064] Mix the first antibody-conjugated microspheres and the second antibody-conjugated microspheres at a volume ratio of 1:1 (5 mL of each), vortex to mix, and obtain a mixed solid suspension loaded with two capture antibodies simultaneously, with a solid content of 5 mg / mL. Dispense into 2.5 mL / bottle and store at 4°C.

[0065] S5: Preparation of enzyme-labeled antibodies (modified sodium periodate method)

[0066] Weigh 20 mg of HRP and dissolve it in 5 mL of deionized water. Slowly add 2 mL of freshly prepared 0.1 M NaIO4 solution while stirring. Stir at room temperature in the dark for 30 minutes until the solution turns yellow-green.

[0067] Add 0.5 mL of ethylene glycol to stop the oxidation and continue stirring for 15 minutes.

[0068] The reaction solution was placed in a dialysis bag (molecular weight cutoff 14 kDa) and dialyzed overnight at 4°C with 1 mM sodium acetate buffer (pH 4.4), changing the buffer 3 times.

[0069] Remove the activated HRP solution after dialysis, and add 10 mg of the first detection antibody and 10 mg of the second detection antibody (pre-dialyzed to pH 9.5 with 0.01 M carbonate buffer). Adjust the pH to 9.5 with 1 M carbonate buffer and stir at room temperature in the dark for 2 hours.

[0070] Add 0.5 mL of freshly prepared NaBH4 solution (4 mg / mL) and let stand at 4°C for 1 hour for reduction.

[0071] Dialyze the PBS (pH 7.4) overnight at 4°C, changing the solution 3 times.

[0072] The enzyme-labeled antibody and free matter were separated using a Superdex 200 gel chromatography column. The first elution peak was collected to obtain purified enzyme-labeled first detection antibody and enzyme-labeled second detection antibody.

[0073] After measuring the protein concentration and HRP activity, the two enzyme-labeled antibodies were mixed in equimolar amounts and adjusted to the working concentration with enzyme-labeled dilution buffer (PBS containing 1% BSA, 0.1% Tween-20, and 50% glycerol). The mixture was then dispensed into 1 mL vials and stored at -20°C in the dark.

[0074] S6: Chemiluminescent substrate solution

[0075] Substrate A solution: Tris-HCl buffer (pH 8.5) containing luminol (5 mM) and p-iodophenol (0.5 mM).

[0076] Substrate B solution: Tris-HCl buffer containing hydrogen peroxide (8 mM).

[0077] Mix in equal volumes before use. Dispense into 5 mL vials and store at 2–8°C protected from light.

[0078] S7: Calibrators and Quality Control Products

[0079] Prepare the following calibrators using standard dilution buffer (PBS containing 4% BSA and 0.1% ProClin 300):

[0080] cTnI: 0, 0.04, 0.2, 1, 5, 25, 50 ng / mL; BNP: 0, 10, 50, 200, 800,2000, 5000 pg / mL.

[0081] The quality control samples are divided into three levels: low, medium, and high, with target values ​​of cTnI (0.1, 1.5, 15 ng / mL) and BNP (30, 400, 3000 pg / mL), respectively.

[0082] After repackaging, freeze-dry or store in liquid.

[0083] S8: Reagent Kit Assembly

[0084] Package the mixed solid-phase reagent (2.5 mL / bottle), enzyme-labeled antibody reagent (1 mL / bottle), substrate solutions A and B, calibrator set, quality control set, and concentrated washing buffer (20×) together with the instruction manual and store at 2~8℃.

[0085] Performance test results

[0086] Limits of detection: cTnI 0.008 ng / mL, BNP 8 pg / mL.

[0087] Linearity: cTnI 0.04~50 ng / mL, BNP 10~5000 pg / mL, r>0.99.

[0088] Precision: Intra-batch CV 3.2%~4.8%, inter-batch CV 5.6%~7.9%.

[0089] After being stored at 4℃ for 12 months, the calibrator signal changed by less than 8%, and there was no significant drift in the detection limit and linearity.

[0090] Example 2

[0091] Co-coupled solid-phase dual-target kit (AP / CDP-Star system)

[0092] Antibody against

[0093] First capture antibody: anti-cTnI (episodes 56-76); First detection antibody: anti-cTnI (episodes 28-36).

[0094] Second capture antibody: anti-BNP (C-terminus 13~32); Second detection antibody: anti-BNP (77~108 circular structure region).

[0095] S1: Activation of magnetic microspheres

[0096] Take 50 mg of γ-Fe2O3 magnetic microspheres with a particle size of 3 μm and carboxyl modification, wash with MES buffer (25 mM, pH 6.0), add EDC (final concentration 3 mg / mL) and NHS (final concentration 8 mg / mL), activate at pH 6.3 for 15 minutes, wash and set aside.

[0097] S2: Mixed antibody co-conjugation

[0098] 1.25 mg of the first capture antibody and 1.25 mg of the second capture antibody were premixed in 2 mL of PBS (pH 7.4) to obtain a total antibody amount of 2.5 mg.

[0099] Resuspend the S1 activated microspheres in PBS, add the above mixed antibody, adjust the total antibody / microsphere mass ratio to 1:20, the total volume is 5 mL, and the pH is 7.4.

[0100] The reaction was carried out at room temperature by rotation for 1 hour. After magnetic separation, the mixture was washed with PBS containing 1% BSA and blocked overnight.

[0101] Resuspend the microspheres in preservation solution to a concentration of 5 mg / mL to obtain a solid-phase reagent co-loaded with dual capture antibodies.

[0102] S3: Preparation of enzyme-labeled antibodies (EDC method)

[0103] Dissolve AP in PBS to a concentration of 5 mg / mL, and take 2.5 mL (12.5 mg).

[0104] Add 12.5 mg of the first detection antibody and the second detection antibody (mass ratio 1:1) to the first detection antibody and the second detection antibody respectively, and then add EDC to the final concentration of 1 mg / mL. Stir gently at 4°C for 16 hours.

[0105] The reaction solution was dialyzed against PBS and purified using a Sephacryl S-300 column to obtain enzyme-labeled detection antibodies.

[0106] Mix the two enzyme-labeled antibodies in equal molar amounts, dilute with Tris buffer containing 1% BSA and 0.05% NaN3, aliquot, and store at 2-8°C.

[0107] S4: Substrate solution

[0108] CDP-Star stock solution was diluted to working concentration with 0.1 M diethanolamine buffer (pH 9.6, containing 1 mM MgCl2) and aliquoted for protection from light.

[0109] S5: Calibrators, quality control products and assembly

[0110] Same as Example 1. The solid-phase reagent was dispensed in 1.5 mL / bottle.

[0111] Performance: cTnI detection limit 0.006 ng / mL, BNP 6 pg / mL; linear range consistent with Example 1.

[0112] Inter-batch CV ≤ 9%. After storage at -20℃ for 18 months, all indicators decreased by < 10%.

[0113] Example 3

[0114] Small-diameter amino microsphere dual-target kit (glutaraldehyde coupling method)

[0115] S1: Activation of amino magnetic microspheres

[0116] 50 mg of Fe3O4 microspheres with a particle size of 0.8 μm and amino-modified surface were washed with PBS, and then 5% glutaraldehyde solution (dissolved in 0.1 M phosphate buffer, pH 7.0) was added. The mixture was rotated at room temperature for 3 hours. After the reaction, the free glutaraldehyde was removed by thorough washing with PBS to obtain aldehyde-activated microspheres.

[0117] S2: Capture antibody conjugation

[0118] 1.67 mg of the first capture antibody (cTnI, epitopes 28-36) (1:30 mass ratio with microspheres) was dissolved in PBS and mixed with half of the activated microspheres; 1.67 mg of the second capture antibody (BNP, epitopes 77-108) was mixed with the other half of the activated microspheres. The pH was adjusted to 7.4, and the reaction was carried out overnight at 4°C. The next day, NaBH3CN (final concentration 5 mM) was added for reduction, and after washing, the microspheres were blocked with ethanolamine solution containing 1% BSA. The first and second antibody-conjugated microspheres were prepared separately and mixed at a volume ratio of 1:1.5 to obtain a mixed solid phase.

[0119] S3: Enzyme-labeled antibody and other components

[0120] The HRP enzyme-labeled detection antibody was prepared using the modified sodium periodate method as in Example 1. The substrates and calibrators were also the same.

[0121] Performance: Sensitivity is comparable to Example 1, magnetic field response time is shortened by 40%, suitable for fast mode of fully automated chemiluminescence analyzer.

[0122] Comparative Example 1

[0123] Single-target detection kit

[0124] preparation

[0125] cTnI kit only: All activated microspheres are conjugated to the first capture antibody (feed ratio 1:20) to prepare a cTnI solid phase; the enzyme-labeled antibody contains only the enzyme-labeled first detection antibody. The remaining substrates and calibrators contain only cTnI.

[0126] BNP-only kit: Similarly, the solid phase is only coupled with the second capture antibody, the enzyme label contains only the second detection antibody, and the calibrator contains only BNP.

[0127] Detection comparison

[0128] Sixty-two samples from suspected SICM patients were collected and tested using both a dual-target kit (Example 1) and a single-target kit. Example 1 required 1.5 hours and consumed 50 μL of sample per test; the two single-target kits required two tests, totaling approximately 3 hours and 200 μL of sample. For prognostic assessment, a double-positive rule (cTnI ≥ 0.04 and BNP ≥ 100) was used. Example 1 provides two results for direct interpretation, while the single-target method requires additional manual combination analysis. 28-day mortality risk prediction: patients with double-positive results had a higher risk than those with only cTnI or only BNP positivity; the combined detection net reclassification index (NRI) was 0.32.

[0129] Comparative Example 2

[0130] Capturing and detecting antibody epitope overlap

[0131] Preparation: The conditions were exactly the same as in Example 1, but the antibodies were changed: the first capture antibody and the first detection antibody both used the same clone that recognizes epitopes 28-36 of cTnI; the second capture antibody and the second detection antibody both used the same clone that recognizes the circular structure 77-108 of BNP.

[0132] The methods for activating, coupling, mixing, and labeling the magnetic microspheres with enzyme-linked antibodies are the same as in Example 1. The assembly methods for all components of the kit are consistent.

[0133] Test results

[0134] Standard assays: The RLU value at the highest cTnI concentration (50 ng / mL) was only 1.3 times the background signal; the RLU at the highest BNP concentration (5000 pg / mL) was only 1.1 times the background signal, making it impossible to establish an effective standard curve. The recovery rate of positive samples was <8% for all samples. This indicates that site overlap leads to competitive inhibition, preventing the formation of sandwich immune complexes and rendering the kit ineffective.

[0135] Comparative Example 3: Physical Adsorption vs. Deviation in Feed Ratio

[0136] preparation

[0137] Physical adsorption instead of covalent coupling: Carboxyl microspheres were taken without EDC / NHS activation, and the first capture antibody (resuspended in PBS) and the second capture antibody were added directly. The microspheres were then adsorbed and coated in pH 9.6 carbonate buffer at 4°C for 16 hours, followed by washing and blocking.

[0138] Change the feed ratio: the mass ratio of the first capture antibody to the microspheres is 1:3, and the mass ratio of the second capture antibody to the microspheres is 1:80.

[0139] The physically adsorbed microspheres and the microspheres with abnormal feed ratios were mixed, labeled and assembled according to the method in Example 1.

[0140] Stability and performance comparison

[0141] In the physical adsorption solid-phase reagent, after accelerated stability testing at 37°C, a large amount of free antibody was detectable in the supernatant after washing at 6 hours, and the RLU signal decreased to 62% (cTnI) and 58% (BNP) at the beginning on day 10. In contrast, the signal of the covalently coupled solid-phase reagent in Example 1 remained above 95% after 10 days.

[0142] A 1:3 cTnI solid phase: Due to steric hindrance caused by excessive antibody density, the signal of the 50 ng / mL calibrator was only 28% of that in Example 1, and the signal even decreased in the high concentration region (hook effect). A 1:80 BNP solid phase: Due to insufficient antibody, the signal plateau above 2000 pg / mL narrowed to 10~800 pg / mL, rendering the kit ineffective. This demonstrates that covalent coupling and the claimed feed ratio range are key to ensuring stability and broad linearity.

[0143] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0144] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A dual-target SICM diagnostic and prognostic combined detection kit, characterized in that... include: (1) A first capture antibody and a first detection antibody, wherein the first capture antibody and the first detection antibody specifically bind to human cardiac troponin I and the binding sites do not overlap; (2) A second capture antibody and a second detection antibody, wherein the second capture antibody and the second detection antibody specifically bind to human B-type natriuretic peptide and the binding sites do not overlap; (3) A magnetic microsphere solid-phase carrier, wherein the first and second capturing antibodies are covalently coupled to the surface of the magnetic microspheres; (4) Enzyme marker, wherein the enzyme marker is horseradish peroxidase or alkaline phosphatase, and the first detection antibody and / or the second detection antibody are coupled with the enzyme marker to form an enzyme-labeled antibody; (5) Chemiluminescent substrate solution; (6) Calibrators containing known concentrations of cTnI and BNP standards; (7) Quality control materials, including cTnI and BNP quality control materials within a preset concentration range; The magnetic microsphere solid-phase carrier is simultaneously loaded with the first capture antibody and the second capture antibody, and the enzyme-labeled antibody contains both the enzyme-labeled first detection antibody and the enzyme-labeled second detection antibody.

2. The dual-target SICM diagnostic and prognostic combined detection kit according to claim 1, characterized in that: The first capture antibody and the first detection antibody target different epitopes of cTnI, wherein the epitopes are selected from the epitopes located at positions 28-36, 56-76, and / or 80-110 of the cTnI amino acid sequence; the second capture antibody and the second detection antibody target different epitopes of BNP, wherein the epitopes are selected from the cyclic region at positions 77-108 and / or the region at position 13-32 of the C-terminus of the BNP molecule.

3. The dual-target SICM diagnostic and prognostic combined detection kit according to claim 1, characterized in that: The magnetic microspheres have a particle size of 0.5~5 μm, a magnetic core of Fe3O4 or γ-Fe2O3, and a surface modified with carboxyl or amino groups.

4. The dual-target SICM diagnostic and prognostic combined detection kit according to claim 1, characterized in that: The first capture antibody and the second capture antibody are immobilized on the surface of the magnetic microspheres in the following manner: first, the two capture antibodies are coupled to separate magnetic microspheres, and then the two antibody-coupled microspheres are mixed in a certain proportion.

5. The dual-target SICM diagnostic and prognostic combined detection kit according to claim 1, characterized in that: The enzyme marker is HRP, and the chemiluminescent substrate solution contains luminol, p-iodophenol, and hydrogen peroxide; or the enzyme marker is AP, and the chemiluminescent substrate solution is APS-5 or CDP-Star.

6. The dual-target SICM diagnostic and prognostic combined detection kit according to claim 1, characterized in that: When used in the kit for detection, the volume ratio of the solid-phase reagent to the sample to be tested is 1:1, and the volume ratio of the enzyme-labeled antibody reagent to the solid-phase complex is 1:1 to 1:

2.

7. The preparation method of the dual-target SICM diagnostic prognostic combined detection kit according to claim 1, characterized in that, Includes the following steps: S1. Take the carboxyl-modified magnetic microspheres, wash them with MES buffer, add EDC and NHS to activate the carboxyl groups, and prepare an activated magnetic microsphere suspension; S2. Mix the first capture antibody with the activated magnetic microspheres and react at room temperature for 0.5 to 2 hours to couple the first capture antibody to the magnetic microspheres, thus obtaining the first antibody-coupled microspheres; Similarly, the second capture antibody was conjugated to obtain a second antibody-conjugated microsphere; S3. Mix the first antibody-conjugated microspheres and the second antibody-conjugated microspheres at a volume ratio of 1:0.5 to 1:2, and resuspend them in phosphate buffer containing BSA and surfactant to obtain a mixed solid suspension. S4. The first detection antibody and the second detection antibody are respectively coupled with HRP to prepare enzyme-labeled first detection antibody and enzyme-labeled second detection antibody; S5. Dispense the mixed solid-phase suspension to obtain solid-phase reagent, and dispense the enzyme-labeled antibody to obtain enzyme-labeled antibody reagent. Seal and assemble the reagent together with the chemiluminescent substrate solution, calibrator and quality control sample to obtain the final product.

8. The preparation method of a dual-target SICM diagnostic prognostic combined detection kit according to claim 7, characterized in that: The MES buffer concentration in S1 is 10-50 mM, pH 5.0-6.5; the concentrations of EDC and NHS are 1-10 mg / mL, and the activation reaction is carried out for 5-30 minutes at pH 5.5-6.

5.

9. The preparation method of a dual-target SICM diagnostic prognostic combined detection kit according to claim 7, characterized in that: In step S2, the mass ratio of the first capturing antibody to the magnetic microspheres is 1:20 to 1:30, and the mass ratio of the second capturing antibody to the magnetic microspheres is 1:20 to 1:

30. The coupling reaction is carried out at pH 7.0 to 7.5 for 1 to 1.5 hours. In step three, the volume ratio of the first antibody-coupled microspheres to the second antibody-coupled microspheres is 1:

1.

10. The preparation method of a dual-target SICM diagnostic prognostic combined detection kit according to claim 7, characterized in that: The coupling of HRP and detection antibody in S4 was performed using a modified sodium periodate oxidation method: HRP was dissolved in deionized water, NaIO4 solution was added, and the mixture was stirred at room temperature in the dark for 30 minutes. After the reaction was terminated, the detection antibody was added, and the mixture was reacted at room temperature for 2 hours. NaBH4 was then added for reduction to obtain the enzyme-labeled antibody.

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