Allergen specificity IgG4 antibody magnetic particle chemiluminescence detection kit

Abstract

The invention relates to an allergen specificity IgG4 antibody magnetic particle chemiluminescence detection kit, which comprises a reagent 1, a reagent 2, a reagent 3, a reagent 4 and a luminescent substrate solution, the first buffer solution component comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, glycosylated bovine serum albumin, glycerol and glycine methyl ester; the reagent 2 is a biotin-labeled allergen solution; the reagent 3 comprises an alkaline phosphatase labeled anti-human IgG4 antibody and a third buffer solution; the third buffer solution comprises MES, sodium chloride, bovine serum albumin, magnesium chloride, zinc chloride and PEG 8400; the reagent 4 comprises a biotin-labeled anti-human IgG4 antibody and a fourth buffer solution, the fourth buffer solution comprises MES, sodium chloride, bovine serum albumin and glycerin, and the kit has excellent accuracy, specificity, stability and repeatability.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagents, specifically relating to an allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit. Background Technology

[0002] The incidence of allergic diseases is on the rise globally. Against this backdrop, specific immunotherapy (AIT) has become an important treatment option for sensitized patients because it is currently the only treatment that can intervene in the disease process.

[0003] In the efficacy assessment of AIT (allergen-specific immunotherapy), elevated serum allergen-specific IgG4 (sIgG4) levels are considered a key biological indicator. Studies have confirmed that increased sIgG4 levels are significantly correlated with improvement in clinical symptoms, and this trend can be observed early in treatment, suggesting that sIgG4 plays a central role in the early stages of inducing immune tolerance. Its mechanism of action lies in the fact that sIgG4, as a functional "blocking antibody," competitively inhibits the binding of allergens to specific IgE bound to effector cells (such as mast cells and basophils), thereby effectively preventing the release of allergic mediators and reducing immediate anaphylactic reactions. Long-term follow-up data further demonstrate that successful AIT can guide the immune response towards a tolerance state, manifested by a decrease in specific IgE (sIgE) levels and a sustained and significant increase in sIgG4 levels, which reinforces the important role of sIgG4 in maintaining the long-term efficacy of AIT.

[0004] Currently, the main detection kits used for detecting serum allergen-specific IgG4 are enzyme-linked immunosorbent assay (ELISA) kits. The advantages of ELISA IgG4 detection kits lie primarily in their cost-effectiveness, universality, and flexibility, making them a practical tool for non-emergency applications with moderate sample sizes and less stringent speed requirements. However, when faced with the demands of high-throughput, rapid reporting, and high-precision standardization in clinical settings, their shortcomings (including poor repeatability, low automation, long processing time, limited linear range, and unsatisfactory accuracy and specificity) become apparent, severely limiting their practical application in precision medicine. In such cases, new technologies such as fully automated chemiluminescence immunoassay become more advantageous.

[0005] Magnetic microparticle chemiluminescence immunoassay (CLIA) is a novel in vitro diagnostic technique based on the principle of immunoreaction, combining magnetic microparticle separation with chemiluminescence detection. This method achieves rapid and efficient separation and enrichment of antigens or antibodies by coating them onto the surface of magnetic microparticles under a magnetic field; simultaneously, a chemiluminescence labeling system amplifies the signal and enables quantitative analysis. Although nanoparticle chemiluminescence immunoassay is a mature technology, commercially available allergen-specific IgG4 antibody nanoparticle chemiluminescence assay kits are not yet widely available. The core limiting factor lies in the multiple high hurdles in the productization process: kit development involves the fine optimization and system matching of multiple key components, including streptavidin magnetic bead screening and formulation optimization, allergen optimization, and antibody optimization and labeling of enzyme conjugates. Deficiencies in any of these aspects can affect the sensitivity, accuracy, and specificity of the detection; furthermore, the long-term stability of each component within its shelf life also faces significant challenges. These technical difficulties collectively lead to long development cycles and high costs, thus greatly limiting the commercialization of this type of kit. Summary of the Invention

[0006] The technical problem solved by this invention is to overcome the shortcomings of the prior art and provide an allergen-specific IgG4 antibody nanoparticle chemiluminescence detection kit with high sensitivity, wide linear range, strong anti-interference ability, excellent repeatability and high accuracy, excellent specificity and good stability.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The first aspect of the present invention provides an allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit, characterized in that it comprises reagent 1, reagent 2, reagent 3, reagent 4 and luminescent substrate solution.

[0008] Reagent 1 is a streptavidin-labeled magnetic nanoparticle suspension, comprising streptavidin-labeled magnetic nanoparticles and a first buffer solution. The first buffer solution is formulated as follows: 5-10 mM disodium hydrogen phosphate, 2-4 mM sodium dihydrogen phosphate, 0.8%-1.2% (w / v) sodium chloride, 0.8%-1.2% (w / v) glycosylated bovine serum albumin (GSA), 0.8%-1.2% (v / v) glycerol, 0.08%-0.12% (v / v) nonionic detergent, 0.03%-0.08% (v / v) preservative, 0.1-0.3 M glycine methyl ester, with the balance being water, and a pH of 7.2-7.6.

[0009] Reagent 2 is a biotin-labeled allergen solution, comprising a biotin-labeled allergen and a second buffer solution. The formulation of the second buffer solution is: 5-10 mM disodium hydrogen phosphate, 2-4 mM sodium dihydrogen phosphate, 0.8%-1.2% (w / v) sodium chloride, 0.001%-0.003% (w / v) 4-aminoantipyrrolidone, 0.8%-1.2% (v / v) glycerol, 0.8%-1.2% (w / v) BSA, 0.01%-0.02% (v / v) nonionic detergent, 0.08%-0.12% (v / v) preservative, with the balance being water, and a pH of 7.2-7.6.

[0010] Reagent 3 is an alkaline phosphatase-labeled anti-human IgG4 antibody solution, comprising alkaline phosphatase-labeled anti-human IgG4 antibody and a third buffer solution. The third buffer solution is formulated as follows: 40-60 mM MES, 0.8%-1.2% (w / v) sodium chloride, 0.8%-1.2% (w / v) bovine serum albumin, 4-8 mM magnesium chloride, 0.08-0.12 mM zinc chloride, 0.8%-1.2% (w / v) PEG8400, 0.8%-1.2% (v / v) nonionic detergent, 0.03%-0.06% (v / v) preservative, with the balance being water, and a pH of 5.8-6.2.

[0011] Reagent 4 is a biotin-labeled anti-human IgG4 antibody solution, which includes biotin-labeled anti-human IgG4 antibody and a fourth buffer solution. The fourth buffer solution has the following formulation: 40~60mM MES, 0.8%~1.2% (w / v) sodium chloride, 0.8%~1.2% (w / v) bovine serum albumin, 0.8%~1.2% (v / v) glycerol, 0.08%~0.12% (v / v) nonionic detergent, 0.03%~0.08% (v / v) preservative, and the balance being water, with a pH of 5.8~6.2.

[0012] In some embodiments of the present invention, the alkaline phosphatase-labeled anti-human IgG4 antibody in reagent 3 is obtained by reacting alkaline phosphatase with anti-human IgG4 antibody purified by an IgG1 adsorption column and / or an IgE adsorption column. The packing material of the IgG1 adsorption column is AminoLink Plus coupling resin conjugated with recombinant human IgG1 Fc fragments, and the packing material of the IgE adsorption column is AminoLink Plus coupling resin conjugated with recombinant human IgE Fc fragments.

[0013] In some embodiments of the present invention, the anti-human IgG4 antibody is first purified by the IgG1 adsorption column and then purified by the IgE adsorption column.

[0014] In some embodiments of the present invention, the concentration of reagent 1 is 0.1~0.5 mg / mL, for example 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, or 0.5 mg / mL.

[0015] In some embodiments of the present invention, the concentration of reagent 2 is 0.5~10 μg / mL, for example 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL.

[0016] In some embodiments of the present invention, the concentration of reagent 3 is 0.1~0.5 μg / mL, for example 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, or 0.5 μg / mL.

[0017] In some embodiments of the present invention, the concentration of reagent 4 is 0.08~0.2 μg / mL, for example 0.08 μg / mL, 0.09 μg / mL, 0.1 μg / mL, 0.11 μg / mL, 0.12 μg / mL, 0.13 μg / mL, 0.14 μg / mL, 0.15 μg / mL, 0.16 μg / mL, 0.17 μg / mL, 0.18 μg / mL, 0.19 μg / mL, or 0.2 μg / mL.

[0018] In some embodiments of the present invention, the nonionic detergents in the first buffer solution, the second buffer solution, the third buffer solution, and the fourth buffer solution are one or more of Triton X-100, Tween-20, Tween-80, Brij35, and Pluronic F-68, respectively.

[0019] In some embodiments of the present invention, the preservatives in the first buffer, the second buffer, the third buffer, and the fourth buffer are one or more of Proclin-200, Proclin-300, and Proclin-950, respectively.

[0020] In some embodiments of the present invention, the luminescent substrate in the luminescent substrate solution is AMPPD.

[0021] In some embodiments of the present invention, it further includes one or more of sample diluent, human IgG4 standard and / or standard diluent.

[0022] The sample diluent was formulated as follows: 5-10 mM disodium hydrogen phosphate, 2-4 mM sodium dihydrogen phosphate, 0.8%-1.2% (w / v) sodium chloride, 0.8%-1.2% (w / v) bovine serum albumin, 0.1% (v / v) nonionic detergent, 0.05% (v / v) preservative, with the balance being water, and a pH of 7.2-7.6.

[0023] The standard diluent is formulated as follows: 40-60 mM tris(hydroxymethyl)aminomethane, 400-600 mM sodium chloride, 0.8%-1.2% (w / v) bovine serum albumin, 0.08%-0.12% (v / v) nonionic detergent, 0.1%-0.3% (w / v) polyoxyethylene lauryl ether, 0.03%-0.06% (v / v) preservative, with the balance being water, pH 7-7.5.

[0024] Furthermore, the nonionic detergents in the sample diluent and / or the standard diluent are one or more of Triton X-100, Tween-20, Tween-80, Brij 35, and Pluronic F-68.

[0025] Furthermore, the preservatives in the sample diluent and / or the standard diluent are one or more of Proclin-200, Proclin-300, and Proclin-950, respectively.

[0026] In some embodiments of the present invention, the allergen is selected from the following allergens: house dust mite allergen, dry dust mite allergen, canine epidermal allergen, artemisia allergen, common ragweed allergen, house dust allergen, cockroach allergen, willow allergen, Alternaria allergen, milk allergen, cod allergen, wheat flour allergen, peanut allergen, soybean allergen, shrimp allergen, beef allergen, mutton allergen, egg allergen, crab allergen, cat epidermal allergen, hop allergen, common birch allergen, sycamore allergen, poplar allergen, cypress allergen, pine allergen, Penicillium allergen, Cladosporium allergen, Aspergillus fumigatus allergen, Candida albicans allergen, Rhizopus nigricans allergen, hazelnut allergen, almond allergen, cashew allergen, pistachio allergen, strawberry allergen, apple allergen, mango allergen, peach allergen, pineapple allergen, and scallop allergen.

[0027] A second aspect of the present invention provides an allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection system, which includes the allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit and the fully automated chemiluminescence analyzer described above.

[0028] Compared with the prior art, the present invention has the following advantages: The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit of the present invention achieves ultra-high sensitivity based on the synergistic effect of nano-magnetic microparticle solid-phase carrier, biotin-streptavidin bridging, and ultra-sensitive chemiluminescent substrate. Through optimization of each reagent component, its excellent accuracy, specificity, stability, and repeatability are ensured.

[0029] The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit of the present invention can overcome the shortcomings of the traditional ELISA method, enabling allergen-specific IgG4 to serve as an objective and reliable biomarker for guiding desensitization treatment and efficacy monitoring of allergic diseases, and truly possessing a realistic basis for large-scale clinical application. Attached Figure Description

[0030] Figure 1 The figure shows a comparison of the methodological results of the kit in Example 1 and Phadia's Immuno CAP for detecting house dust mite-specific IgG4. Figure 2 Linear fitting curve for detecting house dust mite-specific IgG4 using the kit from Example 1. Detailed Implementation

[0031] To develop a high-performance allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit, the applicant conducted extensive research on each component of the kit and verified and compared the detection effects of the combined components, ultimately obtaining the technical solution of this application, as follows: The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit of this application is characterized in that it includes reagent 1, reagent 2, reagent 3, reagent 4 and luminescent substrate solution.

[0032] Reagent 1 is a streptavidin-labeled magnetic nanoparticle suspension, which includes streptavidin-labeled magnetic nanoparticles and a first buffer solution. The first buffer solution has the following formulation: 5-10 mM disodium hydrogen phosphate, 2-4 mM sodium dihydrogen phosphate, 0.8%-1.2% (w / v) sodium chloride, 0.8%-1.2% (w / v) glycosylated bovine serum albumin, 0.8%-1.2% (v / v) glycerol, 0.08%-0.12% (v / v) nonionic detergent, 0.03%-0.08% (v / v) preservative, 0.1-0.3 M glycine methyl ester, and the balance being water, with a pH of 7.2-7.6.

[0033] Reagent 2 is a biotin-labeled allergen solution, which includes a biotin-labeled allergen and a second buffer solution. The formulation of the second buffer solution is as follows: 5-10 mM disodium hydrogen phosphate, 2-4 mM sodium dihydrogen phosphate, 0.8%-1.2% (w / v) sodium chloride, 0.001%-0.003% (w / v) 4-aminoantipyrrolidone, 0.8%-1.2% (v / v) glycerol, 0.8%-1.2% (w / v) BSA, 0.01%-0.02% (v / v) Triton X-100, 0.08%-0.12% (v / v) Proclin-300, with the balance being water, and the pH is 7.2-7.6.

[0034] Reagent 3 is an alkaline phosphatase-labeled anti-human IgG4 antibody solution, comprising alkaline phosphatase-labeled anti-human IgG4 antibody and a third buffer solution. The third buffer solution is formulated as follows: 40-60 mM MES, 0.8%-1.2% (w / v) sodium chloride, 0.8%-1.2% (w / v) bovine serum albumin, 4-8 mM magnesium chloride, 0.08-0.12 mM zinc chloride, 0.8%-1.2% (w / v) PEG8400, 0.8%-1.2% (v / v) nonionic detergent, 0.03%-0.06% (v / v) preservative, with the balance being water, and a pH of 5.8-6.2.

[0035] Reagent 4 is a biotin-labeled anti-human IgG4 antibody solution, which includes biotin-labeled anti-human IgG4 antibody and a fourth buffer solution. The fourth buffer solution is formulated as follows: 40~60mM MES, 0.8%~1.2% (w / v) sodium chloride, 0.8%~1.2% (w / v) bovine serum albumin, 0.8%~1.2% (v / v) glycerol, 0.08%~0.12% (v / v) nonionic detergent, 0.03%~0.08% (v / v) preservative, and the balance being water, with a pH of 5.8~6.2.

[0036] This application achieves ultra-high sensitivity through a three-stage signal amplification system: the synergistic effect between nano-magnetic microparticle solid-phase carrier, biotin-streptavidin bridging, and ultra-sensitive chemiluminescent substrate; it eliminates cross-interference by purifying anti-human IgG4 antibodies through cross-adsorption technology; and it improves stability through the design of various reagent buffer systems, thereby ensuring the accuracy, repeatability, and long-term storage effect of the detection results.

[0037] The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit of this application enables precise quantification of allergen-specific IgG4; its rapid detection characteristics support dynamic monitoring of changes in immune response during desensitization therapy. By establishing a standardized efficacy evaluation system, it can provide objective evidence for adjusting individualized treatment plans in clinical practice, effectively addressing the industry pain points of traditional methods being unable to continuously track treatment effects and guide precise medication, significantly improving the scientific rigor and standardization of allergic disease diagnosis and treatment, and has important practical significance.

[0038] The present invention will be further described below with reference to embodiments and comparative examples. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0039] In the following examples and comparative examples, the implementation conditions not specified are standard conditions in the industry, and the reagents not specified are all commercially available products.

[0040] In the following examples and comparative examples, the water used was laboratory-prepared deionized water.

[0041] In the following examples and comparative examples, the temperature ranged from room temperature to 20±5°C.

[0042] In the following examples and comparative examples, the house dust mite allergen-specific IgG4 antibody detection kit is used as an example. In other examples, other allergens can be selected as needed to prepare other allergen-specific IgG4 antibody detection kits. Only the allergen in reagent 2 needs to be replaced. All other reagents except reagent 2 are universal.

[0043] Example 1: This example provides an allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit, which includes reagent 1, reagent 2, reagent 3, reagent 4, luminescent substrate solution, sample diluent, and standard.

[0044] Reagent 1 is a 0.4 mg / mL streptavidin-labeled magnetic nanoparticle suspension, which is prepared as follows: After mixing 10 mg / mL of streptavidin-labeled magnetic nanoparticles (1 µm in diameter), magnetic separation was performed. The mixture was resuspended in the first buffer solution, washed three times, and magnetically separated again. Finally, it was resuspended in the first buffer solution to obtain reagent 1. The first buffer solution was formulated as follows: 7.2 mM disodium hydrogen phosphate dodecahydrate, 2.8 mM sodium dihydrogen phosphate dihydrate, 0.9% (w / v) sodium chloride, 1% (w / v) glycosylated BSA, 1.0% (v / v) glycerol, 0.1% (v / v) Triton X-100, 0.05% (v / v) Proclin-300, 0.2 M glycine methyl ester, with the balance being water, pH 7.4.

[0045] Reagent 2 is a 1 μg / mL biotin-labeled house dust mite allergen, which includes house dust mite allergen (D1-biotin, purchased from Suzhou Xiruimas Co., Ltd., catalog number MW00325) and a second buffer. The second buffer has the following formulation: 7.2 mM disodium hydrogen phosphate dodecahydrate, 2.8 mM sodium dihydrogen phosphate dihydrate, 0.9% (w / v) sodium chloride, 0.002% (w / v) 4-aminoantipyrrolidone, 1.0% (v / v) glycerol, 1% (w / v) BSA, 0.01% (v / v) Triton X-100, 0.1% (v / v) Proclin-300, and the balance being water, pH 7.4.

[0046] Reagent 3 is a 0.2 μg / mL alkaline phosphatase-labeled anti-human IgG4 antibody solution, and its preparation method is as follows: (1) Purification of anti-human IgG4 antibody: Preparation of IgG1 adsorption column: 5 mg of recombinant human IgG1 Fc fragment (Suzhou Xiruimas Co., Ltd., catalog number CRMGP01) was dissolved in 5 mL of coupling buffer (0.1 M NaHCO3 aqueous solution, pH 8.3), mixed with 1 mL of AminoLink Plus coupling resin (Thermo Fisher Scientific), and incubated overnight at 4°C by rotation; 0.1 M Tris-HCl (pH 8.0) was added for blocking for 1 hour; the AminoLink Plus coupling resin conjugated with the recombinant human IgG1 Fc fragment was loaded into a Tricorn chromatography column (5 × 1 cm), which was designated as the IgG1 adsorption column.

[0047] Preparation of IgE adsorption column: 5 mg of recombinant human IgE Fc fragment (Suzhou Xiruimas Co., Ltd., catalog number CRMEP01) was dissolved in 5 mL of coupling buffer (0.1 M NaHCO3 aqueous solution, pH 8.3), mixed with 1 mL of AminoLink Plus coupling resin (Thermo Fisher Scientific), and incubated overnight at 4 °C by rotation; 0.1 M Tris-HCl (pH 8.0) was added for blocking for 1 hour; the AminoLink Plus coupling resin conjugated with the recombinant human IgE Fc fragment was packed into a Tricorn chromatography column (5 × 1 cm), denoted as IgE adsorption column.

[0048] Initial adsorption (removal of IgG1 cross-reactivity): Equilibrate the column: Wash the IgG1 adsorption column with 5 column volumes of PBS buffer at a flow rate of 0.5 mL / min; Loading the antibody to be purified: Load the anti-human IgG4 antibody (Southern Biotech, catalog number 9200-01) (approximately 5 mg / mL) onto the IgG1 adsorption column at a flow rate of 0.3 mL / min; Collect the breakthrough peak: This part is the antibody component that has not bound IgG1; Regenerating the column: Elute the conjugated proteins with 3 column volumes of elution buffer (glycine-HCl pH 2.5) and equilibrate to baseline with PBS.

[0049] Secondary adsorption (removal of IgE cross-reaction): The breakthrough peak product collected in the initial adsorption step was loaded into the IgE adsorption column using a pulse loading method, with 1 / 3 of the sample volume loaded each time. The breakthrough peak was then collected, which is the purified anti-human IgG4 antibody.

[0050] All operations were performed at 4°C to prevent antibody denaturation and to avoid air bubbles entering the chromatography column.

[0051] (2) Alkaline phosphatase-labeled purified anti-human IgG4 antibody: Antibody pretreatment: The purified anti-human IgG4 antibody was placed in a dialysis bag and dialyzed overnight with PBS buffer (pH 7.4) to remove residual ammonium ions. The antibody was then concentrated to 5 mg / mL by ultrafiltration for later use.

[0052] Enzyme labeling reaction: The purified anti-human IgG4 antibody and alkaline phosphatase (ALP) were mixed in a ratio of 1:2 (mass ratio), and a cross-linking agent (0.1% glutaraldehyde) was added. The mixture was stirred at room temperature in the dark for 2 hours. Glycine (final concentration 0.01M) was added to terminate the cross-linking, and stirring was continued for 30 minutes.

[0053] Purification and collection: The reaction solution was passed through a pre-equilibrated Sephadex G-25 column (PBS equilibrated), and the first elution peak (containing the labeled product) was collected. The mixture was then concentrated by dialyzing with PBS buffer (pH 7.4) containing 1% BSA to obtain alkaline phosphatase-labeled anti-human IgG4 antibody (1 mg / mL) to remove free enzymes and small molecule impurities.

[0054] Cross-reactivity verification: Indirect ELISA method was used for verification. Antigen coating: ELISA plates were coated with human IgG1, IgE, and IgG4 standards (1 μg / mL), respectively. 0.2 μg / mL of anti-human IgG4 antibody containing both purified and unpurified alkaline phosphatase-conjugated antibodies was added, and the plates were incubated at room temperature for 30 min. Then, TMB single-component substrate solution (Jiangsu Haooubo, catalog number MC00130) was added, and the plates were incubated for 10 min. The readings were then taken at 450 nm using a Thermo Multiskan MK3 microplate reader (serial number: 3530912882) (reference wavelength 620 nm or 630 nm). The verification results are shown in Table 1.

[0055] (3) Preparation of reagent 3: The alkaline phosphatase-labeled anti-human IgG4 antibody obtained in step (2) was dialyzed and diluted to 0.2 μg / mL using the third buffer to obtain reagent 3. The third buffer solution is formulated as follows: 50 mM MES, 0.9% (w / v) sodium chloride, 1% (w / v) bovine serum albumin, 5 mM magnesium chloride, 0.1 mM zinc chloride, 1% (w / v) PEG 8400, 0.1% (v / v) Triton X-100, 0.05% (v / v) Proclin-30, with the remainder being water, pH 6.0.

[0056] Reagent 4 is a 0.1 μg / mL biotin-labeled anti-human IgG4 antibody solution, prepared by adding anti-human IgE antibody to Sulfo-NHS-LC-biotin dissolved in dimethylformamide, followed by dialysis with buffer IV to obtain Reagent 4. The fourth buffer solution is formulated as follows: 50 mM MES, 0.9% (w / v) sodium chloride, 1% (w / v) bovine serum albumin, 1.0% (v / v) glycerol, 0.1% (v / v) Triton X-100, 0.05% (v / v) Proclin-300, with the balance being water, pH 6.0.

[0057] Luminescent substrate solution: Alkaline phosphatase-catalyzed luminescent substrate based on AMPPD luminescence (substrate solution for immunoassay system, registration number: Susu Medical Device Registration No. 20141037, product number: MY00073), see invention patent CN104990912B for details.

[0058] The sample diluent was formulated as follows: 7.2 mM disodium hydrogen phosphate dodecahydrate, 2.8 mM sodium dihydrogen phosphate dihydrate, 0.9% (w / v) sodium chloride, 1% (w / v) bovine serum albumin, 0.1% (v / v) Triton X-100, 0.05% (v / v) Proclin-300, with the remainder being water, pH 7.4.

[0059] Standards: Standards with concentrations of 0, 2.5, 12.5, 50, 200, and 500 μg / L were prepared using standard diluent and purchased human IgG4 standard (manufacturer: Abcam, UK, catalog number: ab316091). The standard diluent was formulated as follows: 50 mM tris(hydroxymethyl)aminomethane, 500 mM sodium chloride, 1% (w / v) bovine serum albumin, 0.1% (v / v) Triton X-100, 0.2% (w / v) polyoxyethylene lauryl ether (Brij-35), 0.05% (v / v) Proclin-300, and the balance being water, pH 7.2.

[0060] The reagent kit operation steps are as follows: (1) Mix 30µL of standard, 50µL of streptavidin-labeled magnetic nanoparticle suspension (reagent 1), and 15µL of biotin-labeled anti-human IgG4 antibody (reagent 4), incubate at 37℃ for 15min, remove the supernatant by magnetic separation, and wash three times. (2) Add 135µL of alkaline phosphatase-labeled anti-human IgG4 antibody (reagent 3), mix, incubate at 37℃ for 15min, remove the supernatant by magnetic separation, and wash three times; (3) Add 15µL of luminescent substrate, incubate at 37℃ for 5min, measure the chemiluminescence intensity, and plot a standard curve with the concentration of the standard as the x-axis and the chemiluminescence intensity as the y-axis. (4) Selectively dilute the test sample using sample diluent. Take 30µL of diluted or undiluted test sample and mix it with 50µL of streptavidin-labeled magnetic nanoparticle suspension (reagent 1) and 15µL of biotin-labeled anti-human IgG4 antibody (reagent 4). Incubate at 37℃ for 15min, remove the supernatant by magnetic separation, and wash three times. (5) Add 135µL of alkaline phosphatase-labeled anti-human IgG4 antibody (reagent 3), mix, incubate at 37℃ for 15min, remove the supernatant by magnetic separation, and wash three times; (6) Add 15µL of luminescent substrate, incubate at 37℃ for 5min, measure the chemiluminescence intensity, substitute into the standard curve, and calculate the concentration of allergen-specific IgG4 antibody in the sample to be tested.

[0061] The standard curve can be recorded in the instruction manual, and steps (1) to (3) can be omitted as needed when using it later.

[0062] Alternatively, the SMART 6500 fully automated chemiluminescence analyzer manufactured by Chongqing Cosma Biotechnology Co., Ltd. can be used. The reagent kit is configured to be compatible with the SMART 6500 fully automated chemiluminescence analyzer. Labels containing corresponding information are placed on the reagent kit and each reagent tube. The automated detection method is as follows: 1. Place the reagent kit into the corresponding position in the reagent compartment of the fully automated chemiluminescence immunoassay analyzer. The reagent kit information can be entered into the instrument system via a barcode scanner or set through the instrument's software.

[0063] 2. Identify calibration and quality control information by scanning the master calibration curve card / calibration card / quality control card, and assign the location of the calibration / quality control to the instrument system.

[0064] 3. Place the sample to be tested in the instrument's sample compartment and edit the corresponding test information using the instrument's software.

[0065] 4. Start the program, and all calibrator / quality control / sample processing steps will be executed automatically.

[0066] Calibration procedure Calibration is performed using the IgG4 antibody calibrator from the kit. By measuring the high and low calibrators, each calibration point on the predefined master curve is adjusted (recalibrated) to a new, instrument-specific measurement level, i.e., the working curve.

[0067] Comparative Example 1: This comparative example provides another allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit, which includes reagent 1, reagent 2, reagent 3, reagent 4, luminescent substrate, sample diluent and standard. All reagents in this comparative example except for reagent 1 are the same as those in Example 1.

[0068] In this comparative example, Reagent 1 is a 0.4 mg / mL streptavidin-labeled magnetic nanoparticle suspension. The difference between Reagent 1 in Example 1 and Reagent 1 is the formulation of the first buffer solution. The formulation of the first buffer solution for Reagent 1 in this comparative example is: 7.2 mM disodium hydrogen phosphate dodecahydrate, 2.8 mM sodium dihydrogen phosphate dihydrate, 0.9% (w / v) sodium chloride, 1% (w / v) BSA, 1.0% (v / v) glycerol, 0.1% (v / v) Triton X-100, 0.05% (v / v) Proclin-300, with the balance being water, pH 7.4.

[0069] Comparative Example 2: This comparative example provides another allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit, which includes reagent 1, reagent 2, reagent 3, reagent 4, luminescent substrate, sample diluent and standard. All reagents in this comparative example except for reagent 1 are the same as those in Example 1.

[0070] In this comparative example, Reagent 1 is a 0.4 mg / mL streptavidin-labeled magnetic nanoparticle suspension. The difference between Reagent 1 in Example 1 and Reagent 1 is the formulation of the first buffer solution. The formulation of the first buffer solution for Reagent 1 in this comparative example is: 7.2 mM disodium hydrogen phosphate dodecahydrate, 2.8 mM sodium dihydrogen phosphate dihydrate, 0.9% (w / v) sodium chloride, 1% (w / v) glycosylated BSA, 1.0% (v / v) glycerol, 0.1% (v / v) Triton X-100, 0.05% (v / v) Proclin-300, with the balance being water, pH 7.4.

[0071] Comparison of detection performance of the kits in Example 1, Comparative Example 1, and Comparative Example 2: The kits used in Example 1, Comparative Example 1, and Comparative Example 2 were used to quantitatively detect the content of house dust mite allergen-specific IgG4 in clinical serum samples, including 5 positive samples (samples P1~P4), 10 negative samples (samples N1~N10), and non-specific samples. The software automatically generated the corresponding luminescence value and calculated concentration value for each sample. A detection concentration value ≤2.5 μg / L was interpreted as negative, and a detection concentration value >2.5 μg / L was interpreted as positive. The results were compared with those of the Immuno CAP test from Phadia, and the results are shown in Table 2.

[0072]

[0073] Table 2 shows that the kit from Example 1 had the best accuracy in detecting the results.

[0074] Interference detection of non-specific reactions: Five clinical bed serum samples were taken and the content of house dust mite allergen-specific IgG4 was detected using the kits of Example 1, Comparative Example 1, and Comparative Example 2. Reagent 2 was not added during the test, and the other operations remained unchanged. The test results are shown in Table 3.

[0075]

[0076] Table 3 shows that the kit of Example 1 does not have nonspecific reaction interference.

[0077] Comparative Example 3: This comparative example provides another allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit, which includes reagent 1, reagent 2, reagent 3, reagent 4, luminescent substrate solution, sample diluent and standard. All reagents in this comparative example except for reagent 3 are the same as those in Example 1.

[0078] In this comparative example, reagent 3 is a 0.2 μg / mL alkaline phosphatase-labeled anti-human IgG4 antibody solution. The difference between reagent 3 and reagent 3 in Example 1 is that it has undergone affinity purification with human IgG1 and human IgE purification columns. In the purification of anti-human IgG4 antibody, CNBr activated agarose resin (Thermo Fisher Scientific) was used instead of AminoLink Plus coupling resin. The purification process adopted pulse loading, with three loadings, each time loading 1 / 3 of the sample.

[0079] Comparison of detection performance between the kits in Example 1 and Comparative Example 3: The kits used in Example 1 and Comparative Example 3 were used to quantitatively detect IgE-positive and IgG4-negative samples 1-3, IgG4-positive and IgE-negative samples 1-5, and IgG4-negative and IgE-negative samples 1-3. The results were compared with those from the Immuno CAP assay of Phadia. The results are shown in Table 4. All tested samples were clinical serum samples.

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[0081] Comparative Example 4: This comparative example provides another allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit, which includes reagent 1, reagent 2, reagent 3, reagent 4, luminescent substrate solution, sample diluent and standard. All reagents in this comparative example except for reagent 3 are the same as those in Example 1.

[0082] In this comparative example, reagent 3 is a 0.2 μg / mL alkaline phosphatase-labeled anti-human IgG4 antibody solution. The difference between reagent 3 and reagent 3 in Example 1 is that the formulation of the third buffer used in preparation is different. The formulation of the third buffer used in this comparative example is: 50 mM MES, 0.9% (w / v) sodium chloride, 1% (w / v) bovine serum albumin, 0.1% (v / v) Triton X-100, 0.05% (v / v) Proclin-30, and the balance being water, pH 8.0.

[0083] Comparative Example 5: This comparative example provides another allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit, which includes reagent 1, reagent 2, reagent 3, reagent 4, luminescent substrate solution, sample diluent and standard. All reagents in this comparative example except for reagent 3 are the same as those in Example 1.

[0084] In this comparative example, reagent 3 is a 0.2 μg / mL alkaline phosphatase-labeled anti-human IgG4 antibody solution. The difference between reagent 3 and reagent 3 in Example 1 is that the formulation of the third buffer used in preparation is different. The formulation of the third buffer used in this comparative example is: 50 mM MES, 0.9% (w / v) sodium chloride, 1% (w / v) bovine serum albumin, 0.1% (v / v) Triton X-100, 0.05% (v / v) Proclin-30, and the balance being water, pH 6.0.

[0085] Comparative Example 6: This comparative example provides another allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit, which includes reagent 1, reagent 2, reagent 3, reagent 4, luminescent substrate solution, sample diluent and standard. All reagents in this comparative example except for reagent 3 are the same as those in Example 1.

[0086] In this comparative example, reagent 3 is a 0.2 μg / mL alkaline phosphatase-labeled anti-human IgG4 antibody solution. The difference between reagent 3 and reagent 3 in Example 1 is that the formulation of the third buffer used in preparation is different. The formulation of the third buffer used in this comparative example is: 50 mM MES, 0.9% (w / v) sodium chloride, 1% (w / v) bovine serum albumin, 5 mM magnesium chloride, 0.1 mM zinc chloride, 0.1% (v / v) Triton X-100, 0.05% (v / v) Proclin-30, and the balance being water, pH 6.0.

[0087] Example 1: Comparison of detection performance of reagent kits in Comparative Examples 4-6: The kits used in Example 1 and Comparative Examples 4-6 were used to quantitatively detect the content of house dust mite allergen-specific IgG4 in clinical serum samples, including 10 positive samples (samples P11-P20), 10 negative samples (samples N1-N10), and non-specific samples. The software automatically generated the corresponding luminescence value and calculated concentration value for each sample. A detection concentration value ≤2.5 μg / L was interpreted as negative, and a detection concentration value >2.5 μg / L was interpreted as positive. The results were compared with those of the Immuno CAP test from Phadia, and the results are shown in Table 5.

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[0089] Accelerated stability test: Some kits of Example 1 and Comparative Examples 4-6 were stored at 37°C for 10 days. Three positive samples (samples P16-P18) were quantitatively detected using the kits before and after storage. The signal retention rate was calculated based on the quantitative detection results. The results are shown in Table 6.

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[0091] Precision verification of the kit in Example 1: Low-value and high-value samples were selected and measured twice daily (morning and afternoon) using the kit from Example 1 for 20 consecutive days, resulting in 20 measurements for each concentration level. The average intra-assay precision, inter-day precision, and inter-assay precision for each concentration level were calculated. x The results of the standard deviation (s) and coefficient of variation (CV) are shown in Table 7.

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[0093] The results in Table 7 show that the precision indicators at all concentration levels meet the requirements, and the coefficients of variation (CV) for intra-batch, inter-day, and inter-batch are all less than 10%.

[0094] Methodological comparison: One hundred clinical samples were selected for parallel testing using two methods: the kit described in Example 1 and the Immuno CAP assay from Phadia. The correlation results are shown in [link to results]. Figure 1 The two methods showed a good linear correlation, with a fitted regression equation of y = 1.0948x + 0.1447, a correlation coefficient r of 0.951, and a coefficient of determination R² of 0.9036, indicating a high correlation. This demonstrates that the two methods have a high correlation and good consistency in quantitative results, indicating that the kit in Example 1 has good accuracy and reliability in the quantitative detection of house dust mite-specific IgG4, and can meet clinical testing needs.

[0095] Linear range detection: High-value samples approaching the upper limit of the linear range were diluted with sample diluent to 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256, 1 / 512, and 1 / 1024 concentrations (10 concentrations in total). Each concentration was tested four times using the kit from Example 1. Polynomial regression analysis and linearity tests were performed. The linear fitting curves are shown in [Figure 1]. Figure 2 The linear range is 0.1-50 mg / L.

[0096] The results above show that the kit in Example 1 performed best in terms of specificity, accuracy, stability, and repeatability. The kit in Example 1 overcomes the shortcomings of traditional ELISA methods, enabling allergen-specific IgG4 to serve as an objective and reliable biomarker for guiding desensitization therapy and efficacy monitoring in allergic diseases, thus possessing a realistic basis for large-scale clinical application.

[0097] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A chemiluminescent detection kit for allergen-specific IgG4 antibody magnetic microparticles, characterized in that, It includes reagent 1, reagent 2, reagent 3, reagent 4, and a luminescent substrate solution. Reagent 1 is a streptavidin-labeled magnetic nanoparticle suspension, comprising streptavidin-labeled magnetic nanoparticles and a first buffer solution. The formulation of the first buffer solution is: 5-10 mM disodium hydrogen phosphate, 2-4 mM sodium dihydrogen phosphate, 0.8%-1.2% (w / v) sodium chloride, 0.8%-1.2% (w / v) glycosylated bovine serum albumin, 0.8%-1.2% (v / v) glycerol, 0.08%-0.12% (v / v) nonionic detergent, 0.03%-0.08% (v / v) preservative, 0.1-0.3 M glycine methyl ester, with the balance being water, and a pH of 7.2-7.

6. Reagent 2 is a biotin-labeled allergen solution, comprising a biotin-labeled allergen and a second buffer solution. The formulation of the second buffer solution is: 5-10 mM disodium hydrogen phosphate, 2-4 mM sodium dihydrogen phosphate, 0.8%-1.2% (w / v) sodium chloride, 0.001%-0.003% (w / v) 4-aminoantipyrrolidone, 0.8%-1.2% (v / v) glycerol, 0.8%-1.2% (w / v) BSA, 0.01%-0.02% (v / v) nonionic detergent, 0.08%-0.12% (v / v) preservative, with the balance being water, and a pH of 7.2-7.

6. Reagent 3 is an alkaline phosphatase-labeled anti-human IgG4 antibody solution, comprising alkaline phosphatase-labeled anti-human IgG4 antibody and a third buffer solution. The third buffer solution is formulated as follows: 40-60 mM MES, 0.8%-1.2% (w / v) sodium chloride, 0.8%-1.2% (w / v) bovine serum albumin, 4-8 mM magnesium chloride, 0.08-0.12 mM zinc chloride, 0.8%-1.2% (w / v) PEG8400, 0.8%-1.2% (v / v) nonionic detergent, 0.03%-0.06% (v / v) preservative, with the balance being water, and a pH of 5.8-6.

2. Reagent 4 is a biotin-labeled anti-human IgG4 antibody solution, which includes biotin-labeled anti-human IgG4 antibody and a fourth buffer solution. The fourth buffer solution has the following formulation: 40~60mM MES, 0.8%~1.2% (w / v) sodium chloride, 0.8%~1.2% (w / v) bovine serum albumin, 0.8%~1.2% (v / v) glycerol, 0.08%~0.12% (v / v) nonionic detergent, 0.03%~0.08% (v / v) preservative, and the balance being water, with a pH of 5.8~6.

2.

2. The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit according to claim 1, characterized in that, The alkaline phosphatase-labeled anti-human IgG4 antibody in reagent 3 is obtained by reacting alkaline phosphatase with anti-human IgG4 antibody purified by an IgG1 adsorption column and / or an IgE adsorption column. The packing material of the IgG1 adsorption column is AminoLink Plus coupling resin conjugated with recombinant human IgG1 Fc fragment, and the packing material of the IgE adsorption column is AminoLink Plus coupling resin conjugated with recombinant human IgE Fc fragment.

3. The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit according to claim 2, characterized in that, The anti-human IgG4 antibody was first purified by the IgG1 adsorption column and then purified by the IgE adsorption column.

4. The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit according to claim 1, characterized in that, The concentration of reagent 1 is 0.1~0.5 mg / mL; And / or, the concentration of reagent 2 is 0.5~10 μg / mL; And / or, the concentration of reagent 3 is 0.1~0.5 μg / mL; And / or, the concentration of reagent 4 is 0.08~0.2 μg / mL.

5. The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit according to claim 1, characterized in that, The nonionic detergents in the first buffer solution, the second buffer solution, the third buffer solution, and the fourth buffer solution are one or more of Triton X-100, Tween-20, Tween-80, Brij 35, and Pluronic F-68, respectively. And / or, the preservatives in the first buffer, the second buffer, the third buffer, and the fourth buffer are one or more of Proclin-200, Proclin-300, and Proclin-950, respectively.

6. The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit according to claim 1, characterized in that, The luminescent substrate in the luminescent substrate solution is AMPPD.

7. The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit according to claim 1, characterized in that, It also includes one or more of the following: sample diluent, human IgG4 standard, and / or standard diluent. The sample diluent was formulated as follows: 5-10 mM disodium hydrogen phosphate, 2-4 mM sodium dihydrogen phosphate, 0.8%-1.2% (w / v) sodium chloride, 0.8%-1.2% (w / v) bovine serum albumin, 0.1% (v / v) nonionic detergent, 0.05% (v / v) preservative, with the balance being water, pH 7.2-7.

6. The standard diluent is formulated as follows: 40-60 mM tris(hydroxymethyl)aminomethane, 400-600 mM sodium chloride, 0.8%-1.2% (w / v) bovine serum albumin, 0.08%-0.12% (v / v) nonionic detergent, 0.1%-0.3% (w / v) polyoxyethylene lauryl ether, 0.03%-0.06% (v / v) preservative, with the balance being water, pH 7-7.

5.

8. The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit according to claim 7, characterized in that, The nonionic detergents in the sample diluent and / or the standard diluent are one or more of Triton X-100, Tween-20, Tween-80, Brij 35, and Pluronic F-68, respectively. And / or, the preservatives in the sample diluent and / or the standard diluent are one or more of Proclin-200, Proclin-300, and Proclin-950, respectively.

9. The allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit according to claim 1, characterized in that, The allergens mentioned are selected from house dust mite allergens, dust mite allergens, canine epidermal allergens, artemisia allergens, common ragweed allergens, house dust allergens, cockroach allergens, willow allergens, Alternaria allergens, milk allergens, cod allergens, wheat flour allergens, peanut allergens, soybean allergens, shrimp allergens, beef allergens, mutton allergens, egg allergens, crab allergens, cat epidermal allergens, hop allergens, common birch allergens, sycamore allergens, white poplar allergens, cypress allergens, pine allergens, Penicillium allergens, Cladosporium allergens, Aspergillus fumigatus allergens, Candida albicans allergens, Rhizopus nigricans allergens, hazelnut allergens, almond allergens, cashew allergens, pistachio allergens, strawberry allergens, apple allergens, mango allergens, peach allergens, pineapple allergens, and scallop allergens.

10. An allergen-specific IgG4 antibody magnetic particle chemiluminescence detection system, characterized in that, It includes the allergen-specific IgG4 antibody magnetic microparticle chemiluminescence detection kit and the fully automated chemiluminescence analyzer as described in any one of claims 1 to 9.

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