Ige latex-enhanced immunoturbidimetric reagent and preparation method thereof

Abstract

The application provides an IgE latex enhanced immunoturbidimetric reagent and a preparation method thereof. The IgE latex enhanced immunoturbidimetric reagent comprises a first reagent, the first reagent comprises a mixture of first latex microspheres, second latex microspheres and third latex microspheres, the surfaces of the first latex microspheres, the second latex microspheres and the third latex microspheres are covalently coupled with anti-IgE antibodies, and the remaining active sites on the surfaces are grafted and crosslinked with carboxylated streptomycin and aminated penicillin G as blocking agents through covalent bonds, the particle size of the first latex microspheres is 80-120 nm, the particle size of the second latex microspheres is 160-240 nm, and the particle size of the third latex microspheres is 260-340 nm. The reagent has low background, high stability, ultra-wide linearity, strong anti-interference, low value and high precision, high value and no hook effect, is suitable for existing IVD production lines and mainstream automatic biochemical analyzers, and can be mass-produced.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic (IVD) biochemical immunoassay reagents, and particularly to latex-enhanced immunoturbidimetric assay reagents, especially to IgE latex-enhanced immunoturbidimetric reagents and their preparation methods. Background Technology

[0002] Immunoglobulin E (IgE) is a secretory immunoglobulin composed of two light chains and two heavy chains. It is produced by plasma cells in the lamina propria of the nasopharynx, tonsils, bronchi, and gastrointestinal mucosa, and is the main antibody causing type I hypersensitivity reactions. IgE has extremely strong allotropic affinity, sensitizing only human and monkey cells. It is also the type of immunoglobulin with the worst thermal stability, shortest half-life (approximately 2-3 days), lowest synthesis rate, and highest degradation rate. Therefore, its concentration in normal human serum is extremely low, approximately 150 IU / mL.

[0003] Serum IgE levels in patients with allergies or hypersensitivity can be several to tens of times higher than in normal individuals. Abnormal IgE levels are closely related to allergic rhinitis, extrinsic asthma, urticaria, parasitic infections, immunodeficiency diseases, autoimmune diseases, and multiple myeloma. Therefore, accurate quantitative detection of serum IgE is of great significance for the diagnosis, differential diagnosis, and monitoring of treatment efficacy in clinical diseases.

[0004] Latex immunoturbidimetry is currently the mainstream technique for clinical IgE detection. It involves covalently coupling specific antibodies to the surface of latex microspheres, triggering microsphere aggregation through an antigen-antibody reaction, and achieving quantitative analysis by detecting changes in the turbidity of the reaction solution. This technique offers advantages such as ease of operation, high automation, fast detection speed, and cost-effectiveness.

[0005] Existing commercial reagents and publicly available literature mostly employ the classic process of activation-antibody conjugation-BSA physical blocking, commonly using high-concentration bovine serum albumin (BSA) to physically adsorb and block unreacted active sites on microspheres. This approach has several technical shortcomings, such as poor stability: BSA adheres to the microsphere surface solely through hydrophobic adsorption and weak electrostatic interactions, without chemical bonding. It is highly susceptible to desorption during reagent storage, transportation, opening, and temperature fluctuations, leading to re-exposure of active sites, non-specific self-aggregation, increased background, signal attenuation, and a significantly shortened shelf life. Furthermore, non-specific interference is severe; BSA, being a heterologous large molecule protein, readily cross-reacts with heterophilic antibodies, rheumatoid factor (RF), complement, etc., in the sample, raising the detection background and reducing low-value sensitivity and specificity. Finally, the hook effect is prominent; traditional single-particle and dual-particle latex systems, with high concentrations of IgE, exhibit significant hook effects. Antigens can saturate and occupy antibody binding sites, inhibiting microsphere cross-linking and aggregation, leading to signal decline instead of increase and falsely low results. High-value clinical samples must be diluted and retested, increasing human error and turnaround time. The linear range is narrow: excellent commercial reagents have a linear range of only 25–1000 IU / mL, while IgE levels in severe allergic patients and multiple myeloma patients can reach thousands or even tens of thousands of IU / mL, requiring multiple dilutions and retests outside the linear range. Low-value precision is insufficient: the CV (concentration coefficient) in the low-concentration range below 50 IU / mL is generally >10%, failing to meet the needs of accurate screening for low-value allergies in subclinical settings. Resistance to matrix interference is weak: it has poor tolerance to common clinical conditions such as hemolysis, lipemia, jaundice, and high RF, easily causing detection deviations exceeding clinically acceptable limits. Stability after opening and transportation is poor: the background of traditional BSA-blocked reagents increases significantly after 7 days of opening, and performance deteriorates significantly after temperature fluctuations and long-distance vibration transportation, resulting in high cold chain logistics costs and significant clinical waste. Summary of the Invention

[0006] To address the aforementioned problems in the prior art, this invention provides an IgE latex-enhanced immunoturbidimetric reagent and its preparation method, achieving low background, high stability, ultra-wide linearity, strong anti-interference, low-value high precision, and high-value no hook effect. It is compatible with existing IVD production lines and mainstream fully automated biochemical analyzers, and can be mass-produced on a large scale.

[0007] To achieve the above objectives, the present invention provides an IgE latex-enhanced immunoturbidimetric reagent, characterized in that it includes a first reagent, wherein the first reagent comprises a mixture of a first latex microsphere, a second latex microsphere, and a third latex microsphere. The surfaces of the first, second, and third latex microspheres are all covalently coupled with anti-IgE antibodies, and the remaining active sites on the surface are all covalently grafted with carboxylated streptomycin and aminolated penicillin G as blocking agents. The first latex microsphere has a particle size of 80–120 nm, the second latex microsphere has a particle size of 160–240 nm, and the third latex microsphere has a particle size of 260–340 nm. Preferably, the molar ratio of carboxylated streptomycin to aminolated penicillin G is 1:0.5 to 1:2.0; the anti-IgE antibody is a mouse anti-human IgE monoclonal antibody.

[0008] Preferably, the volume ratio of the first latex microspheres, the second latex microspheres, and the third latex microspheres is 1:1.5-2.5:1.5-2.5.

[0009] Preferably, each latex microsphere is a carboxyl latex microsphere with a solid content of 5%, a particle size CV of <5%, and a surface carboxyl density of 80–120 μmol / g.

[0010] Preferably, after sealing, the surface of each latex microsphere is activated by EDC and NHS and covalently grafted with polysulfobetaine methacrylate (pSBMA), with a final polysulfobetaine methacrylate grafting concentration of 4–6 mg / mL.

[0011] Preferably, the reagent includes a second reagent, which includes Tris-HCl, NaCl, Tween-20, EDTA•2Na, HBR-3 heterophile antibody blocking agent, sodium caseinate, and Proclin 300 preservative.

[0012] Preferably, the first reagent includes Tris-HCl, NaCl, trehalose, glycerol, and BSA protectant.

[0013] This invention also provides a method for preparing the aforementioned IgE latex-enhanced immunoturbidimetric reagent, characterized by modifying three types of carboxylated latex microspheres with different particle sizes to prepare first latex microspheres, second latex microspheres, and third latex microspheres; and then compounding the first latex microspheres, second latex microspheres, and third latex microspheres to obtain the first reagent. The modification method includes the following steps: (1) Wash the carboxylated latex microspheres and add EDC and NHS to carry out the carboxyl activation reaction; (2) Add ethanolamine to quench any remaining unreacted activated carboxyl groups; (3) Add anti-IgE antibody and carry out covalent coupling reaction; (4) Add carboxylated streptomycin derivative and aminolated penicillin G derivative to carry out covalent blocking reaction; (5) Add polysulfobetaine methacrylate, along with EDC and NHS, to carry out zwitterionic grafting reaction.

[0014] Preferably, in step (3), the mass ratio of anti-IgE antibody to latex microspheres is 1:18-22.

[0015] Preferably, in step (4), the pH is 6.0 to 7.0, the reaction temperature is 20 to 30°C, and the reaction time is 1 to 3 h.

[0016] The beneficial effects of the IgE latex-enhanced immunoturbidimetric reagent and its preparation method of the present invention are as follows: Improved stability: The covalent bonding of carboxylated streptomycin and aminolated penicillin G dual antigens eliminates the risk of desorption. Accelerated stability at 37°C, open-bottle stability, and simulated transport stability are 4-5 times better than traditional BSA reagents. Shelf life can reach 12 months at 2-8°C, and up to 18 months under extreme storage conditions, which is 2-3 times that of traditional reagents. There is no significant performance degradation after 30 days of opening, greatly reducing clinical reagent waste.

[0017] Low background and high specificity: It completely abandons BSA xenoprotein blocking and combines pSBMA anti-fouling modification to effectively resist heterophilic antibodies and RF cross-reaction; the anti-interference deviation is ≤±5%, which is far better than the industry standard of ±10%.

[0018] Ultra-wide linear range, complete suppression of the HOOK effect: linear range 2.0–3000 IU / mL, correlation coefficient R²=0.9999; tolerable at ultra-high concentrations ≥10000 IU / mL with no significant signal drop, no need for dilution and retesting, covering the full range of clinical samples.

[0019] Low value, high precision: 50 IU / mL intra-batch CV < 3.5%, 100 IU / mL CV < 1.5%, far exceeding the industry requirement of CV < 10%, meeting the requirements for accurate screening of subclinical allergies with low values.

[0020] The process is adapted for industrial mass production: the entire reaction is carried out at room temperature and pressure, without the need for special high temperature and high pressure equipment. The process is highly tolerant of errors, and small fluctuations in feed, pH, and temperature do not affect the performance of the finished product. It is fully compatible with existing IVD reagent production lines.

[0021] The instrument is highly versatile: it is compatible with mainstream fully automated biochemical analyzers such as Hitachi, Beckman Coulter, Mindray, Abbott, Roche, Dirui, and Cooper, requiring no hardware modifications and only minor parameter adjustments before it can be used. Attached Figure Description

[0022] Figure 1 This is the calibration curve of the immunoglobulin E assay reagent in this embodiment of the invention.

[0023] Figure 2 This is a graph showing the correlation between the measured values ​​of this invention and the measured values ​​of Siemens reagents.

[0024] Figure 3This is a graph showing the correlation between the measured values ​​of this invention and the measured values ​​of the bioreactor.

[0025] Figure 4 The graph shows the linearity verification results of the reagent of this invention. Detailed Implementation

[0026] To more clearly describe the technical content of the present invention, the following description is provided in conjunction with specific embodiments.

[0027] This invention relates to a fully automated biochemical reagent kit for the quantitative detection of human serum immunoglobulin E (IgE), focusing on: the dual-antigen covalent backbone blocking of carboxylated latex microspheres, pSBMA zwitterionic grafting, and the integrated modification process of three-particle-size latex compound.

[0028] Dual-antigen covalent backbone blocking technology: Carboxylated streptomycin and amino-modified penicillin G are selected. These two small molecule antigens have active amino and carboxyl groups. After activation by EDC and NHS, they are covalently grafted and cross-linked to form a dense three-dimensional network molecular backbone on the surface of latex microspheres. This can completely replace traditional BSA physical blocking, and no BSA is added during the entire blocking process. The covalent bonds are strong, and there is no desorption during storage, temperature fluctuations, or vibration transport. The small molecule antigens do not have cross-reactions with different macromolecules, which greatly reduces non-specific background.

[0029] Small molecule antigen derivatives are covalently bonded to replace the physical adsorption of BSA, permanently occupying the active site without desorption or interference from foreign proteins; Three-particle-size compound optimization: microspheres of different sizes fill the space, increasing the density of reaction sites. Different particle sizes complement each other in reaction, resulting in sensitive response at low concentrations, linear and stable response at medium concentrations, and no drop at high concentrations. There is no obvious hook effect at ≥10000 IU / mL, and the linearity is broadened to 2.0~3000.0 IU / mL.

[0030] pSBMA zwitterionic chemical grafting: A dense superhydrophilic hydration layer is formed on the surface of the microspheres, which blocks the non-specific adsorption of proteins and lipids and enhances the ability to resist hemolysis, lipemia, jaundice and RF interference.

[0031] In this invention, polysulfobetaine methacrylate (pSBMA) is covalently grafted onto the carboxyl groups on the surface of microspheres via EDC / NHS activation after the dual-antigen covalent blocking step. The core grafting sites are the exposed carboxyl groups on the microsphere surface. The exposed, activatable carboxyl groups on the microsphere surface mainly originate from two parts: the residual carboxyl groups of the latex microspheres themselves (the basic microspheres used in this invention are carboxylated polystyrene latex microspheres (surface carboxyl density 80–120 μmol / g), where the initial antibody conjugation and dual-antigen blocking only occupy a portion of the carboxyl sites, leaving unbound native carboxyl groups exposed on the microsphere surface); and the active carboxyl groups on the blocking agent molecules: the carboxylated streptomycin molecules covalently grafted onto the microsphere surface themselves possess active carboxyl functional groups, which can serve as additional grafting sites. By adding EDC (carbodiimide) and NHS (N-hydroxysuccinimide), the exposed carboxyl groups are reactivated to generate a highly reactive succinimide ester intermediate. The activated carboxyl groups form stable amide bonds with the reactive amino groups on the pSBMA molecular chain, thereby covalently grafting pSBMA onto the surface of the microspheres, ultimately forming a dense, superhydrophilic, hydration-resistant antifouling layer.

[0032] It is compatible with mainstream fully automated biochemical analyzers from domestic and international manufacturers such as Hitachi, Beckman Coulter, Mindray, Abbott, Roche, Dirui, and Cooper, and is widely used in clinical screening, differential diagnosis, and efficacy monitoring of allergic diseases, parasitic infections, autoimmune diseases, immunodeficiency diseases, and multiple myeloma.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] The specific specifications of the raw materials involved in the following embodiments are as follows: Carboxylated latex microspheres: solid content 5%, particle size CV < 5%, surface carboxyl density 80–120 μmol / g; manufactured by JSR Corporation of Japan, low particle size microspheres model IMMUTEX P0001, medium particle size microspheres model IMMUTEX P0219, and large particle size microspheres model IMMUTEX P0322.

[0035] Specific antibody: Mouse anti-human IgE monoclonal antibody, purified antibody, purity ≥90%; purchased from HaiTai Biotechnology (Shanghai) Co., Ltd., catalog number: 1E4cc, clone number: E411cc, specifically recognizes human IgE, Fc-region, is a monoclonal mouse anti-human IgE antibody, the hybridoma clone is obtained by hybridization of Sp2 / 0 myeloma cells with Balb / c mouse spleen cells immunized with IgE from human myeloma serum.

[0036] Carboxylated streptomycin is prepared by succinylation, which involves the acylation reaction between succinic anhydride and the primary amino group in the streptomycin molecule to introduce a single carboxyl functional group. The reaction conditions are mild and there are few byproducts.

[0037] Preparation steps: Preparation of reaction system: Dissolve 1g of streptomycin sulfate (purity ≥99%, CAS No.: 3810-74-0, brand: Shanghai Maclean Reagent) in 50mL of 0.1M PBS buffer (pH 8.0) and stir in an ice bath until completely dissolved.

[0038] Add acylation agent: Dissolve 0.5g of succinic anhydride in 10mL of anhydrous dimethyl sulfoxide (DMSO) and slowly add it dropwise to the streptomycin solution. The addition time should be controlled at 30min, and the entire process should be carried out in an ice bath to avoid light.

[0039] Constant temperature reaction: After the addition is complete, raise the temperature to 25°C, stir the reaction in the dark for 2 hours, and maintain the pH at 7.5-8.0 with 0.1M NaOH.

[0040] Purification: The reaction solution was transferred to a dialysis bag (molecular weight cutoff 1 kDa) and dialyzed with 0.01 M PBS (pH 7.4) for 72 h. The dialysis solution was changed every 6 h to remove unreacted succinic anhydride and byproducts.

[0041] Freeze-drying preservation: The dialysis solution is sterilized by passing it through a 0.22μm filter membrane, freeze-drying it to obtain a white powder, which is then sealed and stored at -20℃. It has a shelf life of 12 months.

[0042] Aminated penicillin G is prepared by ethylenediamine amidation. It uses 6-aminopenicillanic acid (6-APA, CAS No.: 551-16-6, brand: Aladdin Reagent) as the starting material. Its carboxyl group is activated by EDC / NHS and then undergoes a monoamidation reaction with ethylenediamine to introduce a terminal amino group, thus retaining the core antigenic epitope of penicillin G.

[0043] Preparation steps: Carboxyl activation: Dissolve 1g of 6-APA (purity ≥99%, Sigma A9518) in 50mL of 0.1M MES buffer (pH 6.0), add 2g of EDC and 1g of NHS, and activate at 25℃ in the dark for 30min with stirring.

[0044] Amide reaction: The activation solution was slowly added dropwise to 100 mL of 0.1 M MES buffer (pH 6.5) containing 5 mL of ethylenediamine over a period of 1 hour. The mixture was stirred at 25 °C in the dark for 3 hours.

[0045] To terminate the reaction, add 1 mL of 1 M glycine solution and stir for 10 min.

[0046] Purification: The reaction solution was purified using cation exchange resin (SP Sepharose Fast Flow), and the target product was eluted with 0.1M NaCl. The elution peak was collected and dialyzed (molecular weight cutoff 500 Da) for 48 h.

[0047] Lyophilization: After sterilization of the dialysis solution, lyophilization was performed to obtain a white crystalline powder. It was then stored in a sealed container at -20°C for 18 months. pSBMA: Polysulfobetaine methacrylate, molecular weight 10000 Da; EDC and NHS: Biochemical grade; MES, PBS, Tris, EDTA•2Na, Trehalose, Glycerol, and Proclin 300 are all reagent grade. Calibration and quality control traceability: WHO 75 / 502 IgE standard was used, and the matrix was normal human serum matrix without IgE.

[0048] The following are common process control parameters in the embodiments: Centrifugation conditions: 8000 rpm, 4℃, 10 min; Stirring routine: Stir gently at 100-250 rpm, and keep away from light throughout the process; Double antigen grafting fixation: pH 6.5, temperature 25℃, reaction time 2 h; Ultrasound parameters: power 100 W, ultrasound for 2 seconds, interval for 2 seconds, cycle for 5 minutes; Sterilization filtration: 0.22 μm PES filter membrane is used uniformly.

[0049] Example 1 Preparation of latex dual antigen backbone with a small particle size of 100 nm Latex washing: Take 1 mL of 100 nm carboxylated latex microspheres (5% solid content), add 9 mL of 0.1 M MES buffer (pH 6.0), invert and mix 10 times, transfer to a 15 mL centrifuge tube; centrifuge at 8000 rpm and 4℃ for 10 min, discard the supernatant; repeat the washing twice, and finally resuspend in 1 mL of 0.1 M MES (pH 6.0) for later use.

[0050] Latex activation: Prepare 100 μL of each of the following pure water solutions: 20 mg / mL EDC and 10 mg / mL NHS. Add the solution to the latex suspension and activate by gentle stirring at 25°C and 100 rpm in the dark for 30 min.

[0051] Quenching residual activation sites: After activation, centrifuge and discard the supernatant, add 1 mL of 50 mM ethanolamine solution (pH 7.4), and let it stand at room temperature for 20 min to block unreacted activated carboxyl groups and avoid subsequent non-specific coupling.

[0052] Specific antibody conjugation: Centrifuge and discard the supernatant, resuspend in 1 mL 10 mM PBS (pH 7.4); add 1 mg of mouse anti-human IgE monoclonal antibody at an antibody:microsphere mass ratio of 1:20; finely adjust the pH to 8.2 with 0.1 M NaOH, and react at 25℃ and 250 rpm in the dark for 90 min.

[0053] Double antigen covalent blocking: Prepare a 1:1 mixture of carboxylated streptomycin and aminolated penicillin G, with a total concentration of 0.5 mg / mL (solvent: 10 mM PBS, pH 7.4); add 300 μL of the mixture and adjust the pH to 6.5 with 0.1 M HCl; react at 25℃ and 200 rpm for 2 h with constant temperature stirring, replacing physical blocking with BSA.

[0054] pSBMA zwitterionic antifouling grafting: Add pSBMA monomer to a final concentration of 5 mg / mL; add EDC to a final concentration of 10 mg / mL and NHS to a final concentration of 5 mg / mL, and adjust the pH to 6.5; react at room temperature in the dark for 30 min to form a dense hydrated antifouling layer on the surface of the microspheres.

[0055] Wash and store the reaction solution by centrifugation, wash three times with 10 mM PBS (pH 7.4) to remove ungrafted small molecules and free pSBMA; finally, resuspend in 1 mL R2 latex preservation solution and store in a sealed container at 4°C protected from light.

[0056] The specific preparation method for R2 latex preservation solution (1 L) is as follows: Measure 800 mL of purified water and add the following in sequence: 20 mM Tris-HCl, pH 7.4; NaCl: 8.5 g; Trehalose: 50 g; Glycerol: 100 mL; BSA: 1.0 g; Proclin 300: 1.0 mL; Bring the volume to 1 L, stir to dissolve, filter through a 0.22 μm filter, and store at 2–8 °C.

[0057] Trehalose and glycerol provide low-temperature antifreeze and protect the surface modification layer of the microspheres, preventing protein denaturation and microsphere aggregation.

[0058] Trace amounts of BSA act only as protein protective stabilizers and do not participate in latex surface sealing.

[0059] Example 2 Preparation of latex with medium particle size of 200 nm Following the method described in Example 1, the latex microspheres were replaced with 200 nm carboxyl latex microspheres (solid content 5%, particle size CV < 5%). The remaining raw material ratios, buffer system, pH, temperature, reaction time, stirring speed, centrifugation parameters, activation / coupling, blocking, pSBMA grafting steps, etc., were completely consistent with Example 1 and will not be repeated here. A 200 nm single-component latex reagent was thus prepared.

[0060] Example 3 Preparation of large-particle-size (300 nm) latex modified Following the method described in Example 1, the latex microspheres were replaced with 300 nm carboxyl latex microspheres (solid content 5%, particle size CV < 5%). The remaining raw material ratios, buffer system, pH, temperature, reaction time, stirring speed, centrifugation parameters, activation / coupling, blocking, pSBMA grafting steps, etc., were completely consistent with Example 1 and will not be repeated here. A 300 nm single-component latex reagent was thus prepared.

[0061] Example 4 Preparation of R2 latex reagent (1 L system) using a three-particle-size compound Measure the latex: According to the volume ratio of 1:2:2 for the 100 nm single-component latex reagent obtained in Example 1: 200 nm single-component latex reagent obtained in Example 2: 300 nm single-component latex reagent obtained in Example 4, accurately measure: 100 mL for 100 nm single-component latex reagent; 200 mL for 200 nm single-component latex reagent; 200 mL for 300 nm single-component latex reagent.

[0062] Mix thoroughly: Add the three single-component latex reagents sequentially to a 1 L glass preparation container, and stir at low speed of 100 rpm for 15 min under ice bath conditions to mix thoroughly.

[0063] Ultrasonic dispersion: Turn on the circulating ultrasonic treatment for 5 min (power 100 W, ultrasonic 2 s, intermittent 2 s) to prevent microspheres from agglomerating.

[0064] Sterilization filtration: Finally, filter the solution through a 0.22 μm PES (polyether sulfone) membrane for sterilization. The filtrate is the final reagent R2 and should be stored at 2–8°C in the dark.

[0065] Example 5 R1 buffer solution preparation (1 L) Measure 800 mL of purified water and place it in a 1 L beaker. Stir mechanically at 25°C and 300 rpm, then add the following components sequentially, stirring until completely dissolved: Tris (tris(hydroxymethyl)aminomethane): 6.06 g (prepared as 50 mM Tris-HCl), pH adjusted to 7.4 with concentrated HCl; NaCl: 9.0 g; Tween-20: 1.0 g; EDTA•2Na (disodium ethylenediaminetetraacetate): 1.0 g; Heterophilic antibody blocker HBR-3: 100 mg; Sodium caseinate: 5.0 g; Proclin 300 (preservative): 1.0 mL Add purified water to a final volume of 1.0 L and continue stirring for 10 min. Measure the conductivity and maintain it within the range of 14.0–14.5 mS / cm. Filter through a 0.22 μm PES membrane to obtain the R1 buffer, and store at 2–8 °C.

[0066] Example 6 Preparation of calibrators and quality control materials Add the following to the calibrator matrix solution (1 L): 20 mM Tris-HCl, pH 7.4; NaCl: 8.8 g; Trehalose: 2.5 g; Sodium caseinate: 10 g; Glycerol: 100 mL; Triton X-100: 10 mL; Proclin 300: 0.2 mL.

[0067] Make up to 1 L, stir to dissolve, and filter through a 0.22 μm filter.

[0068] Calibration preparation Add WHO-traceable pure IgE, accurately prepared into the following concentration gradients: 0 IU / mL, 50 IU / mL, 150 IU / mL, 500 IU / mL, 1500 IU / mL, 3000 IU / mL, aliquoted into 1 mL vials, and stored at -20℃.

[0069] Preparation of quality control products Add WHO-traceable pure IgE, formulated as follows: Low-value control sample: 100 IU / mL; High-value control sample: 500 IU / mL, dispensed into 1 mL vials, stored at -20℃.

[0070] Example 7 Detection methods and calibration curve preparation Applicable instruments Fully automated biochemical analyzers such as Hitachi 7180 / 7600, Beckman AU580, and Mindray BS800.

[0071] Detection parameters Measurement method: Two-point endpoint method Main wavelength: 570 nm Sample size: 5 μL R1 reagent volume: 200 μL R2 reagent volume: 100 μL Incubation temperature: 37℃ Incubation time: 5 min Reading points: Point 1 (reaction 1 min), Point 2 (reaction 5 min) Fitting method: Employ nonlinear calculation models, such as Logit-log (4P) and Spline. Calibration curve creation The calibrators obtained in Example 6 were tested sequentially from low to high concentration. The calibration curve was plotted with the calibrator concentration as the x-axis and the difference in absorbance between the two points as the y-axis.

[0072] The results are as follows Figure 1 As shown, the horizontal axis represents the concentration of immunoglobulin E standard, and the vertical axis represents the absorbance (OD value) of the reaction. The instrument used was a Hitachi 7180 fully automated biochemical analyzer.

[0073] Example 8 The reagent kit of the present invention The kit of this invention consists of R1 buffer, R2 latex reagent, calibrators, and quality control products. The entire process can be mass-produced at room temperature and pressure.

[0074] R1 buffer solution: prepared according to the method described in Example 5.

[0075] R2 latex reagent: prepared according to the method described in Example 4.

[0076] Calibrators and quality control samples: prepared according to the method described in Example 6.

[0077] Assembly and storage: After dispensing each component, assemble the kit and store at 2-8℃ protected from light.

[0078] Sensitivity and specificity The reagent kit of this invention was compared with samples from imported reagents (manufacturers: Japan Sanken, Siemens) using the following method, the specific method of which is as follows: Sample preparation: Collect remaining anonymous serum samples from the laboratory of a tertiary hospital, including 46 qualified human serum samples. Focus on enriching samples near the medical decision level of 150 IU / mL and blind-number them.

[0079] Unified testing: On the same Hitachi 7180 biochemical analyzer, each sample was calibrated and quality-controlled according to the parameters in the instructions of each reagent (as shown in Example 7). Each sample was tested once, and the quality control was kept under control throughout the process.

[0080] Data analysis: After removing outliers, linear regression analysis was performed to calculate the correlation coefficient R²; the sensitivity and specificity of this kit were statistically analyzed with 150 IU / mL as the cutoff.

[0081] The samples used already had Siemens IgE assay values ​​at the time of acquisition, and the detection equipment was a Siemens BNⅡ protein analyzer, used solely for methodological validation, and containing no personally identifiable information. Samples were strictly stratified according to IgE concentration ranges, covering the entire linear range of the kit, with 52% of samples near the medically decisive level of 150 IU / mL, ensuring the reliability of sensitivity and specificity validation. The Japanese bioreactor was purchased externally; its registration number in China is 20152403244.

[0082] The results are shown in Tables 1 and 2 below. Figure 1 , Figure 2 As shown.

[0083] Table 1

[0084]

[0085] Table 2

[0086] The clinical decision level of this method is 150 IU / mL. As can be seen, in 46 samples, the sensitivity and specificity of this invention compared with Siemens reagents and Shengyan reagents were both 100%, and the sample correlation was all above 0.99.

[0087] Therefore, it can be seen that the present invention has good sample correlation with imported reagents, and no false positive or false negative values ​​have been found.

[0088] Ultra-wide linear detection range The kit of this invention has an ultra-wide linear detection range, with good linear regression in the range of 2.0 to 3000.0 IU / mL.

[0089] The testing method is as follows: Eight dilution concentrations (Xi) (0%, 5%, 10%, 20%, 40%, 60%, 80%, 100%) were prepared by mixing high-concentration samples (human serum matrix samples) close to the upper limit of the linear interval (3000.0 IU / mL) and low-concentration samples (human serum matrix samples) close to the lower limit of the linear interval (2.0 IU / mL) in a certain proportion. Each dilution concentration was measured twice, and the mean value (Yi) of the test results for each dilution concentration was calculated. The linear regression equation was obtained with (Xi) as the independent variable and Yi as the dependent variable, and the correlation coefficient (r) of the linear regression was calculated according to formula (1).

[0090] ...(1) The test results are shown in Table 3 below. Figure 4 As shown.

[0091] Table 3

[0092] The linearity verification results of the kit of the present invention are shown in Table 3 above. Figure 4 As shown, within the concentration range of 1.9 IU / mL to 3064.5 IU / mL, the correlation coefficient R between the kit's detection results and the theoretical values ​​was 0.9999. The absolute deviation at the lowest concentration point (1.9 IU / mL) was 0.6 IU / mL, and the relative deviations at all other concentration points were within ±5%. The results indicate that the kit of this invention has an extremely wide linear range and excellent linear correlation, which can meet the direct detection needs of high-value clinical samples without the need for dilution and retesting.

[0093] Low value repeatability The specific method for determining the repeatability of the kit of the present invention when measuring samples of 50 IU / mL and 100 IU / mL is as follows.

[0094] Instrument: Hitachi 7180 fully automated biochemical analyzer. Calibration, routine maintenance, and performance verification were completed in advance to ensure the instrument's stable condition.

[0095] Reagents: IgE kits (R1+R2) from the same batch of this invention, along with matching calibrators and high and low value quality control samples, equilibrated to room temperature at 2–8°C.

[0096] Samples: Laboratory-prepared mixed human serum samples. Multiple qualified human serum samples were collected, thoroughly mixed, and then confirmed using imported reference reagents for intra-batch precision verification. Two concentration levels were used: a low-value sample of approximately 50 IU / mL and a medium-value sample of approximately 100 IU / mL.

[0097] Sample requirements: no hemolysis, lipemia, jaundice, no repeated freeze-thaw cycles, and stored at 2–8°C for no more than 24 hours.

[0098] Testing process The instrument should be powered on and warmed up for 30 minutes. Calibration should be performed using the matching calibrators (R²≥0.999). High and low value quality control samples should be tested. Experiments can only begin if the results are within ±2SD of the target value.

[0099] The two concentrations of samples were tested 10 times consecutively, without inserting other samples during the testing process, and the instrument operating conditions were kept consistent.

[0100] Record the raw concentration values ​​from 10 tests.

[0101] Data processing Calculate the mean (AV), standard deviation (SD), and coefficient of variation (CV) for 10 tests at each of the two concentrations: Coefficient of variation (CV) = (SD / AV) × 100% Result determination Intra-assay CV < 3.5% for 50 IU / mL; Intra-assay CV < 1.5% for 100 IU / mL. The criteria are far superior to the industry-standard requirement of CV < 10%, meeting the needs for accurate screening of subclinical low-value allergies.

[0102] The results are shown in Table 4 below.

[0103] Table 4

[0104] Therefore, the repeatability (CV) of this invention when measuring 50 IU / mL and 100 IU / mL samples is 3.32% and 1.35%, respectively, which is far superior to the CV < 10% requirement of excellent reagents on the market.

[0105] HOOK effect IGE samples with an actual value of 10000 IU / mL were serially diluted, and their values ​​were measured using the kit of this invention. The measurement results are shown in Table 5 below.

[0106] Table 5

[0107] It can be seen that when measuring a sample of 10,000 IU / mL, the measured value of this invention is still greater than the upper limit of the linear range of the reagent of 3,000 IU / mL, which greatly overcomes the false negative results caused by the HOOK effect.

[0108] Anti-interference experiment Experimental preparation Instruments and reagents: Hitachi 7180 fully automated biochemical analyzer, the same batch of the IgE reagent kit of this invention, and matching calibrators and quality control materials (equilibrated to room temperature at 2-8℃).

[0109] Base samples: Collect 10 human mixed serum samples with an IgE concentration of approximately 250 IU / mL (no hemolysis, no lipemia, no jaundice, and endogenous rheumatoid factor (RF) <20 IU / mL), confirm the values ​​by 3 repeated tests, and use them as blank controls and base solutions for preparing interference samples of various gradients; store at 2-8℃, use within the experimental period, and do not repeatedly freeze and thaw.

[0110] Interfering substance stock solution: Hemolytic interference: Preparation of hemoglobin solution (concentration 49000 mg / dL) Bilirubin interference: Prepare stock solutions of conjugated bilirubin (F) (2150 mg / dL) and free bilirubin (C) (2070 mg / dL). Chyle interference: Preparation of fat emulsion stock solution (189180 FTU) RF interference: Pure human RF solution was prepared into a stock solution of 20,000 IU / mL. Experimental Design and Operation Sample preparation: Mix the interfering substance stock solution with the basic sample at volume ratios of 0%, 10%, 20%...100% to prepare 11 gradient concentrations of interfering samples (with the same total volume), while setting up the pure basic sample as a blank control.

[0111] Calibration quality control: The instrument is powered on and warmed up for 30 minutes. The reagent kit is calibrated to be qualified (R²≥0.999). The detection high and low values ​​are within the range of ±2SD of the target value.

[0112] Sample testing: Each interference gradient sample is tested twice, and samples are processed sequentially from low to high concentration. A quality control test is performed every 5 samples to ensure that the entire process is under control.

[0113] Replication verification: All interference experiments were independently repeated twice, and the average value was taken as the final result.

[0114] Data processing and judgment Calculate the average value of each interfering concentration sample and compare it with the blank control value to calculate the relative deviation: Relative deviation (%) = [(interfering sample value - control value) / control value] × 100% Judgment criteria: A relative deviation of ≤±10% is clinically acceptable.

[0115] The results of the hemolysis (hemoglobin) interference test are shown in Table 6 below; the results of the bilirubin F (conjugated bilirubin) interference test are shown in Table 7 below; the results of the bilirubin C (free bilirubin) interference test are shown in Table 8 below; the results of the chyle interference test are shown in Table 9 below; and the results of the rheumatoid factor (RF) interference test are shown in Table 10 below.

[0116] Table 6

[0117] Table 7

[0118] Table 8

[0119] Table 9

[0120] Table 10

[0121] The above results show that, within the range of interference concentration, all relative deviations are ≤±5%, far below the industry standard of ±10%, demonstrating excellent anti-interference capability.

[0122] Stability verification All stability tests included parallel controls: Experimental group: Reagents of this invention

[0123] Control group: Traditional 2% BSA physical blocking reagent (the rest of the formulation, process, and detection parameters are exactly the same as the reagent of this invention). Detection indicators: signal retention rate (with a concentration of 3000 IU / mL on day 0 as 100%), background absorbance (OD570), and CV precision at 50 IU / mL.

[0124] Judgment criteria: The calibrator with a signal retention rate of ≥90%, background rise ≤10%, and CV ≤10% is considered qualified.

[0125] The stability verification experiment used laboratory-prepared mixed human serum samples. Multiple clinical residual anonymous human serum samples were collected, mixed evenly, and then confirmed by imported reference reagents. The concentration was approximately 50 IU / mL. The sample screening criteria were no hemolysis, no lipemia, and no jaundice. The samples were aliquoted at -20℃ for single use.

[0126] Accelerated stability (thermal stability) at 37℃ Simulate extreme storage conditions to quickly assess reagent shelf life.

[0127] Experimental preparation Instruments: Hitachi 7180 fully automated biochemical analyzer, 37℃ constant temperature incubator (protected from light) Reagents and Samples Experimental group: IgE kits (R1+R2) from the same batch of this invention Control group: Traditional 2% BSA physical sealing reagent (formula, process, and batch were completely identical to the experimental group). Matching calibrators and high / low value quality control samples (store in separate containers at -20℃ to avoid repeated freeze-thaw cycles). 50 IU / mL serum sample, 3000 IU / mL calibrator Dispensing process: Dispense the two groups of reagents into separate 4mL / vial sealed tubes, with ≥15 tubes per group, and label the group and date to avoid repeated opening of the caps.

[0128] Experimental Procedure Day 0 baseline testing After the instrument is powered on, preheated, calibrated, and quality controlled, the initial values ​​of the two sets of reagents are measured: ① Background absorbance. (Add only R1+R2, no sample) ② 3000 IU / mL calibrator's reaction absorbance (as initial signal 100%) ③ 50 IU / mL sample, detect 10 times consecutively, calculate initial CV All indicators were tested in parallel three times, and the average value was taken as the baseline value.

[0129] Accelerate training Place all dispensed reagents in a 37°C constant temperature incubator and store them in a sealed container away from light to avoid temperature fluctuations.

[0130] Regular sampling and testing Three parallel reagents from each of the two groups were taken out on days 3, 7, and 14, and after equilibration at room temperature for 30 minutes, the above three indicators were tested again using the same procedure as on day 0.

[0131] Use the same batch of calibrators and quality control materials for each test to ensure consistent testing conditions.

[0132] Data processing and judgment Calculate the core metrics: Signal retention rate = (Absorbance at 3000 IU / mL on detection day / Absorbance on day 0) × 100% Background elevation rate = [(Detected Japanese baseline OD - Day 0 baseline OD) / Day 0 baseline OD] × 100% Acceptance criteria: Simultaneously meeting the following requirements: signal retention ≥90%, background elevation ≤10%, and CV ≤10% at 50 IU / mL.

[0133] The results are shown in Table 11 below.

[0134] Table 11

[0135] Therefore, the accelerated stability of the reagent of the present invention at 37°C is more than 5 times that of traditional BSA blocking reagents, which can significantly extend the shelf life of the reagent.

[0136] Bottle-opening stability (usage stability) Simulates clinical laboratory bottle opening scenarios; store at 2–8°C.

[0137] The experimental preparation, reagents and samples, data processing and judgment are exactly the same as those for the thermal stability experiment described above.

[0138] Pretreatment: Remove both groups of reagents simultaneously from the refrigerator at 2-8℃, allow them to equilibrate to room temperature for 30 minutes, unscrew the original bottle caps (keeping the caps loose, and use the appropriate instrument's sampling needle for puncture), and mark the group and the date of opening.

[0139] Experimental Procedure Day 0 baseline testing The instrument should be powered on and warmed up for 30 minutes to complete routine maintenance and blank testing. Place the two sets of opened reagent bottles, R1 and R2, directly into the corresponding refrigerated reagent compartments of the instrument, and do not remove them during the entire process. Calibrate using the matching calibrators (R² ≥ 0.999), and ensure high and low value quality control is within ±2 SD of the target value. The initial properties of the two sets of reagents were measured in parallel three times: ① Background absorbance (Add only R1+R2, without sample) ② 3000 IU / mL calibrator's reaction absorbance (defined as initial signal 100%) ③ Calculate the initial intra-batch CV after 10 consecutive tests with 50 IU / mL sample. Record all baseline data.

[0140] Simulated clinical continuous use The instrument is turned on and runs for 8 hours at a fixed time every day (simulating a clinical workday), and automatically completes routine operations such as sample addition and cleaning. After the instrument is turned off, the reagents should remain in the refrigerated reagent compartment without being removed or the caps tightened; keep them loose. Daily instrument maintenance was performed during the experiment to keep the reagent compartment clean and avoid cross-contamination.

[0141] Regular performance testing The above three indicators were tested again on days 7, 14, and 30 after opening, using the same instrument parameters, calibrators, and quality control materials as on day 0. Before each test, high and low value quality control was run to confirm that the instrument and reagents were under control. The same batch of calibrators and quality control materials were used throughout the process to eliminate batch-to-batch errors.

[0142] The results are shown in Table 12 below.

[0143] Table 12

[0144] Therefore, the reagent of this invention has more than 4 times the opening stability of traditional BSA-sealed reagents, which can significantly reduce clinical reagent waste.

[0145] Long-term stability (validity period verification) Store at 2–8℃ away from light, simulating normal storage conditions.

[0146] Experimental preparation Instruments and Environment Testing instrument: Hitachi 7180 Fully Automated Biochemical Analyzer Storage equipment: calibrated 2-8℃ refrigerator / freezer Environmental requirements: Laboratory temperature 20–25℃, relative humidity 40%–60%. Reagents and Samples Experimental group: 10 sets of the same batch of IgE reagent kits of this invention (R1+R2, original unopened and intact packaging) were prepared (2 sets of parallel samples at each time point). Control group: Traditional 2% BSA physical blocking reagent (original, unopened, same specification, formula, process, and production batch completely identical to the experimental group), 10 sets prepared in total. Matching calibrators and high / low value quality control samples: dispensed at -20℃ for single use only, avoiding repeated freeze-thaw cycles throughout the process, and using samples from the same batch. Standardized samples: 50 IU / mL low-value serum samples (aliped and stored at -20℃), 3000 IU / mL high-value calibrators. Preprocessing All reagents are simultaneously removed from the production line and immediately placed in a refrigerated box at 2–8°C. The group, production batch number, and experiment start date are marked. The reagents are stored in a sealed, light-proof container throughout the process. Opening or moving the container midway is prohibited. The temperature is continuously monitored by a temperature recorder to ensure that the temperature fluctuation does not exceed ±1°C.

[0147] Experimental Procedure Month 0 baseline test Take two sets of reagents from each of the experimental and control groups and allow them to equilibrate at room temperature for 30 minutes. Turn on the instrument for warm-up and calibration, ensuring the high and low absorbance values ​​are within ±2 SD of the target value. Perform three parallel tests and record the initial performance indicators: ① Background absorbance. (Add only R1+R2, without adding samples) ② 3000 IU / mL calibrator reaction absorbance (defined as 100% of the initial signal) ③ 50 IU / mL sample is tested 10 times consecutively, and the initial intra-batch CV is calculated; the average value of 2 sets of parallel reagents is taken as the baseline value.

[0148] Regular sampling and testing On fixed dates in months 3, 6, 12, and 18, two sets of unopened reagents were taken from both the experimental and control groups. The above three indicators were tested repeatedly using the same instrument parameters, operating procedures, and testing system as in month 0. Before each test, high and low value quality control was run to confirm that the instrument was under control. If the quality control was out of control, the instrument was recalibrated before testing. All tests were completed on the same day, and any remaining reagents were not returned to the refrigerator for further storage.

[0149] Data processing and judgment Calculate the core evaluation indicators: Signal retention rate = (Absorbance of 3000 IU / mL in the detection month / Absorbance in month 0) × 100% Background elevation rate = [(Base OD in the month of detection - Base OD in month 0) / Base OD in month 0] × 100% Pass / Fail Criteria: All three of the following requirements must be met simultaneously. Signal retention rate ≥90% at 3000 IU / mL; background absorbance increase ≤10%; intra-batch CV ≤10% at 50 IU / mL.

[0150] The results are shown in Table 13 below.

[0151] Table 13

[0152] Therefore, the reagent of the present invention has a shelf life of up to 12 months at 2-8°C, and up to 18 months at the longest, which is 2-3 times that of the traditional BSA blocking reagent (6 months).

[0153] Simulated transport stability Simulates road transport vibration and temperature fluctuations, cycling and vibrating for 72 hours at -10℃ to 40℃.

[0154] Experimental preparation Instruments and equipment Testing instrument: Hitachi 7180 Fully Automated Biochemical Analyzer Transportation simulation equipment: High and low temperature alternating test chamber (temperature control range -20℃~50℃), vertical reciprocating vibration table (frequency adjustable from 1~50Hz) Monitoring equipment: High-precision temperature recorder (accuracy ±0.1℃, recording once every 30 minutes) Auxiliary equipment: foam boxes for reagent transportation, shockproof bubble wrap Reagents and Samples Experimental group: 6 sets of the same batch of IgE reagent kits of this invention (R1+R2, original unopened and intact packaging). Control group: Traditional 2% BSA physical blocking reagent (original, unopened, same specification, formula, process, and batch number completely identical to the experimental group), 6 sets in total. Matching calibrators and high / low value quality control samples: repackaged at -20℃ for single use only, and all samples should be from the same batch. Fixed-value samples: 50 IU / mL low-value serum sample, 3000 IU / mL high-value calibrator Preprocessing Place the temperature recorder into the reagent packaging for the experimental and control groups respectively, ensuring that the probe is in close contact with the reagent bottle wall; pack all reagents according to the actual transportation method: place them in a special foam box, fill with shockproof bubble wrap, and seal the box lid; mark the group and the start date of the experiment, and calibrate all equipment parameters in advance.

[0155] Experimental Procedure Day 0 baseline testing Take out two sets of unpackaged reagents from each of the experimental and control groups, and allow them to equilibrate at room temperature for 30 minutes. After the instrument is turned on, preheated, and calibrated, and quality control is in place, perform three parallel tests on the initial performance: ① Background absorbance. ② 3000 IU / mL calibrator reaction absorbance (defined as 100% of the initial signal) ③ 50 IU / mL sample was tested 10 times consecutively, and the initial intra-batch CV was calculated; the average value was taken as the baseline value.

[0156] Transportation simulation processing The remaining 4 sets of experimental reagents and 4 sets of control reagents were divided into two groups: Simulated transport group: 2 sets of experimental groups + 2 sets of control groups, simultaneously placed in a high and low temperature alternating test chamber + vibration table. Normal storage control group: 2 sets of experimental group + 2 sets of control group, stored in a sealed refrigerator at 2-8℃. The simulation program was started, with temperature cycling and vibration occurring simultaneously, running continuously for 72 hours: Temperature cycling: -10℃ for 4 hours → increase temperature to 40℃ at 1℃ / min and hold for 4 hours → decrease temperature to -10℃ at 1℃ / min, repeat 9 times. Vibration parameters: frequency 20Hz, amplitude 1mm, vertical vibration, simulating the working conditions of a third-level highway. The temperature is continuously monitored by a temperature recorder to ensure that temperature fluctuations meet the set values.

[0157] Post-processing performance testing After the 72-hour simulation, all reagents were immediately removed and allowed to equilibrate at room temperature for 30 minutes. Using the same instrument parameters and operating procedures as on day 0, three performance indicators were tested simultaneously for both the simulated transport group and the normal storage control group. Before each test, high and low value quality control was run to confirm that the instrument was in control.

[0158] Data processing and judgment Calculate the core evaluation indicators: Signal retention rate = (Absorbance of simulated transport group 3000 IU / mL / Absorbance on day 0) × 100% Baseline rise rate = [(Simulated transport group baseline OD - Day 0 baseline OD) / Day 0 baseline OD] × 100% Between-group bias = |Simulated transport group measurement - Normal storage control group measurement| / Normal storage control group measurement × 100% Acceptance criteria: Simultaneously meet the following requirements: signal retention rate ≥90% at 3000 IU / mL; background absorbance increase ≤10%; intra-batch CV ≤10% at 50 IU / mL; and inter-group deviation ≤±5% from the normal storage control group.

[0159] The results are shown in Table 14 below.

[0160] Table 14

[0161] Therefore, the reagent of this invention can withstand conventional transportation conditions, does not require cold chain transportation, and greatly reduces logistics costs.

[0162] This invention completely solves the core problem of easy desorption in traditional BSA physical sealing through dual antigen covalent sealing technology, and improves thermal stability, opening stability, long-term stability and transportation stability, fully meeting the stringent requirements of clinical laboratories for reagent stability.

[0163] Comparative Example 1 100nm single particle size latex reagent Only the 100 nm carboxylated latex microspheres obtained in Example 1 were used, without adding 200 nm and 300 nm latex. All other processes, formulations, and detection parameters were exactly the same as the reagents of this invention. The results are shown in Table 15 below.

[0164] Table 15

[0165] As shown in the table above, a severe hook effect occurs at high concentrations, resulting in a narrow linearity and making it impossible to achieve a wide detection range of 3000 IU / mL.

[0166] Comparative Example 2 100nm and 200nm dual-particle-size latex reagent The 100 nm single-component latex reagent obtained in Example 1 and the 200 nm single-component latex reagent obtained in Example 2 were compounded at a volume ratio of 1:2 according to the method described in Example 4. No 300 nm latex was added. All other processes, formulations, and detection parameters were exactly the same as those of the reagent of the present invention. The results are shown in Table 16 below.

[0167] Table 16

[0168] The results above show that it is impossible to cover 3000 IU / mL, the high value drops back, and there is still a significant hook effect.

[0169] Comparative Example 3 Comparison of different proportions of three particle sizes Keeping the total latex content constant, the volume ratios of the 100 nm single-component latex reagent obtained in Example 1, the 200 nm single-component latex reagent obtained in Example 2, and the 300 nm single-component latex reagent obtained in Example 3 were adjusted. All other processes, formulations, and detection parameters were exactly the same as those of the reagent of this invention. The results are shown in Table 17 below.

[0170] Table 17

[0171] The results above show that when the three particle sizes of latex are compounded in a volume ratio of 1:2:2, they can simultaneously achieve comprehensive performance including low-value sensitivity, mid-range smoothness, high-value hook-free performance, and ultra-wide linearity.

[0172] Comparative Example 4 Comparison of sealing methods Four parallel control groups were set up, with identical latex particle size, antibody, buffer system, and detection method, differing only in the blocking method. The results are shown in Table 18 below.

[0173] Control 1: Traditional 2% BSA physical sealing for 1 h Control 2: Blocking with only carboxylated streptomycin Control 3: Blocking with only aminopyrine G This invention: Covalent blocking of carboxylated streptomycin-aminolated penicillin G dual antigens Table 18

[0174] The results above show that Control 1 and Control 2 have better blocking effects than traditional BSA, but still have problems such as insufficient stability and high interference. The carboxylated streptomycin-aminopenicillin G double antigen of the present invention forms a synergistic covalent backbone, which achieves significant performance improvement in terms of stability, anti-interference and precision.

[0175] Comparative Example 5 Control experiment with and without pSBMA zwitterion modification Under the premise of the same latex particle size ratio, the same dual-antigen covalent backbone blocking, the same antibody conjugation process, the same R1 / R2 buffer system, and the same detection parameters, only the two groups of ungrafted pSBMA and pSBMA-grafted were compared to verify the independent contribution of pSBMA zwitterionic modification to background absorbance, precision, anti-interference, thermal stability, and open-bottle stability. This proves that pSBMA is a necessary technical feature to achieve low background, strong anti-interference, and high stability.

[0176] Control group: Three-particle-size compound (100:200:300=1:2:2) + dual-antigen covalent backbone blocking, without pSBMA grafting modification. Experimental group: Three-particle-size compound (100:200:300=1:2:2) + dual-antigen covalent backbone blocking + pSBMA grafting modification Except for the pSBMA grafting step, all other raw materials, formulations, process parameters, reaction times, centrifugation speeds, stirring conditions, compounding processes, filtration and sterilization, and testing parameters were kept completely consistent. The results are shown in Table 19.

[0177] Table 19

[0178] While using a three-particle-size compound with dual antigen covalent blocking is superior to traditional BSA blocking reagents, it still has shortcomings: the reagent background is relatively high, the repeatability of low-value detection is poor, the anti-interference ability of samples with hemolysis, lipemia, jaundice and rheumatoid factor is average, the thermal stability, storage and open-bottle stability are still lacking, and there is still a slight hook effect in high-concentration samples.

[0179] The performance was improved after introducing pSBMA zwitterionic modification. pSBMA can form a hydrophilic protective hydration layer on the surface of latex microspheres, stabilizing the microsphere structure, inhibiting the non-specific adsorption of impurities and lipids, and maintaining the monodisperse state of the microspheres. Experimental results show that the addition of pSBMA significantly reduces reagent background, greatly improves low-value precision, enhances resistance to interference from various clinical matrices, significantly optimizes thermal stability and open-bottle stability, and provides a superior linear range, completely resolving the hook effect and false negative issues associated with ultra-high concentration samples.

[0180] In summary, the three core technologies of dual antigen covalent blocking, three-particle-size compounding, and pSBMA antifouling modification are indispensable. Neither of these three technologies alone can achieve the comprehensive performance of this invention, which features low background, high precision, wide linearity, strong anti-interference, and high stability.

[0181] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, the specification and drawings should be considered illustrative rather than restrictive.

Claims

1. An IgE latex-enhanced immunoturbidimetric reagent, characterized in that, Includes a first reagent, which comprises a mixture of a first latex microsphere, a second latex microsphere, and a third latex microsphere; The surfaces of the first, second, and third latex microspheres are all covalently coupled with anti-IgE antibodies, and the remaining active sites on the surface are all covalently grafted with carboxylated streptomycin and aminolated penicillin G as blocking agents. The first latex microspheres have a particle size of 80–120 nm, the second latex microspheres have a particle size of 160–240 nm, and the third latex microspheres have a particle size of 260–340 nm.

2. The IgE latex-enhanced immunoturbidimetric reagent according to claim 1, characterized in that, The molar ratio of carboxylated streptomycin to aminolated penicillin G is 1:0.5 to 1:2.0; the anti-IgE antibody is a mouse anti-human IgE monoclonal antibody.

3. The IgE latex-enhanced immunoturbidimetric reagent according to claim 1, characterized in that, The volume ratio of the first latex microsphere, the second latex microsphere, and the third latex microsphere is 1:1.5-2.5:1.5-2.

5.

4. The IgE latex-enhanced immunoturbidimetric reagent according to claim 1, characterized in that, Each latex microsphere is a carboxyl latex microsphere with a solid content of 5%, a particle size CV of <5%, and a surface carboxyl density of 80–120 μmol / g.

5. The IgE latex-enhanced immunoturbidimetric reagent according to claim 1, characterized in that, After sealing, the surface of each latex microsphere is activated by EDC and NHS and covalently grafted with polysulfobetaine methacrylate, with a final graft concentration of 4-6 mg / mL.

6. The IgE latex-enhanced immunoturbidimetric reagent according to claim 1, characterized in that, The reagents include a second reagent, which includes Tris-HCl, NaCl, Tween-20, EDTA•2Na, HBR-3 heterophile antibody blocking agent, sodium caseinate, and Proclin 300 preservative.

7. The IgE latex-enhanced immunoturbidimetric reagent according to claim 1, characterized in that, The first reagent includes Tris-HCl, NaCl, trehalose, glycerol, and BSA protectant.

8. A method for preparing an IgE latex-enhanced immunoturbidimetric reagent according to any one of claims 1 to 7, characterized in that, Three types of carboxylated latex microspheres with different particle sizes were modified to prepare first latex microspheres, second latex microspheres, and third latex microspheres. The first latex microspheres, second latex microspheres, and third latex microspheres were then compounded to obtain the first reagent. The modification method includes the following steps: (1) Wash the carboxylated latex microspheres and add EDC and NHS to carry out the carboxyl activation reaction; (2) Add ethanolamine to quench any remaining unreacted activated carboxyl groups; (3) Add anti-IgE antibody and carry out covalent coupling reaction; (4) Add carboxylated streptomycin derivative and aminolated penicillin G derivative to carry out covalent blocking reaction; (5) Add polysulfobetaine methacrylate, along with EDC and NHS, to carry out zwitterionic grafting reaction.

9. The preparation method according to claim 8, characterized in that, In step (3), the mass ratio of anti-IgE antibody to latex microspheres is 1:18-22.

10. The preparation method according to claim 8, characterized in that, In step (4), the pH is 6.0 to 7.0, the reaction temperature is 20 to 30°C, and the reaction time is 1 to 3 h.

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