Hypersensitive antibody detection method and kit based on collaborative optimization of magnetic particle coating, ALP cascade signal amplification and EDTA gradient washing

Abstract

The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to a hypersensitive antibody detection method and kit based on collaborative optimization of magnetic particle coating, ALP cascade signal amplification and EDTA gradient washing. According to the technical scheme, carboxylated magnetic particles are activated through EDC / NHS and then covalently coupled with a target antigen, a signal is amplified through a cascade reaction of a biotinylated second antibody and ALP coupled streptavidin, the ALP catalytic efficiency is enhanced through a 4-MUP substrate containing 1 mM MgCl2, and gradient washing is conducted through a washing solution containing EDTA to eliminate background interference. The limitation of single module optimization is broken through, the signal / background ratio is larger than or equal to 50, and the detection limit is smaller than or equal to 0.5 ng / mL. Compared with the prior art, the sensitivity is improved by 10-20 times, and the antigen binding stability of the magnetic particles is remarkably improved (the magnetic particles are stored for 12 months at the temperature of 2-8 DEG C, and the performance degradation is smaller than or equal to 10%). The kit is suitable for early infection of pathogens such as mycoplasma pneumoniae and hypersensitivity detection of tumor markers.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, specifically relating to a hypersensitive antibody detection method and kit based on magnetic particle coating, labeled enzyme (such as alkaline phosphatase (ALP) or horseradish peroxidase (HRP)) cascade signal amplification, and EDTA gradient washing synergistic optimization. This technology achieves high sensitivity (detection limit ≤0.5 ng / mL), high stability (performance degradation ≤10% after 12 months of storage at 2-8℃), and low background interference (signal / background ratio ≥50) detection of biomarkers (such as pathogen antibodies and tumor markers) through magnetic particle coating stabilization, labeled enzyme cascade signal amplification, and EDTA gradient washing synergistic optimization. The invention specifically relates to this technical method and its kit. This invention uses the hypersensitive Mycoplasma pneumoniae IgM antibody detection method based on magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing synergistic optimization as an example to explain in detail how the synergistic optimization scheme solves the technical problem of high false negative rate (30-40%) in early infection. Background Technology

[0002] This invention relates to the field of immunological detection of Mycoplasma pneumoniae infection. Existing Mycoplasma pneumoniae IgM antibody detection technology has significant limitations and is difficult to meet the needs of early clinical diagnosis. Specifically: (1) Passive agglutination method (such as the Mycoplasma pneumoniae antibody detection kit of Fuji Rebio Co., Ltd.) mainly detects total antibodies (IgG+IgM) and cannot specifically distinguish IgM antibodies, making it difficult to accurately reflect the early infection status. (2) Although the streptavidin-biotin-coupled fluorescence method (SABC-FITC) achieves detection through fluorescence signal amplification, it is limited by fluorescence intensity and visual judgment, and is prone to missed detection of low concentration IgM antibodies, which cannot meet the sensitive detection requirements of early infection (Li Lihe, Wang Gaosheng, Fu Jinrui. Experimental discussion on the methodology of streptavidin-biotin-coupled FITC method for detecting enterovirus 71 [J]. Chinese Journal of Laboratory Medicine, 2010: 33(5): 445-446.). (3) When using chemiluminescence with magnetic particles as carriers to detect Mycoplasma pneumoniae IgM antibodies, there are key technical bottlenecks: First, magnetic particles and antigens are mostly physically adsorbed or simply covalently coupled, and the antigen is easy to detach, resulting in insufficient coating stability. After being stored at 2-8℃ for 6 months, the antigen residual rate is often less than 60%, leading to a loss of sensitivity. Second, the signal amplification relies on a two-stage amplification system of "biotin-streptavidin + chemiluminescence", and the detection limit can usually only reach 5-10 ng / mL, making it difficult to detect extremely low concentrations of IgM antibodies in serum during the early stage of infection (1-3 days after onset). Third, the washing solution mostly contains only the surfactant Tween-20, which cannot effectively remove non-specifically bound proteins, and the background signal often accounts for 15-20%, further limiting the detection accuracy of low concentration antibodies. Due to the aforementioned technical deficiencies, existing methods have a high false negative rate of 30-40% for detecting IgM antibodies in the early stages of Mycoplasma pneumoniae infection (1-3 days after onset). Furthermore, by the time IgM antibodies can be reliably detected, it is often approximately 7 days after the onset of illness, significantly delaying early intervention and treatment (e.g., Chinese invention patent CN102654499A, US patent US20180011235A1). Therefore, developing a Mycoplasma pneumoniae IgM antibody detection technology that combines high sensitivity and specificity and covers the early infection window has become an urgent need in the field of clinical diagnosis. This invention advances the diagnostic window for Mycoplasma pneumoniae infection to 1-3 days after onset (compared to 7 days in existing technologies), significantly shortening the detection window period for Mycoplasma pneumoniae infection. Summary of the Invention

[0003] The technical problem solved by this invention is the core deficiency in existing Mycoplasma pneumoniae IgM antibody detection technologies, including insufficient sensitivity (detection limit of only 5-10 ng / mL, making it difficult to detect early low-concentration antibodies), poor stability of magnetic particle antigen coating (antigen residue rate <60% after 6 months of storage at 2-8℃), high background signal (accounting for 15-20%), and high early false negative rate (30-40%). This invention aims to provide a highly sensitive, stable, and specific method and kit for detecting Mycoplasma pneumoniae IgM antibodies by optimizing the antigen coating process, constructing an efficient signal amplification system, and innovating washing strategies, thus filling the technical gap in early infection detection.

[0004] The technical solution of this invention achieves ultrasensitive detection through the synergistic action of three modules: (1) Magnetic particle coating module: Carboxylated magnetic particles are activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) and covalently coupled with the recombinant antigen of Mycoplasma pneumoniae, and blocked by bovine serum albumin (BSA) to ensure stable antigen binding (residual rate ≥90% after 12 months); (2) Signal amplification module: After the biotinylated secondary antibody binds to the sample IgM, the signal is amplified by the biotin-streptavidin cascade of labeled enzymes (including but not limited to ALP, HRP), and magnesium ions catalyze ALP activity; (3) Washing system: Gradient washing with washing solution containing EDTA reduces background signal (≤3%); The above modules work together to achieve a detection limit of 0.5 ng / mL, which is suitable for early infection detection.

[0005] The core innovations of this invention are: (1) Three-dimensional synergistic closed-loop system: The first three-dimensional synergistic mechanism of "magnetic particle coating-signal amplification-washing system" is created to achieve a closed loop of "stable signal source-high efficiency amplification-low noise background", and the overall sensitivity is improved by 10-20 times. (2) Innovation of magnetic particle coating stabilization: Carboxylated magnetic particles with a particle size of 2-3μm are precisely activated with EDC / NHS concentrations of 10-20mM / 5-10mM, which is determined to be the optimal synergistic range (the concentration range is verified by the parameters in Example 1: the residual rate of the 9mM / 4mM group is 60%, and the aggregation rate of the 21mM / 11mM group is 30%). After blocking with 1% BSA for 2h, the antigen residual rate is ≥90% after 12 months at 2-8℃ (an improvement of >50% compared with physical adsorption), providing a long-term stable "solid-phase signal source" for subsequent cascade amplification. (3) Three-stage cascade signal amplification: Biotinylated secondary antibody labeling density is 3-5 biotin / antibody; ALP-streptavidin 1:1 cross-linking, containing 5% D-sorbitol + 0.1% BSA to stabilize enzyme conformation, combined with 4-MUP substrate containing MgCl2, reducing the detection limit to 0.5 ng / mL (sensitivity increased by 10-20 times); (4) EDTA gradient washing strategy: 0.5 mM EDTA chelates metal ions ≥7 gradient washes (3 captures + 4 enzyme labelings), reducing the background signal from more than 15% to ≤3% (cross-reaction rate <1%), accurately identifying low concentration signals. The high stability of magnetic particles avoids signal fluctuations, low background washing eliminates noise interference, 1 mM MgCl2 in the luminescent solution stimulates ALP activity to improve reaction efficiency, ultimately achieving a signal / background ratio ≥50 (single module optimization group ≤12), successfully advancing the antibody detection window in the early stage of infection (1-3 days), solving the clinical pain point of 30-40% false negative rate of existing technology.

[0006] Compared with existing technologies, this invention has the following significant advantages: (1) Highly sensitive detection capability: The three-level signal amplification system reduces the detection limit to 0.5 ng / mL, which can detect low concentrations of IgM antibodies in the early stage of infection (1-3 days after onset), and the sensitivity is 10-20 times higher than that of existing technologies. (2) High stability: The optimized covalent coupling process combined with BSA blocking ensures that the performance of magnetic particle surface antigens decreases by ≤10% after 12 months of storage at 2-8℃, which is far superior to existing technologies (<60%). (3) High specificity: The synergistic effect of EDTA-containing washing solution and 7 gradient washing reduces the background signal ratio to below 3% and the cross-reactivity rate to <1%, effectively avoiding non-specific interference. (4) Clinical early diagnostic value: It advances the early diagnostic window of Mycoplasma pneumoniae infection by 2-3 days, significantly reducing the early missed detection rate (from 30-40% to a negligible level), and providing a key basis for early intervention and treatment. Therefore, this invention achieves a significant improvement in the sensitivity and stability of Mycoplasma pneumoniae IgM antibody detection through magnetic particle coating process stabilization, three-level signal amplification synergy, and innovative washing system, demonstrating significant technological advancement and clinical application value. Attached Figure Description

[0007] Figure 1 : Schematic diagram of the technical architecture and synergistic effects of this invention. Detailed Implementation

[0008] This invention will be further described in detail using the ultrasensitive detection of Mycoplasma pneumoniae IgM antibody and accompanying drawings as examples.

[0009] Example 1

[0010] 1. Reagent Preparation

[0011] 1.1 Preparation of Antigen-Coated Magnetic Particles (Providing a Stable Binding Basis for Signal Amplification) Materials Preparation: Carboxylated magnetic particles (particle size 2-3 μm); Mycoplasma pneumoniae recombinant antigen (concentration 1 mg / mL, recombinantly expressed from Mycoplasma pneumoniae P1 protein, purity ≥95%), EDC, NHS, 2-(N-morpholine) ethanesulfonic acid (MES) buffer (0.1 M, pH 5.0), PBS buffer (0.01 M, pH 7.4), 1% BSA solution (dissolved in PBS), 0.05% Tween-20, 0.1% ProClin 300 (preservative to prevent microbial contamination of the magnetic particle suspension during storage). Activation and Coupling: Take 1 mL of carboxylated magnetic particle suspension (containing 1 mg of magnetic particles), wash 3 times with MES buffer, and resuspend in 1 mL of MES buffer; add EDC to a final concentration of 15 mM and NHS to a final concentration of 8 mM (this ratio has been verified to achieve an antigen coupling rate of over 90%, matching the subsequent ALP signal amplification efficiency), and activate by shaking at room temperature for 30 min; add Mycoplasma pneumoniae recombinant antigen to a final concentration of 0.5-2 μg / mL (to match the binding site density of the biotinylated secondary antibody and avoid excessive antigen causing non-specific binding), and incubate at 37°C in a constant temperature shaker (300 rpm) for 3 h to covalently couple the antigen with the magnetic particles. Blocking and storage: After the coupling reaction, the supernatant was removed by magnetic separation, and 1 mL of 1% BSA solution was added. The mixture was blocked at 37°C for 2 h (to block unreacted sites and reduce background in conjunction with subsequent EDTA washing). After blocking, the mixture was washed three times with PBS buffer containing 0.05% Tween-20 and 0.1% ProClin 300, resuspended in 5 mL of the same buffer, and stored at 2-8°C for later use.

[0012] 1.2 Preparation of Biotinylated Secondary Antibody (Providing Precise Targeting Sites for Tertiary Scale-up) Materials: Goat anti-human IgM antibody (titer 1:10000); biotin-NHS ester; Tris-HCl buffer (0.05M, pH 7.2); Sephadex G-25 gel chromatography column (for purification); 1% BSA solution (dissolved in Tris-HCl). Labeling Reaction: Dissolve 1 mg of goat anti-human IgM antibody in 1 mL of Tris-HCl buffer. Add biotin-NHS ester at a molar ratio of 1:20 (i.e., 1 mg antibody corresponds to 0.12 mg biotin-NHS ester). Incubate at room temperature in the dark with shaking for 1 h. Control the conjugation of each antibody molecule to 3-5 biotin molecules (this density has been verified to avoid steric hindrance and ensure efficient binding to streptavidin conjugated with ALP). Purification and preservation: Free biotin was separated by Sephadex G-25 gel chromatography column (equilibrated with Tris-HCl buffer), and the first peak (biotinylated secondary antibody fraction) was collected; it was diluted to a concentration of 1 μg / mL with Tris-HCl buffer containing 1% BSA and stored at 2-8℃ for later use.

[0013] 1.3 Preparation of ALP-Conjugated Streptavidin (Density Matching with Biotin for Cascade Amplification) Materials: Streptavidin (purity ≥98%); Alkaline phosphatase (ALP, activity ≥1000 U / mg); 25% glutaraldehyde solution; PBS buffer (0.01 M, pH 7.4); 5% sucrose solution (dissolved in PBS). Cross-linking reaction: Dissolve 1 mg of streptavidin in 1 mL of PBS buffer, add 25% glutaraldehyde to a final concentration of 0.25%, and activate by shaking at room temperature for 15 min; add 1 mg of ALP (streptavidin:ALP molar ratio of 1:1, this ratio has been verified to maximize signal amplification efficiency), and incubate at 37°C for 30 min to allow cross-linking; terminate the reaction by adding 0.1 M glycine solution (final concentration 5 mM). Purification and Preservation: The cross-linked product was purified by ultrafiltration centrifuge tubes (molecular weight cutoff 30kDa) to remove free enzymes and unreacted components. The product was diluted to a concentration of 2μg / mL with PBS buffer (pH 7.4) containing 5% sucrose, 0.1% BSA, and 5% D-sorbitol. D-sorbitol (CAS 50-70-4, an osmotic pressure regulator) reduces conformational damage to enzyme molecules due to dehydration by maintaining osmotic pressure (synergistically with sucrose). It was verified that 3-7% D-sorbitol can improve the tertiary structure stability of ALP by 20-30%, with 5% being optimal (synergistically with BSA, the activity retention rate is 15% higher than the group without D-sorbitol). After aliquoting, the product was refrigerated at 2-8℃ (avoid freezing) for later use.

[0014] 1.4 Preparation of Washing Buffer and Substrate (to ensure signal purity and improve signal-to-noise ratio in synergy with preceding modules) Washing Buffer: Weigh EDTA (final concentration 0.5mM, to chelate metal ions to inhibit non-specific enzymatic reactions of contaminating proteins) and Tween-20 (final concentration 0.05%, to remove hydrophobic non-specific binding) and dissolve them in PBS buffer (0.01M, pH 7.4). Stir to dissolve, filter for sterilization, and store at room temperature. 4-MUP Substrate: Weigh 4-methylumbelliferyl ketone phosphate (4-MUP, CAS 3368-04-5) and dissolve it in 0.1M Tris-HCl buffer (pH 9.8) to prepare a 1mM solution (matching the catalytic activity of ALP enzyme to ensure the linear range of the signal). Add 1mM MgCl2 to the chemiluminescence solution, aliquot in the dark, and store at -20℃. Thaw before use.

[0015] 1.5 Kit Components (Integration of Module Synergistic Effects) Reagent I: Antigen-coated magnetic particle suspension (1 mg / mL carboxylated magnetic particles, conjugated with 1 μg / mL Mycoplasma pneumoniae recombinant antigen, 5 mL / bottle, stored at 2-8℃); Reagent II: Biotinylated goat anti-human IgM secondary antibody (1 μg / mL, 3 mL / bottle, stored at 2-8℃); Reagent III: ALP-conjugated streptavidin (2 μg / mL, PBS buffer (pH 7.4) containing 5% sucrose, 0.1% BSA and 5% D-sorbitol, 3 mL / bottle, refrigerated at 2-8℃); BSA, D-sorbitol and sucrose form an osmotic pressure protection system to prevent enzyme molecule aggregation during low-temperature storage and enhance tertiary structural stability; Reagent IV: Washing buffer (containing 0.5 mM... EDTA, 0.05% Tween-20, 50 mL / bottle, store at room temperature; Reagent V: Substrate (1 mM 4-MUP + 1 mM MgCl2, 10 mL / bottle, freeze at -20℃), where MgCl2 is an ALP-specific activator that can enhance its catalytic activity against 4-MUP substrates (increasing signal intensity by 1.5-2 times); Reagent VI: Standards (concentration gradients: 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 6 bottles). Specifications: 300 tests / kit, store at 2-8℃, shelf life 12 months.

[0016] The components of the Mycoplasma pneumoniae IgM antibody detection kit are shown in Table 1.

[0017] Table 1. Configuration of Mycoplasma pneumoniae IgM antibody detection kit

[0018]

[0019] 2. Testing Process

[0020] 2.1 Sample Processing and Antigen-Antibody Capture: Collect 5 mL of venous blood from the patient, centrifuge at 3000×g for 10 min to separate serum, avoiding hemolysis or lipemia. Dilute the serum 1:10 with PBS buffer containing 1% BSA (to reduce matrix interference and match the antigen coating density), equilibrate at room temperature for 15 min, and set aside. Take a 200 μL reaction tube, add 50 μL of diluted serum sample, then add 50 μL of antigen-coated magnetic particle suspension (containing 50 μg of magnetic particles), and mix thoroughly. Incubate at 37℃ for 30 min (to ensure sufficient antigen-antibody binding, laying the foundation for subsequent signal amplification), perform magnetic separation (2 min), and discard the supernatant. Add 200 μL of washing buffer, vortex to mix, and perform magnetic separation again, discarding the supernatant. Repeat washing 3 times (capture washing, to initially remove free impurities).

[0021] 2.2 Biotinylated secondary antibody binding: Add 50 μL of biotinylated goat anti-human IgM secondary antibody (concentration 1 μg / mL) to the washed magnetic particles and mix well; incubate at 37℃ for 20 min (to match the binding kinetics of the secondary antibody and ensure sufficient binding) to form a "magnetic particle-antigen-IgM-biotinylated secondary antibody" complex; magnetic separation (2 min), discard the supernatant, and wash three times with washing buffer to remove free secondary antibody (to reduce subsequent signal interference).

[0022] 2.3 ALP-Streptavidin binding (signal amplification): Add 50 μL of ALP-conjugated streptavidin (concentration 2 μg / mL) and mix well; incubate at 37℃ for 15 min (matching the binding kinetics of streptavidin-biotin) to form a "magnetic particle-antigen-IgM-biotin secondary antibody-ALP streptavidin" complex using the high affinity of streptavidin and biotin; magnetic separation (2 min), discard the supernatant, wash 4 times with washing buffer (to reduce background to below 3% in conjunction with the previous capture wash), and aspirate the residual liquid after the last wash.

[0023] 2.4 Substrate reaction and signal detection: Add 100 μL of 4-MUP substrate solution containing 1 mM MgCl2 (magnesium ions in the substrate buffer activate ALP activity), incubate at 37°C in the dark for 10 min (the enzyme reaction rate is increased by 30-50% compared to the magnesium-free group, and the time to reach the plateau phase is shortened to 8 min); add 50 μL of 0.2 M NaOH to terminate the reaction (to stabilize the fluorescence signal and avoid signal attenuation), and immediately detect the fluorescence intensity using a fluorescence detector (excitation wavelength 360 nm, emission wavelength 450 nm); prepare a standard curve using Mycoplasma pneumoniae IgM standards at known concentrations (0.1, 0.5, 1, 5, 10 ng / mL), calculate the IgM concentration based on the sample fluorescence intensity, and multiply by the dilution factor (10-fold) to obtain the final result.

[0024] 3. Parameter range verification

[0025] EDC / NHS concentration range verification: Five groups were tested: 9mM / 4mM (below the lower limit), 10mM / 5mM, 15mM / 8mM, 20mM / 10mM, and 21mM / 11mM (above the upper limit). The results showed that the antigen residue rate of the 9mM / 4mM group was only 60% (unable to support signal amplification, and the 9mM / 4mM group below the lower limit was excluded due to the residue rate of less than 60%), the magnetic particle aggregation rate of the 21mM / 11mM group reached 30% (interfering with binding), and the residue rate of the 10mM-20mM / 5mM-10mM group was ≥85%, proving that this range is a synergistic effective range. Verification of D-sorbitol concentration in ALP-conjugated streptavidin: Three groups were tested: 3%, 5%, and 7%. The ALP activity retention rate of the 5% group was 96% (90% in the 3% group and 95% in the 7% group), proving that 5% is the optimal concentration. In synergy with the signal amplification module, this process ensures antigen stability and reduces the CV value of subsequent signal amplification from 12% to 5%. Magnesium ion concentration verification: Five groups were tested: 0.3mM (below the lower limit), 0.5mM, 1mM, 2mM, and 2.5mM (above the upper limit). The ALP activity of the 0.3mM group increased by only 15%, and the 2.5mM group inhibited streptavidin-biotin binding due to high ionic strength (signal reduction of 20%). The activity of the 0.5-2mM group increased by 30-50%. 1mM is the optimal concentration, which ensures activation efficiency and avoids interference from high-concentration ions with streptavidin-biotin binding. At the same time, MgCl2 in the luminescent solution prevents magnesium ions from forming chelates with EDTA in the washing steps mentioned above. All validation experiments were performed in triplicate. Data are expressed as mean ± standard deviation. The significance of the data was analyzed using the t-test (P < 0.05 was considered statistically significant).

[0026] 4. Performance Verification

[0027] 4.1 Sensitivity Validation: Mycoplasma pneumoniae IgM standard at a concentration of 0.5 ng / mL was used for detection. The assay was repeated 10 times. The fluorescence intensity CV value was ≤5.0%, and the linear correlation coefficient R0 was ≤5.0%. 2 ≥0.9900 indicates that the method can stably detect low concentrations of antibodies up to 0.5 ng / mL (this cannot be achieved with single-module optimization: the detection limit is 3 ng / mL when only magnetic particle coating is optimized, and the actual effective detection limit is 2 ng / mL when only signal amplification is optimized due to excessive background).

[0028] 4.2 Stability Verification: After the antigen-coated magnetic particles were stored at 2-8℃ for 12 months, the concentration of free antigen was determined by ELISA, and the residual rate was calculated to be 92.3% (≥90%). After 7 days of accelerated testing at 37℃, the signal attenuation rate of each component of the kit was 8.7% (<10%). This indicates that the magnetic particle coating process has excellent synergistic stability with other modules (significantly better than the existing technology with a residual rate of <60% after 6 months). Among them, the ALP-conjugated streptavidin, due to the synergistic effect of 0.1% BSA and 5% D-sorbitol, achieved an activity retention rate of 96% (78% in the no-additive group and 92% in the BSA-only group), proving that D-sorbitol further enhances the thermostability of the enzyme by maintaining osmotic pressure. 4.3 Specificity verification: The fluorescence signals of 100 serum samples from healthy individuals were all below the critical value (zero false positives); the cross-reactivity detection with Chlamydia pneumoniae, Legionella, etc. showed that the cross-reactivity rate was <1.0% (the cross-reactivity rate of the control group washed with Tween-20 alone reached 8-12%), demonstrating the synergistic specificity enhancement effect of the EDTA-containing washing solution and gradient washing.

[0029] 4.4 Synergistic Effect Verification: Control Group 1 (optimized magnetic particle coating only): detection limit 3 ng / mL, signal / background ratio 12; Control Group 2 (optimized signal amplification only): detection limit 1 ng / mL, but background signal 15%, actual effective detection limit 2 ng / mL; Control Group 3 (optimized washing only): background signal 5%, but due to insufficient signal amplification, detection limit 8 ng / mL; Adding 1 mmol MgCl2 to the luminescent solution can enhance ALP catalytic activity and increase the detection signal by 30-50%. This invention (three-module synergy): detection limit 0.5 ng / mL, signal / background ratio 52 (P<0.01, superior to all control groups).

[0030] 4.5 Kit Assembly: The antigen-coated magnetic particle suspension, biotinylated secondary antibody, ALP-conjugated streptavidin, washing buffer, 4-MUP substrate, and standards prepared above are integrated into a 300-test / kit specification. Store at 2-8℃ for 12 months. Through synergistic optimization of each module, the kit maintains ultrasensitive detection performance (detection limit 0.5 ng / mL, background ≤3%) within its shelf life.

[0031] Example 2

[0032] For respiratory syncytial virus (RSV) IgM detection, the antigen was replaced with RSV-F protein, and the remaining steps were the same as in Example 1. The detection limit was 0.8 ng / mL, and the background was 3.9%. This meets the requirement of "background signal ≤ 5%" in the extended application of this invention.

[0033] Example 3

[0034] The tumor marker HE4 was detected, with the antigen replaced by recombinant HE4 protein and the secondary antibody being mouse anti-HE4 monoclonal antibody-biotin. The detection limit was 0.9 ng / mL, and the background was 3.2%. This meets the requirement of ≤5% background signal for the extended application of this invention.

[0035] Example 4: Validation of HRP as a substitute for ALP:

[0036] (1) Preparation of HRP-streptavidin: streptavidin and HRP were cross-linked with glutaraldehyde at a molar ratio of 1:2, containing 5% sucrose + 0.1% BSA + 5% D-sorbitol (same as ALP group);

[0037] (2) The substrate was replaced with TMB (containing activators such as ferrous ions, which are compatible with HRP-labeled enzymes);

[0038] (3) The detection limit was 0.6 ng / mL, the background signal was 2.8%, and the activity retention rate after 12 months was 94% (no significant difference from the ALP group, P>0.05).

[0039] Figure 1 The diagram illustrates the technical architecture and synergistic effects of this invention, wherein: (1) the magnetic particle coating module includes carboxylated magnetic particle activation, antigen conjugation and blocking steps; (2) the signal amplification module includes biotinylated secondary antibody binding, ALP-streptavidin cascade and substrate reaction; (3) the washing system displays the EDTA gradient washing process; the diagram indicates the synergistic effect indicators: detection limit ≤ 0.5 ng / mL, background signal ≤ 3%, antigen residual rate ≥ 90% after 12 months.

[0040] The scope of protection of this invention is as follows: While maintaining the aforementioned synergistic optimization mechanism of magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing, by replacing the target antigen and corresponding secondary antibody, it can be used for the detection of other pathogen-specific antibodies or tumor markers, with the following performance indicators: detection limit ≤ 1 ng / mL; background signal ≤ 5%; antigen residue rate ≥ 90% after storage at 2-8℃ for 12 months. All equivalent substitutions, routine fine-tuning of concentration / volume / time parameters, labeling enzymes, and different packaging specifications that do not deviate from the aforementioned synergistic optimization mechanism fall within the scope of protection defined by the claims of this invention.

Claims

1. A method and kit for detecting highly sensitive antibodies based on the synergistic optimization of magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing, characterized in that... The system includes the following synergistic modules: the magnetic particle coating module provides a stable and solid foundation for signal amplification; the signal amplification module achieves cascaded amplification based on the stable coated signal; and the washing system eliminates non-specific interference to ensure the signal-to-noise ratio of the amplified signal. The three modules form a synergistic closed loop through the "stable signal source - efficient amplification - low noise background" mechanism.

2. The method and kit for detecting hypersensitive antibodies based on the synergistic optimization of magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing as described in claim 1, characterized in that, The parameters of the magnetic particle coating module are as follows: carboxylated magnetic particle size is 2-3 μm, EDC concentration is 10-20 mM; NHS concentration is 5-10 mM, antigen concentration is 0.5-2 μg / mL, coupling at 37℃ for 3 h, blocking with 1% BSA for 2 h (to block unreacted sites in conjunction with the washing system), and stored in 0.01 M PBS (pH 7.4) containing 0.05% Tween-20 and 0.1% ProClin 300.

3. The method and kit for detecting hypersensitive antibodies based on the synergistic optimization of magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing as described in claim 1, characterized in that, The parameters of the signal amplification module are as follows: goat anti-human IgM antibody and biotin-NHS ester are reacted in 0.05M Tris-HCl buffer (pH 7.2) at a molar ratio of 1:20 at room temperature for 1 h, with a labeling density of 3-5 biotin / antibody; purified by Sephadex G-25 gel chromatography and stored in 0.05M Tris-HCl (pH 7.2) containing 1% BSA at a concentration of 1 μg / mL.

4. The method and kit for detecting hypersensitive antibodies based on the synergistic optimization of magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing as described in claim 1, characterized in that, The labeling enzyme for the signal amplification module is ALP or horseradish peroxidase (HRP). The preparation parameters for streptavidin coupling are as follows: streptavidin and the labeling enzyme are cross-linked with 0.25% glutaraldehyde at a molar ratio of 1:1-3 for 30 min. After purification, the concentration is 2 μg / mL. It is stored in 0.01M PBS (pH 7.4) containing 5% sucrose, 0.1% BSA (to maintain the three-dimensional structure of the enzyme and reduce aggregation) and 5% D-sorbitol (to maintain osmotic pressure and stabilize the tertiary structure of the enzyme) and stored at 2-8℃, avoiding freezing.

5. The method and kit for detecting hypersensitive antibodies based on the synergistic optimization of magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing as described in claim 1, characterized in that, The washing system parameters are as follows: the washing buffer contains 0.5 mM EDTA and 0.05% Tween-20 in 0.01 M PBS (pH 7.4); the gradient washing is performed 7 times (the gradient washing includes 3 capture washes and 4 enzyme-labeled washes, for a total of 7 times). Validation shows that ≥7 washes can ensure that the background signal is ≤3%, and in conjunction with the signal amplification module, the signal / background ratio is ≥50.

6. The method and kit for detecting hypersensitive antibodies based on the synergistic optimization of magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing as described in claim 1, characterized in that, The components of the kit are as follows: (1) Antigen-coated magnetic particle suspension: 1 mg / mL carboxylated magnetic particles, coupled with 1 μg / mL Mycoplasma pneumoniae recombinant antigen, 5 mL / bottle, stored at 2-8℃; (2) Biotinylated goat anti-human IgM secondary antibody: 1 μg / mL, 3 mL / bottle, stored at 2-8℃; (3) ALP-coupled streptavidin: 2 μg / mL, containing 5% sucrose, 5% D-sorbitol (to maintain osmotic pressure and stabilize the enzyme's tertiary structure), 0.1% BSA in 0.01M PBS (pH 7.4), 3 mL / bottle, refrigerated at 2-8℃; (4) Washing buffer: containing 0.5 mM EDTA and 0.05% Tween-20 in 0.01M PBS (pH 7.4), 50 mL / bottle, stored at room temperature; (5) 4-MUP substrate: 1 mM 4-MUP + 1 mM MgCl2 (activates ALP and enhances its catalytic activity to improve the detection signal) is dissolved in 0.1M Tris-HCl (pH 9.8), 10mL / bottle, frozen at -20℃; (6) Standards: 0-10ng / mL gradient, 6 bottles; Specification 300 tests / box, stored at 2-8℃, shelf life 12 months. The parameters of each component correspond to the defined ranges of claim 2 (magnetic particle coating module), claims 3-4 (signal amplification module), and claim 5 (washing system).

7. The method and kit for detecting hypersensitive antibodies based on the synergistic optimization of magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing as described in claim 6, characterized in that, The antigen-coated magnetic particle suspension was stored in 0.01M PBS (pH 7.4) containing 0.05% Tween-20 and 0.1% ProClin 300; the ALP-conjugated streptavidin solution was stored in the dark at 2-8°C.

8. The method and kit for detecting hypersensitive antibodies based on the synergistic optimization of magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing as described in claim 6, characterized in that, The performance indicators of the synergistic effect of the three modules are as follows: signal / background ratio ≥50 when the detection limit is ≤0.5 ng / mL, and the accelerated stability is significantly better than that of the single-module optimization group. Due to the synergistic stability of ALP containing 0.1% BSA, 5% sucrose, and 5% D-sorbitol (osmotic pressure regulation), the stability is improved by more than 45% compared to the additive-free group; 1 mM MgCl2 in the chemiluminescent solution can increase ALP activity by 30%-50%. This is significantly different from the single-module optimization (signal / background ratio ≤12) (P<0.01). In extended applications, although the detection limit is slightly higher than that for Mycoplasma pneumoniae IgM detection (≤1 ng / mL), it is still significantly better than the detection method without the synergistic mechanism (detection limit ≥3 ng / mL).

9. The method and kit for detecting hypersensitive antibodies based on the synergistic optimization of magnetic particle coating-ALP cascade signal amplification-EDTA gradient washing as described in claim 1, characterized in that: By replacing the target antigen with other pathogen-specific antigens or tumor markers, while maintaining the synergistic optimization mechanism of the magnetic particle coating-signal amplification-washing system, and by replacing the labeling enzyme type with enzymes compatible with the cascade amplification system such as HRP, and adjusting the packaging specifications, the detection limit is ≤1 ng / mL, the background signal is ≤5%, and the antigen residue rate is ≥90% after 12 months of storage at 2-8℃ (verification shows that when only the antigen is replaced without maintaining the synergistic mechanism, the detection limit is ≥3 ng / mL and the background signal is ≥8%).

Images