A specific IgE calibrator for the dust mite allergen component Der p 10, its preparation method, and its application.

Abstract

The application discloses a kind of dust mite allergen component Der p 10 specific IgE calibrators and its preparation method and application, the active ingredient of the calibrators is recombinant humanized IgE antibody, which is recombined by a specific Der p 10 monoclonal antibody, and the IgE constant region thereof is modified: artificial disulfide bond is introduced by site-directed mutagenesis, and non-essential glycosylation site is mutated to reduce glycoform diversity.Preparation method includes: expression and purification Der p 10 protein;Preparation and screening monoclonal antibody;Construction of recombinant humanized IgE antibody and modification;Expression and purification of recombinant antibody;Formulation calibrator.The binding affinity of the calibrator with Der p 10 protein KD Value ≤1×10 ‑9 mol / L, detection sensitivity ≤0.1 IU / mL, can be applied to construct dust mite allergen component Der p 10 specific IgE standard curve for quantitative detection.

Description

Technical Field

[0001] This invention relates to a specific IgE calibrator for the dust mite allergen component Der p 10, its preparation method, and its application, belonging to the field of allergen detection. Background Technology

[0002] As a major allergen component of house dust mites, Der p10's specific IgE antibody detection is of great significance for the diagnosis and treatment of allergic diseases. Currently, commonly used clinical detection methods include enzyme-linked immunosorbent assay (ELISA), fluorescence enzyme immunoassay (FEIA), and chemiluminescent immunoassay (CLIA).

[0003] In the field of allergen-specific IgE detection, the quality of calibrators directly affects the accuracy and reliability of test results. Traditional allergen-specific IgE calibrators are mostly prepared using human serum pools, which suffers from problems such as large batch-to-batch variations and insufficient specificity. CN121410254A discloses an allergen-specific IgE and IgG antibody composite quality control or calibrator and its preparation method. This method constructs a fusion protein and chemically conjugates it with purified antibodies, allowing for simultaneous detection of allergen IgE and IgG. However, the calibrators prepared by this method still need improvement in terms of specificity and batch-to-batch consistency.

[0004] CN105353133A discloses a kit and method for detecting IgE antibodies specific to house dust mite allergens. This kit includes components such as calibrators, quality controls, and a biotin-labeled house dust mite allergen solution. However, the calibrators used in this kit are still prepared based on traditional serum pools, making it difficult to achieve high standardization and traceability.

[0005] The following technical problems still exist in the field of Der p 10 specific IgE detection in the current technology: 1. Poor comparability of results between testing systems: Test results from different manufacturers and batches of test reagents are difficult to compare directly due to different sources of calibrators, which affects the consistency of clinical diagnosis; 2. Insufficient calibrator specificity: calibrators prepared from traditional serum pools often contain multiple allergen-specific IgE antibodies, making it difficult to achieve accurate calibration of a single allergen component, Der p 10; 3. Large batch-to-batch variability: Due to the limited availability of raw materials, calibrators based on human serum exhibit significant batch-to-batch variability, making it difficult to guarantee the long-term stability of test results; 4. Lack of a standardized traceability system: Existing calibrators are difficult to trace accurately to international standards, which limits the international recognition of test results; 5. Insufficient detection sensitivity: Existing technologies are insufficient to meet the requirements for accurate detection of low-concentration Der p 10-specific IgE, affecting the accuracy of early diagnosis.

[0006] Therefore, there is an urgent need to develop a highly specific, stable, and traceable Der p 10 specific IgE calibrator to improve the accuracy, comparability, and sensitivity of detection and meet the needs of clinical diagnosis. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of existing Der p 10 specific IgE detection calibrators, such as insufficient specificity, large batch-to-batch variability, low sensitivity, and lack of standardized traceability system. The invention provides a highly specific, highly stable, and traceable Der p 10 specific IgE calibrator for dust mite allergen components, as well as its preparation method and application.

[0008] The first technical solution provided by this invention is a specific IgE calibrator for the dust mite allergen component Der p 10, wherein the active ingredient of the calibrator is a recombinant humanized IgE antibody; the amino acid sequences of the heavy and light chain variable regions of the recombinant humanized IgE antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, and the heavy chain constant region of the amino acid sequence shown in SEQ ID NO.3 is modified by at least one of the following: a) Site-directed mutagenesis introduces artificial disulfide bonds; b) Mutate non-essential glycosylation sites to reduce glycoform diversity, wherein the non-essential glycosylation sites are N49 and N99.

[0009] In some embodiments, the natural cysteine ​​C at position 193 of the heavy chain constant region is mutated to alanine A to avoid mismatch between the natural disulfide bond and the artificial disulfide bond; at the same time, at least one of the amino acids at positions 195, 196, and 197 is mutated to cysteine ​​to form an artificial interchain disulfide bond.

[0010] The second technical solution provided by this invention is to encode the gene of the Der p10 specific IgE calibrator of the dust mite allergen component described in the first technical solution.

[0011] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.

[0012] The fourth technical solution provided by the present invention is to express the Der p10 specific IgE calibrator of the dust mite allergen component described in the first technical solution, or the gene described in the second technical solution, or to transform a host cell with the recombinant vector described in the third technical solution.

[0013] In some embodiments, the host cell is an animal cell, plant cell, or microbial cell.

[0014] In some embodiments, the animal cells are HEK293 cells or CHO cells.

[0015] The present invention provides a fifth technical solution, which is a method for preparing a specific IgE calibrator for the dust mite allergen component Der p 10. The method is to culture the host cells described in the fourth technical solution to obtain a culture containing the specific IgE calibrator for the dust mite allergen component Der p 10 described in the first technical solution.

[0016] In some implementations, the following steps are included: (1) Expression and purification of Der p 10 protein: A recombinant expression vector of Der p 10 gene was constructed, and it was transfected into mammalian cells for expression. The expression product was collected and purified to obtain high-purity Der p 10 protein. (2) Preparation and screening of monoclonal antibodies: New Zealand rabbits were immunized with Der p 10 protein obtained in step (1), and spleen cells of the immunized rabbits were electrofused with myeloma cells to screen for a monoclonal antibody with high affinity and high sensitivity. (3) Construction and modification of recombinant humanized IgE antibody: The monoclonal antibody obtained in step (2) was recombinantly constructed and humanized IgE was modified. Then, the following modifications were carried out in sequence: ① site-directed mutagenesis to introduce artificial disulfide bonds; ② mutation of non-essential glycosylation sites to reduce glycoform diversity. (4) Expression and purification of recombinant antibodies: The recombinant expression vector was transfected into mammalian cells for expression, and the expression product was collected and purified to obtain recombinant humanized IgE antibody; (5) Preparation of calibrators: The recombinant antibody was diluted to a preset concentration gradient with calibrator diluent, and after aliquoting and lyophilization, Der p 10 specific IgE calibrators were obtained.

[0017] Preferably, the animal cells in step (1) are HEK293 cells or CHO cells; the Der p 10 protein is purified by nickel column affinity chromatography combined with gel filtration chromatography, and the protein purity after purification is ≥98%.

[0018] Preferably, the monoclonal antibody screening in step (2) uses the ELISA method, with the following criteria: OD value ≥1.5 when binding to Der p 10 protein, and cross-reactivity rate ≤5% with Der p 2 and Der p 3.

[0019] Preferably, the humanized IgE modification in step (3) adopts the CDR transplantation method.

[0020] Preferably, in step (5), the calibrator diluent is a PBS buffer (pH 7.4) containing 10% fetal bovine serum and 0.1% NaN3; the concentration gradient is 0.1, 0.5, 2.0, 10.0, and 50.0 IU / mL.

[0021] The sixth technical solution provided by the present invention is a reagent kit containing the Der p 10 specific IgE calibrator for the dust mite allergen component described in the first technical solution.

[0022] The seventh technical solution provided by the present invention is the application of the Der p10 specific IgE calibrator for detecting the dust mite allergen component described in the first technical solution, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the host cell described in the fourth technical solution, or the method described in the fifth technical solution in the preparation of a product for detecting the Der p10 specific IgE component for detecting the dust mite allergen component.

[0023] In some embodiments, the product includes reagents, kits, detection chips, or biosensors.

[0024] This invention provides the application of the calibrator, which is used in a magnetic microparticle chemiluminescence quantitative detection reagent for Der p 10-specific IgE, a component of house dust mite allergens, to construct a detection standard curve and achieve absolute quantitative detection of Der p 10-specific IgE. The magnetic microparticle chemiluminescence quantitative detection reagent comprises magnetic microparticles coupled to Der p 10 protein, enzyme-labeled secondary antibody, luminescent substrate, and the calibrator; the detection linear range is 0.1-50.0 IU / mL, with a linear correlation coefficient R² ≥ 0.995, intra-assay coefficient of variation ≤ 4.2%, and inter-assay coefficient of variation ≤ 7.5%.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. High specificity: The active ingredient of the calibrator of the present invention is a recombinant humanized IgE antibody, which is completely identical in structure to human IgE. It has high binding specificity to Der p 10 and significantly reduces the cross-reactivity rate (≤2.1%) with other dust mite allergen components (Der p 1, Der p 2). This effectively avoids cross-reactivity caused by the structural differences between animal-derived antibodies and human IgE, and significantly improves the accuracy of detection results.

[0026] 2. High affinity and high sensitivity: This invention screens and obtains high-affinity monoclonal antibodies (KD value ≤ 8.2 × 10⁻⁶). -10 After humanization and site-directed mutagenesis, the affinity was further increased to 4.5 × 10⁻⁶ mol / L. -10With a detection sensitivity of 0.08 IU / mL, it can accurately detect low concentrations of Der p 10-specific IgE, effectively reducing the false negative rate.

[0027] 3. Excellent batch-to-batch consistency and stability: This invention utilizes a mammalian cell expression system to produce recombinant antibodies on a large scale with a purity >98% and a batch-to-batch coefficient of variation ≤7.5%, significantly superior to naturally extracted human calibrators (batch-to-batch CV ≥19.4%). Furthermore, through modifications such as the introduction of artificial disulfide bonds, the calibrators maintain an activity retention rate ≥97.2% after 12 months of storage at 4°C, demonstrating excellent long-term stability.

[0028] 4. Achieve standardized traceability: The calibrators of this invention can construct a precise quantitative standard curve (R²≥0.997), which enables standardized traceability of Der p 10 specific IgE detection results, unifies the detection standards of different laboratories and different detection systems, and improves the comparability of detection results.

[0029] 5. Simple preparation method and scalable production: The preparation method of this invention adopts a mature mammalian cell expression system and genetic engineering technology, which is simple to operate, has good reproducibility, and can realize the large-scale production of recombinant antibodies, reducing the production cost of calibrators. Attached Figure Description

[0030] Figure 1 Purity analysis of recombinant Der p 10 protein denaturation by polyacrylamide gel electrophoresis.

[0031] Figure 2 The binding affinity of McAb-1 to Der p 10 protein was determined.

[0032] Figure 3 Purity analysis of six recombinant humanized IgE antibodies by denaturation and non-denaturation polyacrylamide gel electrophoresis.

[0033] Figure 4 Mass spectrometry analysis of the N-glycoform at the N49 site of the wild-type antibody.

[0034] Figure 5 Mass spectrometry analysis of N-glycoform at N49 site of mutant 5 antibody. Detailed Implementation

[0035] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0036] Raw materials used in the examples: 1. Plasmid pCDNA3.1: Purchased from Thermo Fisher Scientific, catalog number V79020.

[0037] 2. HEK293 cells: purchased from Thermo Fisher Scientific, catalog number R79007.

[0038] 3. FreeStyle™ 293 expression medium: purchased from Thermo Fisher Scientific, catalog number 12338018.

[0039] 4. New Zealand rabbits: purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0040] Example 1: Preparation of a specific IgE calibrator for a dust mite allergen component, Der p 10. Specifically, the following steps are included: (1) Expression and purification of Der p10 protein: First, based on the Der p 10 amino acid sequence in UniProt (accession number: O18416), the full-length Der p 10 gene (encoding 284 amino acids, with a 6×His tag added to the C-terminus) was artificially synthesized and cloned into the pCDNA3.1 vector. BamH I and Xho Between the restriction enzyme sites, a recombinant expression vector pCDNA3.1-Der p10 was constructed. The recombinant expression vector was transformed into DH5α competent cells, plated on LB medium containing ampicillin, and incubated overnight at 37°C. Single colonies were picked for PCR identification and sequencing verification. The sequencing results showed 100% homology with the Der p10 gene sequence in GenBank.

[0041] The successfully constructed recombinant expression vector pCDNA3.1-Der p10 was transfected into HEK293 cells and cultured in suspension on serum-free medium at 37°C, 5% CO2, and a rotation speed of 120 rpm for 6-7 days. The cell culture supernatant was then collected. The collected supernatant was centrifuged at 8000 rpm for 15 min at 4°C to remove cell debris. The supernatant was then filtered through a 0.22 μm filter to remove impurities.

[0042] The filtered supernatant was loaded onto a nickel affinity chromatography column and equilibrated with equilibration buffer (20 mmol / L Tris-HCl, 500 mmol / L NaCl, pH 8.0) until the baseline stabilized. The column was then eluted with a gradient of elution buffer (20 mmol / L Tris-HCl, 500 mmol / L NaCl, 500 mmol / L imidazole, pH 8.0), and the elution peaks were collected. The collected eluent was loaded onto a gel filtration chromatography column (Sephadex G-75) and eluted with PBS buffer (pH 7.4). A single elution peak was collected to obtain the purified Der p 10 protein.

[0043] The purity of Der p10 protein was determined by SDS-PAGE electrophoresis. The stacking gel concentration was 5%, the separating gel concentration was 12%, and the sample loading amount was 20 μg. After electrophoresis, Coomassie brilliant blue staining was performed, and the results were observed after destaining. The results showed that the purified Der p10 protein exhibited a single band at approximately 36 kDa, with no impurities. ImageJ software analysis indicated a protein purity of 98.5% (see [link to image]). Figure 1 ).

[0044] (2) Fusion and screening of hybridoma cell lines: Three healthy female New Zealand rabbits (weighing 2.0-2.5 kg) were selected and immunized with purified Der p 10 protein at a dose of 100 μg per rabbit. For the initial immunization, an equal volume of Der p 10 protein was mixed with Freund's complete adjuvant, thoroughly emulsified, and then injected subcutaneously at multiple sites (back and abdomen). After the initial immunization, booster immunizations were administered every two weeks for a total of three booster immunizations. For each booster immunization, an equal volume of Der p 10 protein was mixed with Freund's incomplete adjuvant and emulsified.

[0045] Seven days after the last immunization, blood was collected from the marginal ear vein of rabbits. Serum was separated by centrifugation, and the titer of anti-Derp 10 antibody in the serum was detected by ELISA. Derp 10 protein was diluted to 0.5 μg / mL with coating buffer (0.05 mol / L carbonate-bicarbonate buffer, pH 9.6), and 100 μL / well was added to a 96-well microplate. The plate was incubated overnight at 4°C. The next day, the coating solution was discarded, and the plate was washed three times with PBST buffer (PBS + 0.05% Tween-20) for 5 min each time. Blocking buffer (PBST containing 5% skim milk powder) was added at 200 μL / well, and the plate was blocked at 37°C for 2 h. Discard the blocking solution, wash three times, and add serially diluted rabbit serum (1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000), 100 μL / well, and incubate at 37°C for 1 h. Discard the serum, wash three times, and add HRP-labeled goat anti-rabbit IgG secondary antibody (1:5000 dilution), 100 μL / well, and incubate at 37°C for 1 h. Discard the secondary antibody, wash five times, add TMB chromogenic buffer, 100 μL / well, and incubate at 37°C in the dark for 15 min. Add 2 mol / L H2SO4 stop solution, 50 μL / well, and measure the absorbance (OD value) at 450 nm using a microplate reader. 450 A value ≥0.2 and greater than twice that of the negative control is considered a positive result.

[0046] Table 1 Serum antibody titer detection after four immunizations

[0047] The results showed that the serum antibody titers of all three New Zealand rabbits reached over 1:128000 (see Table 1), with rabbit No. 1 exhibiting the highest antibody titer. Rabbit No. 1 was selected for subsequent hybridoma cell fusion. Rabbit No. 1 was euthanized, and its spleen was aseptically harvested, ground into a single-cell suspension, and mixed with myeloma cells at a ratio of 2:1. The cell suspension was centrifuged at 1000 r / min for 5 minutes, and the cell pellet was washed twice with PM solution to adjust the cell density to 1×10⁻⁶. 7 Electrofusion was performed. Cells were transferred to electrode pools, and the following electric field conditions were applied: AC electric field frequency of 1 MHz, amplitude of 250 V / cm, for 30 seconds for electrolyte electrophoresis. When a small number of cells were observed to have formed beaded strings under a microscope, a perforation pulse was applied. The parameters were: pulse amplitude of 5 kV / cm, time constant of 20 ms, number of pulses of 5, and interval of 1 second. After electrofusion, cells were screened using HAT medium. After culturing for 7-10 days, the growth of hybridoma cells was observed. Wells with well-grown hybridoma cells were selected for initial screening using ELISA to identify hybridoma cells that were positive for Der p10 protein binding.

[0048] Hybridoma cells that initially tested positive were cloned using a limiting dilution method, and this process was repeated three times to obtain monoclonal hybridoma cell lines. A second screening was performed using ELISA, and the binding ability of the hybridoma cell culture supernatant to Derp10, Derp1, and Derp2 proteins was also measured. The screening criterion was the binding OD of Derp10 protein. 450 ≥1.5, binding OD to Der p1 and Der p2 proteins 450 ≤0.15 (cross-reactivity rate ≤5%). Finally, a monoclonal hybridoma cell line with high titer was obtained through screening, named McAb-1, which can specifically bind to Der p10 protein and has no obvious cross-reactivity.

[0049] The binding affinity of McAb-1 to Der p 10 protein was detected using surface plasmon resonance (SPR) technology on a Biacore T200 (Cytiva) instrument. Antibodies were conjugated to the surface of a CM5 chip, and the kinetic parameters of antibody-antigen interactions were determined using a multi-cycle kinetic method. McAb-1 antibody samples were diluted to 5 μg / mL with HBS-EP+Buffer (1×) and fixed on the chip surface for 3 min at a flow rate of 5 μL / min. Subsequently, the target antigen was serially diluted using HBS-EP+Buffer (1×) to a concentration range of 7.813–500 nmol / L, with seven dilutions. The testing conditions were 25 ℃, 30 μL / min, binding for 3 min, and dissociation for 5 min. One cycle of repeated measurements was used for quality control, and two cycles of blank control were used to correct for systematic bias. Regeneration conditions were: 10 mmol / L Gly-HCl (pH 2.5), 30 μL / min, regeneration for 3 min. The results were analyzed using BIAevaluation Software, with a 1:1 combination mode selected to obtain the fitted curve and calculate the equilibrium dissociation constant (K). D McAb-1 antibody K D The value is 8.22 × 10 -10 mol / L (see Figure 2 ).

[0050] (3) Construction and modification of recombinant humanized IgE antibodies: McAb-1 hybridoma cells were collected, and total RNA was extracted using Trizol reagent and reverse transcribed into cDNA. Primers were designed based on the conserved sequence of the rabbit IgG variable region gene, and the VH and VL genes of McAb-1 were amplified by PCR. The PCR products were purified by agarose gel electrophoresis and cloned into the pMD19-T vector. Sequencing verification was performed, and the amino acid sequences were deduced based on the sequencing results. The amino acid sequence of the McAb-1 heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2. Human IgE heavy chain constant region gene (IgE-constant, GenBank accession number: M12528.1, containing Cε1, Cε2, Cε3, and Cε4 domains) and human IgE kappa light chain constant region gene (IgE kappa-constant, UniProt accession number: P01834) were artificially synthesized. Using overlap extension PCR, the McAb-1 heavy chain variable region gene (SEQ ID NO.14) was chimeric with the human IgE heavy chain constant region gene, and the light chain variable region gene (SEQ ID NO.15) was chimeric with the human IgE light chain constant region gene, respectively. The resulting chimeric genes were cloned into the pcDNA3.1 vector to construct the heavy chain recombinant expression vector McAb-1-H-IgE-constant and the light chain recombinant expression vector McAb-1-L-IgE kappa-constant.

[0051] The following modifications were then carried out: The desired amino acid mutation was introduced into the Cε2 domain of the McAb-1-H-IgE-constant region using site-directed mutagenesis. First, primers containing the target mutation site were designed based on the synthetic gene sequence. Then, the mutation was introduced using overlap extension PCR or a commercially available site-directed mutagenesis kit. Finally, the correctness of the mutation was verified by DNA sequencing.

[0052] 1) Site-directed mutagenesis was performed on the disulfide bond region of the Cε2 domain in the IgE constant region. First, the natural cysteine ​​at position 193 of the wild type was knocked out and a C193A mutation was performed to avoid mismatch between the natural and artificial disulfide bonds. Then, one of the sites at positions 195, 196, and 197 was mutated to cysteine ​​(V195C, T196C, Y197C) to introduce a disulfide bond and improve the stability of the antibody.

[0053] 2) Site-directed mutagenesis of non-essential glycosylation sites in the IgE constant region to reduce glycoform diversity: For mutants (N49Q / N99Q), two mutations can be introduced at once or in stages. This embodiment uses a single introduction.

[0054] Table 2 Primer Information

[0055] The recombinant expression vectors were sequenced to verify that the mutation sites were correct and that there were no other base mutations. The final light chain recombinant expression vector McAb-1-L-IgE kappa-constant and heavy chain recombinant expression vectors McAb-1-H-IgE-constant-mut-1, McAb-1-H-IgE-constant-mut-2, McAb-1-H-IgE-constant-mut-3, McAb-1-H-IgE-constant-mut-4, and McAb-1-H-IgE-constant-mut-5 were obtained (see Table 3).

[0056] Table 3 Heavy chain recombination expression mutation sites

[0057] (4) Expression and purification of recombinant humanized IgE antibody: The recombinant expression vectors McAb-1-H-IgE-constant, McAb-1-H-IgE-constant-mut-1, McAb-1-H-IgE-constant-mut-2, McAb-1-H-IgE-constant-mut-3, McAb-1-H-IgE-constant-mut-4, and McAb-1-H-IgE-constant-mut-5 were co-transfected with McAb-1-L-IgE-kappa-constant at a 1:1 ratio into 293 cells. The cells were then cultured in suspension using serum-free medium at 37°C, 5% CO2, and a rotation speed of 120 r / min for 6–7 days. When cell viability dropped below 60%, the cell culture supernatant was collected and centrifuged at 10,000 r / min for 20 min at 4℃ to remove cell debris. The supernatant was filtered through a 0.22 μm filter and purified by Protein L affinity chromatography. The chromatography column was first equilibrated with equilibration buffer (20 mmol / L PBS, pH 7.4) until the baseline stabilized, and then the target protein was eluted with elution buffer (0.1 mol / L glycine-HCl, pH 3.2). The elution peak was collected and neutralized to pH 7.4 with neutralization buffer (1 mol / L Tris-HCl, pH 8.5) to obtain the purified recombinant humanized IgE antibody.

[0058] (5) Identification of recombinant humanized IgE antibodies: The purity of recombinant antibodies was detected by SDS-PAGE electrophoresis. The stacking gel concentration was 5%, the separating gel concentration was 12%, and the sample loading amount was 20 μg. After electrophoresis, Coomassie brilliant blue staining was performed, and the results were observed after destaining. Under reducing conditions, the light chain of the six recombinant antibodies was approximately 25 kDa, consistent with the theoretical size, while the heavy chain was approximately 90 kDa, slightly larger than the theoretical size of 75 kDa calculated based on the amino acid composition. This indicates that the antibodies are highly glycosylated, and due to the influence of glycosylation, their molecular weight is larger than the theoretical size. Under non-reducing conditions, the molecular weight of the six recombinant antibodies was approximately 170 kDa, consistent with the theoretical size. ImageJ software analysis showed that the purity of all antibodies was >98% (see [link to image]). Figure 3 ).

[0059] The specificity of six recombinant antibodies was detected by Western blot. Der p 10, Der p 1, and Der p 2 proteins were subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked, and then each of the six recombinant antibodies (1:1000 dilution) was added and incubated overnight. HRP-labeled anti-human IgE secondary antibody (1:5000 dilution) was then added and incubated for 1 hour. ECL staining was then performed, and the results were observed. The results showed that the six recombinant antibodies bound only to Der p 10 protein, showing a specific band, and did not bind to Der p 2 or Der p 3 proteins, indicating excellent specificity that meets the specificity requirements of the calibrators.

[0060] The binding affinity of recombinant antibodies to Der p10 protein was detected using SPR technology, as described above. The Kb of six recombinant antibodies to Der p10 protein was determined. D The values ​​are 4.52 × 10 -10 mol / L, 4.38×10 -10 mol / L, 4.23×10 -10 mol / L, 4.12×10 -10 mol / L, 4.15×10 -10 mol / L, 4.05×10 -10 The affinities were all higher than those of the parental monoclonal antibody (mol / L, see Table 4).

[0061] Table 4. SPR assay for antibody binding ability

[0062] (6) Verification of glycosylation heterogeneity of recombinant humanized IgE antibody: Recombinant humanized antibodies were denatured with 8 M urea, reduced with dithiothreitol, and alkylated with iodoacetamide, then dialyzed to 25 mM ammonium bicarbonate buffer (pH 7.8) for later use. Trypsin (targeting the N99 site) was added to the reduced alkylated sample at an enzyme:substrate ratio of 1:30 (w / w), and digestion was carried out at 37°C for 4 h. Subsequently, chymotrypsin (targeting the N49 site) was added to bring the total enzyme:substrate ratio to 1:50 (w / w), and digestion continued at 37°C for 12 h. The reaction was terminated by adding formic acid to a final concentration of 1%. The digested peptides were desalted by C18 solid-phase extraction, vacuum dried, reconstituted with 85% acetonitrile aqueous solution, loaded onto a hydrophilic interaction chromatography (HILIC) column, and eluted with 0.1% trifluoroacetic acid aqueous solution. The eluent was collected and vacuum dried.

[0063] The dried peptides were reconstituted with 0.05% formic acid aqueous solution and loaded onto a C18 nano-column (0.75 μm × 50 cm, Thermo Fisher Scientific). Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. Linear gradient elution was performed at a constant flow rate of 300 nL / min. The separated peptides were then connected online to a Q Exactive™ quadrupole Orbitrap™ high-resolution mass spectrometer (Thermo Fisher Scientific) using positive ion mode data-dependent acquisition (DDA). Secondary fragmentation was performed using precursor ions carrying glycosylation characteristic ions (m / z 204.08, 366.14, etc.) in HCD mode. The glycopeptide structure was resolved by comparing characteristic sugar fragments with peptide backbone fragments, and the glycoform and modification abundance of each N-glycosylation site were identified.

[0064] The results showed that the glycosylation occupancy rates at the N49 and N99 sites of the wild-type antibody were 99.4% and 98.7%, respectively, with more than 10 distinguishable N-glycoforms detected (see Figure 4 for some mass spectrometry analyses), and the glycoform heterogeneity index was 78.3%. No glycosylated peptides were detected at either of the two target sites of mutants 3, 4, and 5, with a glycosylation occupancy rate of 0. Only two low-abundance residual glycoforms at non-target sites were detected (see Figure 4 for details). Figure 5 The glycoform heterogeneity index decreased to 3.1%, indicating that mutants 3, 4, and 5 almost completely eliminated antibody glycoform heterogeneity. Glycosylation heterogeneity of IgE antibodies is one of the key factors leading to batch-to-batch differences: on the one hand, the glycosylation site occupancy, glycoform types, and abundance of different batches of antibodies are prone to fluctuations, thus affecting the spatial conformation, biological activity, and stability of the antibody; on the other hand, high heterogeneity in glycosylation modifications can lead to inconsistencies in antibody product quality, increasing the difficulty of batch-to-batch quality control. The mutants of this invention significantly reduce glycoform heterogeneity by eliminating glycosylation modifications at target sites, thereby reducing batch-to-batch fluctuations caused by glycosylation differences from the source.

[0065] (7) Preparation of calibrators: Prepare a PBS buffer (pH 7.4) containing 10% fetal bovine serum and 0.1% NaN3, with a PBS buffer concentration of 0.01 mol / L. After preparation, autoclave at 121°C for 15 min and cool to room temperature before use. Dilute the purified six recombinant humanized IgE antibodies to five concentration gradients using calibrator diluent: 0.1 IU / mL, 0.5 IU / mL, 2.0 IU / mL, 10.0 IU / mL, and 50.0 IU / mL, with three replicates for each concentration gradient.

[0066] The diluted calibrator was aliquoted into 100 μL / vial and pre-frozen at -80℃ for 2 h. Then it was placed in a lyophilizer and the lyophilization conditions were: pre-freezing temperature -50℃, pre-freezing time 2 h, sublimation drying temperature -20℃, sublimation time 12 h, and resolution drying temperature 25℃, resolution time 4 h. After lyophilization, the sample was sealed and packaged to obtain Der p 10 specific IgE calibrator.

[0067] Example 2 This embodiment verifies the key performance indicators of the Der p 10-specific IgE calibrator for dust mite allergen components prepared according to the method of the present invention (hereinafter referred to as "the calibrator of the present invention"), and compares it with typical calibrators in the prior art (mouse-derived Der p 10-specific IgG-human IgE Fc fusion protein calibrator (Chondrex Inc, Catalog No. 7113) and naturally extracted human-derived Der p 10-specific IgE calibrator (InBios International, Inc., catalog number E-7E8).

[0068] Three calibrators from the same batch (each concentration gradient) were used, and their luminescence intensity was measured using the magnetic microparticle chemiluminescence method for quantitative detection. The intra-batch coefficient of variation (CV) was calculated. The results showed that the intra-batch CV values ​​for each concentration gradient were ≤4.2%, meeting the requirement of ≤5% for calibrators. Three different batches of calibrators (each batch, each concentration gradient, three calibrators) were used, and their luminescence intensity was measured using the same method, and the inter-batch coefficient of variation was calculated. The results showed that the inter-batch CV values ​​for each concentration gradient of the calibrators of this invention were ≤7.5%, meeting the requirement of ≤8% for calibrators. However, the intra-batch and inter-batch CV values ​​for both the mouse-derived Der p 10-specific IgE calibrators and the naturally extracted human Der p 10-specific IgE calibrators for each concentration gradient were ≥15.2%, significantly higher than the variation levels of the calibrators of this invention.

[0069] The above results demonstrate that the calibrators of the present invention are significantly superior to typical calibrators in the prior art in terms of detection sensitivity, intra-batch consistency, and inter-batch consistency.

[0070] Table 5 Comparison of key performance indicators between the calibrators of the present invention and those of existing technologies

[0071] The calibrators of this invention were stored at 4℃, -20℃, and -80℃, respectively, and samples were taken at 0, 3, 6, and 12 months of storage. The luminescence intensity was measured using a magnetic microparticle chemiluminescence quantitative detection method, and the activity retention rate was calculated. The results showed that after 12 months of storage at 4℃, the activity retention rates of calibrators 4, 5, and 6 were all ≥97.2%; after 12 months of storage at -20℃, the activity retention rates were all ≥98.4%; and after 12 months of storage at -80℃, the activity retention rates were all ≥99.3%. These results confirm that calibrators 4, 5, and 6 of this invention can be stably stored at 4℃ for more than 12 months, exhibiting excellent long-term storage stability, which can, to a certain extent, meet the stability requirements of calibrators in in vitro diagnostic applications. The comparison shows that the stability of calibrators 1, 2, and 3 of this invention is weaker than that of calibrators 4, 5, and 6 of this invention, proving that the novel artificial disulfide bond modification and non-essential glycosylation site mutation can effectively improve the structural stability of antibodies, thereby significantly improving the overall stability of calibrators.

[0072] Table 6. Long-term stability of the calibrators of this invention at different temperatures.

[0073] In a preferred embodiment, the calibrator diluent is a PBS buffer (pH 7.4) containing 10% fetal bovine serum and 0.1% NaN3; the concentration gradient is 0.1 IU / mL, 0.5 IU / mL, 2.0 IU / mL, 10.0 IU / mL, and 50.0 IU / mL.

[0074] Example 3 This embodiment provides an application of the Der p 10 specific IgE calibrator for house dust mite allergen components. Specifically, the calibrator is used in a magnetic particle chemiluminescence quantitative detection reagent for Der p 10 specific IgE to construct a detection standard curve and achieve absolute quantitative detection of Der p 10 specific IgE.

[0075] This quantitative assay reagent comprises four main components: magnetically conjugated Der p 10 protein, enzyme-labeled secondary antibody, luminescent substrate, and the aforementioned calibrators. When using this assay system, different concentrations of calibrators are first added to a 96-well chemiluminescence plate, with three replicates for each concentration. Then, the magnetically conjugated Der p 10 protein is added, and the plate is incubated at 37°C for 30 minutes to allow the IgE antibody in the calibrators to bind to the Der p 10 protein. After washing, enzyme-labeled anti-human IgE secondary antibody is added, and the plate is incubated at 37°C for 20 minutes to allow the secondary antibody to bind to the already bound IgE antibody. After washing again, the luminescent substrate is added, and the plate is incubated in the dark for 5 minutes. The luminescence intensity of each well is then measured using a chemiluminescence analyzer. A standard curve is constructed using a four-parameter fitting method, with the concentrations of calibrators 4, 5, and 6 as the x-axis and the luminescence intensity as the y-axis.

[0076] The fitting results showed that the linear correlation coefficient R² of the standard curve constructed using calibrators 4, 5, and 6 was ≥0.997, with a linear range of 0.1–50.0 IU / mL. Backtesting of clinical serum samples using this standard curve yielded intra-batch coefficient of variation ≤3.5% and inter-batch coefficient of variation ≤5.5%, both meeting quality control requirements.

[0077] Table 7. Standard curve data established using the calibrators of this invention.

[0078] Compared with existing technologies, the calibrators provided in this embodiment have several advantages: First, the calibrators based on recombinant humanized IgE antibodies have a binding affinity as high as 4.5 × 10⁻⁶. -10 The concentration is mol / L, which is far superior to traditional mouse-derived calibrators; secondly, the cross-reactivity with Derp 1 and Derp 2 is only 2.1%, and the specificity is significantly better than existing animal-derived calibrators; thirdly, the inter-batch coefficient of variation is controlled within 5.32%, which is better than existing technologies; fourthly, the detection sensitivity reaches 0.08 IU / mL, ensuring accurate detection capability in low-concentration samples.

[0079] By using the standard curve constructed with this calibrator, absolute quantitative detection of Der p 10-specific IgE can be achieved, providing reliable technical support for the accurate diagnosis and individualized treatment of house dust mite allergies.

[0080] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A specific IgE calibrator for the dust mite allergen component Der p 10, wherein the active ingredient of the calibrator is a recombinant humanized IgE antibody; the amino acid sequences of the heavy and light chain variable regions of the recombinant humanized IgE antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, and the heavy chain constant region of the amino acid sequence shown in SEQ ID NO.3 is modified in (a) and (b): (a) The natural cysteine ​​C at position 193 is mutated to alanine A, and the amino acid at any of positions 195, 196, and 197 is mutated to cysteine ​​C; (b) Mutating non-essential glycosylation sites by mutating asparagine N at positions 49 and 99 to glutamine Q.

2. The gene encoding the Der p 10 specific IgE calibrator of the dust mite allergen component of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Expressing the Der p 10 specific IgE calibrator of the dust mite allergen component of claim 1, or the gene of claim 2, or transforming a host cell with the recombinant vector of claim 3.

5. The host cell according to claim 4, characterized in that, The host cell can be an animal cell, plant cell, or microbial cell.

6. The host cell according to claim 5, characterized in that, The animal cells are HEK293 cells or CHO cells.

7. A method for preparing a specific IgE calibrator for the dust mite allergen component Der p 10, characterized in that, The method is to culture the host cells according to any one of claims 4 to 6 to obtain a culture containing the Derp 10 specific IgE calibrator of the dust mite allergen component according to claim 1.

8. A kit comprising the Der p 10 specific IgE calibrator for the dust mite allergen component of claim 1.

9. The use of the Der p 10 specific IgE calibrator for the dust mite allergen component according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3, or the host cell according to any one of claims 4 to 6 in the preparation of a product for detecting the Der p 10 specific IgE component of the dust mite allergen component.

10. The application according to claim 9, characterized in that, The products include reagents, kits, detection chips, or biosensors.

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