A quality control kit for preparing a cannabis sativa pollen allergen extract

Abstract

The application discloses a quality control kit for preparing Humulus pollen allergen extract, which comprises (a) Humulus pollen allergen extract as a control sample, which is used for qualitative or quantitative detection by comparing characteristic protein signals of the sample to be detected; (b) allergen monomers contained in characteristic bands of the extract and combinations thereof, which can be derived from natural purification or recombinant expression purification, and solid-phase or liquid-phase carriers to which the standard samples are attached; (c) antibodies or specific probes or other specific binding substances capable of specifically binding to the allergens in (b), which are used for quantitatively detecting effective allergen levels in the prepared Humulus pollen allergen extract.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a quality control kit for preparing hop pollen allergen extract and its application. Background Technology

[0002] The incidence of pollen-induced allergic diseases has been on the rise over the past decade. Hops pollen allergies are widespread throughout my country, with hop pollen being a major allergen in northern regions. Therefore, the detection, prevention, and treatment of hop pollen allergies are particularly important.

[0003] Allergen extracts prepared from hop pollen are widely used in clinical practice. They can be used as antigen raw materials to immobilize on solid-phase carriers for quantitative or qualitative detection of serum immunoglobulin E (IgE) in patients (Cui L, Yin J. Association of serum specific IgE levels with asthma inautumn pollen-induced allergic rhinitis: A retrospective analysis [J]. JAsthma, 2019, 56(5): 505-11.). They can also be used as biological agents for allergen-specific immunotherapy, enabling the patient's immune system to gradually tolerate the reaction to hop pollen allergens, reducing allergy symptoms and improving quality of life (Guan Kai, Wei Qingyu, Yin Jia. Efficacy and safety evaluation of hop pollen allergen immunotherapy [J]. Chinese Journal of Clinical Immunology and Allergy, 2012, 6 (04): 279-284.). As a clinical diagnostic and therapeutic agent, the quality control of its raw materials and formulation is particularly important to ensure its safety and effectiveness in clinical application.

[0004] Currently, some quality control methods exist for crude extracts of pollen allergens, but some shortcomings remain. For example, nitrogen content determination can calibrate the total protein content in the crude extract, but it cannot reflect the content of allergens and non-allergens in the extract, nor can it reflect the overall sensitization of the extract. Serum IgE reaction assays or cell challenge assays can calibrate the overall sensitization of the crude extract, but these require specific allergy patient samples to perform IgE reactivity assays on the extract. However, this quality control strategy faces the problems of a lack of standard serum samples and the inability to reflect the content of allergens and non-allergens in the crude extract. Patent CN201910210654A discloses an extraction quality control method for crude extracts of hop pollen. In addition to the commonly used physicochemical property tests and overall sensitization assays, it further uses SDS-PAGE detection based on internal reference materials and patient serum immunoblotting to determine 24 allergenic proteins in the crude extract. This is clearly a significant improvement in the quality control of crude allergen extracts. Currently, there is a lack of corresponding combinations of natural or recombinant allergen molecular standards in the field, and the quantitative detection of multiple allergen molecules is insufficient. SDS-PAGE detection also requires further objective quantitative indicators. Although CN117783539A discloses a mass spectrometry-based method for allergen quantification, it requires specialized technicians and expensive equipment.

[0005] In summary, there is still a need for a quality control kit for the preparation of crude extracts of hop pollen allergens that covers more effective molecules. This kit should be able to perform simple, rapid, and low-cost objective comparisons between crude extract reference standards and test samples, as well as qualitative or quantitative analysis of multiple allergen molecules, using conventional equipment, thereby achieving effective control of allergen components in crude extract raw materials or preparations. Summary of the Invention

[0006] Purpose of the invention: To address the shortcomings of existing technologies, the technical problem to be solved by this invention is to provide a quality control method and kit for preparing hop pollen allergen extract. The kit can not only objectively compare the extract reference standard and the prepared test sample qualitatively or quantitatively, but also further perform multiple quantifications on the monomers or combinations of allergen molecular standards. It can be used for the quality control of raw materials or preparations required for the clinical diagnosis and treatment of hop pollen allergy.

[0007] Technical Solution: To solve the above-mentioned technical problems, this invention provides a quality control method for preparing hop pollen allergen extract, which adopts the following technical solution:

[0008] S1: Prepare a reference standard of hop pollen allergen extract. The reference standard is subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to establish a characteristic gel pattern. The pattern contains multiple characteristic protein bands with molecular weights of 10~115 kDa. The molecular weight and gray value (area under curve AUC) of the bands are recorded by image analysis software as the overall quality control benchmark.

[0009] S2: Purify specific allergen molecules from hop pollen, the molecules including disease-associated protein 1 and 11kDa allergen, and prepare allergen molecule standards with gradient concentrations; the encoding gene sequence of the 11kDa allergen is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2;

[0010] S3: Using enzyme-linked immunosorbent assay (ELISA), the reference standard from step S1 is reacted with the allergen molecular standard from step S2 to quantitatively detect the content of the specific allergen molecules in the reference standard and establish a molecular-level quality control benchmark.

[0011] S4: Perform SDS-PAGE gel mapping and ELISA quantitative detection on the extract to be tested. By comparing with the characteristic gel mapping in step S1 and the molecular content benchmark in step S3, determine whether the quality of the extract to be tested is qualified. Among them, SDS-PAGE comparison requires that the characteristic band positions of the test sample and the reference sample match and the difference of the key band AUC is ≤±20%. ELISA quantitative detection requires that the content of specific allergen molecules in the test sample is within ±15% of the corresponding molecular content of the reference sample.

[0012] Furthermore, the SDS-PAGE operation conditions in S1 are as follows:

[0013] The reference standard and loading buffer were mixed at a volume ratio of 4:1 and heated at 100℃ for 10 min. 10 μl of the mixture was loaded into a 12% polyacrylamide gel and run at a constant voltage of 70V for 30 min. The voltage was then increased to 110V and run until the bromophenol blue reached the bottom of the gel. After staining with Coomassie Brilliant Blue for 2 h and destaining, the Rolling Disk Size parameter was adjusted to 5-20 using Image Lab software to eliminate the background. The bands were automatically identified and the molecular weight and AUC were calculated.

[0014] Furthermore, the specific allergen molecule is an 11kDa allergen, and the coding gene sequence of the 11kDa allergen is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0015] Furthermore, the specific allergen molecule is disease-associated protein 1.

[0016] Further, the preparation method of the allergen molecular standard in S2 is as follows: the 11kDa allergen gene is amplified by RT-PCR, cloned into the pET28a+ vector and transformed into BL21(DE3) competent cells, induced by 1mM IPTG, and then purified by ultrasonic disruption, 8M urea dissolution of inclusion bodies, nickel column affinity chromatography, gradient dialysis refolding and HiTrap Q HP anion exchange chromatography to obtain a molecular standard with a purity ≥95%; the concentration range of the molecular standard is 1ng / ml~10mg / m, and it is attached to a polystyrene microplate or nitrocellulose membrane.

[0017] Furthermore, the operation steps of the ELISA in S3 are as follows:

[0018] (1) Coating: Dilute the control to 10 μg / ml with 10 mM phosphate coating buffer (pH 7.2), and add 100 μl / well of allergen molecular standards (0.15 μg / ml, 0.075 μg / ml, 0.0375 μg / ml, 0.01875 μg / ml) to a 96-well microplate, and incubate overnight at 4°C.

[0019] (2) Blocking: After washing with PBST 3 times, add 1% BSA-PBST blocking solution, 200 μl / well, and incubate at 37℃ for 2 h;

[0020] (3) Primary antibody incubation: After washing 3 times with PBST, add 100 μl of anti-11 kDa polyclonal antibody diluted 1:10000 per well and incubate at 37°C for 2 h;

[0021] (4) Secondary antibody incubation: After washing 3 times with PBST, add 1:5000 diluted horseradish peroxidase (HRP) labeled goat anti-rabbit IgG, 100 μl / well, and incubate at 37℃ for 1 h;

[0022] (5) Color development and calculation: Add 100 μl of TMB color development solution per well, react in the dark for 5 min, add 50 μl of 2M sulfuric acid stop solution per well, read the absorbance value at 450 nm using an ELISA reader, fit the standard curve using the Excel TREND function, and calculate the content of specific allergen molecules in the control.

[0023] Furthermore, the specific criteria for determining whether the quality of the extract to be tested is qualified in S4 are as follows: SDS-PAGE comparison requires that the characteristic band positions of the test sample and the control sample match and the difference in AUC of the key band is ≤±20%; ELISA quantification requires that the content of specific allergen molecules in the test sample is within ±15% of the content of the corresponding molecules in the control sample.

[0024] The kit obtained by the above quality control method includes:

[0025] Humulus scandens pollen allergen extract reference standard, wherein the reference standard is the extract prepared above, and is accompanied by the corresponding SDS-PAGE characteristic gel pattern and band molecular weight and AUC data;

[0026] Allergen molecular standards, wherein the aforementioned disease-associated protein 1 and 11kDa allergens are attached to polystyrene microplates at gradient concentrations of 10ng / ml to 100μg / ml;

[0027] Specific detection reagents include rabbit polyclonal antibodies against 11kDa allergens and HRP-labeled goat anti-rabbit IgG;

[0028] Auxiliary reagents include 1% BSA-PBST blocking solution, PBST washing solution, TMB chromogenic solution (Beyotime Biotechnology Co., Ltd., catalog number P0209), 2M sulfuric acid stop solution, and SDS-PAGE electrophoresis reagent kit (12% polyacrylamide gel, loading buffer, Coomassie brilliant blue staining solution).

[0029] The instruction manual describes the operating parameters and judgment criteria of the quality control method described in claim 1.

[0030] In a first aspect, the present invention provides a reference standard for the extract of *Humulus scandens* pollen allergen, comprising proteins dissolved from the pollen that exhibit multiple characteristic protein bands with molecular weights ranging from 10 to 115 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This reference standard can be used for quality control by comparing the protein signal or characteristic features of the test sample. The method is characterized by comparing the signal similarity of the standard extract and the test sample extract after processing them under the same technique, including but not limited to: characteristic scanning of SDS-PAGE gel spectra, qualitative analysis of band molecular weight or quantitative matching analysis of grayscale intensity; fingerprint matching analysis of proteins in the extract based on affinity, reverse chromatography, size exclusion, ion exchange, and hydrophobic chromatography or mass spectrometry; and comparison of total protein concentration determination, etc.

[0031] In a preferred embodiment of the present invention, the preparation of the hop pollen allergen extract is to resuspend the pollen in different buffer solutions, stir and mix overnight, and then centrifuge and filter. The electrophoresis result analysis is to compare the consistency of data obtained by SDS-PAGE of the same volume of crude allergen extract control and test sample under reducing and denaturing conditions, followed by gel staining, scanning imaging, and lane analysis.

[0032] The second aspect of this invention relates to allergen molecular standards. These allergen molecules are distinct from allergen extracts; they are monomeric molecules purified, identified, and fully characterized from pollen, representing effective functional molecules. The molecules can be monomers or combinations thereof, including disease-associated protein 1 (DMA1) and 11 kDa allergens. These molecules can be obtained through purification or recombinant expression from pollen and applied in a gradient concentration range in a detection system to generate a standard curve signal. The concentration range is 1 ng / ml to 10 mg / ml, preferably 10 ng / ml to 100 μg / ml. The gradient concentrations of allergen molecular monomers or combinations are attached to a solid or liquid carrier, preferably a nitrocellulose membrane, polyvinylidene fluoride membrane, or polystyrene microplate.

[0033] In one embodiment of the present invention, the composition also includes, but is not limited to, other conventional components used in the industry for immunoreaction detection, such as diluents, detergents, enzymes or fluorescent or biotin-labeled secondary antibodies, developing agents, etc.

[0034] The third aspect of the invention also includes specific binding probes for the allergen molecules described in the second aspect, including but not limited to polyclonal antibodies, monoclonal monomers, single-domain antibodies, nanobodies, nucleic acid aptamers, peptides, or other molecules that can specifically bind to allergen proteins. The probes can be attached to a solid or liquid carrier as a first probe for capturing the target allergen in a "sandwich method," or as a detection probe for an "indirect method." The detection signal can be generated by directly or indirectly labeling substances that can produce the detected signal, such as fluorescence, enzymes, or biotin. Detection methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunofluorescence, and other specific detection methods based on immune reactions.

[0035] In one embodiment of the present invention, the specific binding probe is a rabbit polyclonal antibody of allergen molecules, which is used as a detection probe to label allergen molecule standards adsorbed in a solid-phase carrier and allergen molecules in the extract to be tested. The detection signal is indirectly generated by enzyme-labeled goat anti-rabbit second antibody and used to determine the content of specific allergen molecules in the allergen extract.

[0036] Beneficial Effects: Compared with existing technologies, the kit provided by this invention can perform quality control on Hops pollen allergen extracts at both the overall and molecular levels. At the overall level, it allows for objective quantitative comparison of the signal similarity between the control and test samples of the allergen extract, offering simple operation, low cost, and high detection efficiency. At the molecular level, the allergen molecular standard monomers and combinations provided in this kit can accurately quantify the effective molecules in Hops pollen allergen extracts without relying on expensive and complex instruments. Based on this kit, the quality and stability of Hops pollen allergen extracts can be more accurately assessed, ensuring their safety and efficacy in clinical applications. Attached Figure Description

[0037] Figure 1A The SDS-PAGE gel image characteristics of the hop pollen allergen extract phosphate (PBS) buffer standard at 6h, 12h, 24h, and 48h are shown.

[0038] Figure 1B The SDS-PAGE gel image characteristics of the Tris buffer solution for the hop pollen allergen extract at 6h, 12h, 24h, and 48h are shown.

[0039] Figure 1C The SDS-PAGE gel image characteristics of the reference standard of hop pollen allergen extract in ammonium bicarbonate (NH4) buffer at 6h, 12h, 24h, and 48h are shown.

[0040] Figure 2 The results of quantitative matching analysis of grayscale intensity of the reference standard of hop pollen allergen extract.

[0041] Figure 3 This is the quality control result of using allergen molecular standards as a reference for hop pollen allergen extract.

[0042] Figure 4 This image shows the expression of an 11 kDa recombinant protein in Hops pollen. Columns M, 1, 2, 3, and 4 represent the protein marker, uninduced whole cell control, induced whole cell control, supernatant after ultrasonic lysis of cells, and inclusion bodies after ultrasonic lysis of cells, respectively.

[0043] Figure 5 A is the spectrum of recombinant 11kDa protein separated by nickel column affinity chromatography. Columns M and 1 are protein markers, and the arrows point to the purified recombinant 11kDa protein.

[0044] Figure 5B is the chromatogram of the recombinant 11kDa protein separated by anion exchange chromatography. Columns M and 1 represent the protein marker and the purified recombinant 11kDa protein, respectively.

[0045] Figure 6 This is an application example of comparing the SDS-PAGE gel chromatogram characteristics of the hop pollen allergen extract reference standard with different samples.

[0046] Figure 7 This is an application example of using an 11 kDa allergen molecule to quantify the reference standard and the test sample of hop pollen allergen extract. Detailed Implementation

[0047] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0048] Example 1: Preparation of a control standard for hop pollen allergen extract and establishment of SDS-PAGE gel chromatography.

[0049] Hops pollen was mixed with phosphate buffer (10 mM anhydrous disodium hydrogen phosphate, 1.76 mM potassium dihydrogen phosphate, 137 mM sodium chloride, 2.68 mM potassium chloride, pH 7.2), Tris-HCl buffer (50 mM Tris, titrated to pH 8.5 with HCl), and ammonium bicarbonate buffer (50 mM ammonium bicarbonate, titrated to pH 8.0). The buffers were mixed at a volume ratio of 1:20, and then 0.1 mM of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) was added. The mixture was then ultrasonically dispersed. Crude extracts of Hops pollen were obtained at 4℃ for 6 h, 12 h, 24 h, and 48 h using different buffers. All the extracted extracts were added to centrifuge tubes, equilibrated, and centrifuged at 15000 rpm for 10 minutes at 4℃. The supernatant was collected, aliquoted, and stored at -20℃ for subsequent experiments. The control standards of *Humulus scandens* pollen extracts obtained under different conditions were separated by polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis conditions were 70V for 30 min, followed by 110V until the electrophoresis was completed. The gel was peeled off, stained with Coomassie Brilliant Blue for 2 hours, and after destaining until the background was clear, it was imaged using a chemiluminescence imaging system (Bio-rad ChemiDoc XRS+) to construct the SDS-PAGE gel map. The specific results are shown in Figure 1, and the details are as follows:

[0050] The three sub-figures correspond to three different buffer solutions. Each sub-figure shows four extraction times horizontally (6h, 12h, 24h, 48h), and the molecular weight marker (kDa) is labeled vertically. The lane numbers (1~10 correspond to different bands) are on the left side.

[0051] Figure 1A: Extraction group in 10mM phosphate buffer (PBS, pH 7.2);

[0052] Figure 1B: Extraction group in 50mM Tris-HCl buffer (pH 8.5);

[0053] Figure 1C: Extraction group in 50mM ammonium bicarbonate buffer (NH4HCO3, pH 8.0).

[0054] In all subplots, the extracts showed stable characteristic bands in the range of 10~115 kDa, with the 11kDa, 15kDa, and 25kDa bands being the clearest (corresponding to the 11kDa allergen, disease-associated protein 1, and other core sensitizing molecules).

[0055] Figure 1A (PBS group): The bands are clearly resolved, and the gray intensity of the bands increases with the extension of extraction time. The 11kDa band has the highest brightness, which proves that the PBS buffer has the best extraction efficiency.

[0056] Figure 1B (Tris group): The bands became lighter overall and decreased with prolonged extraction time (e.g., the 11kDa band became blurred), indicating that Tris-HCl caused some sensitizing proteins to be easily hydrolyzed;

[0057] Figure 1C (NH4 group): The bands became lighter with prolonged extraction time, and the band brightness was lower than that of the PBS group, indicating the second highest extraction efficiency.

[0058] The optimal preparation process for the control standard was determined to be "PBS buffer + 24h extraction" (highest band integrity and sensitizing protein content). Simultaneously, a characteristic band pattern of 10-115 kDa was established for rapid identification of whether it is *Humulus scandens* pollen extract.

[0059] Using Quantity One software, click Lane in the menu bar to generate lanes and set the lane number. Adjust the position of the red line (adjustable left, right, up, and down) as it passes through the lane, trying to pass through the middle of the lane. Adjust the Rolling Disk Size to 5-20, observe the image on the left, and try to make the black line just touch the red line, removing as much background as possible. Click Band to identify and set the band parameters, ensuring that each band on the SDS-PAGE gel image is accurately framed within the red box to obtain the characteristic peak positions and grayscale values ​​of the entire band. Select Match—Standards, click New Standard, enter the name of the created marker, and then enter the molecular weight (kDa) and corresponding name (Name) of the marker. Click Archive to save and create the marker. Click the Match menu again, select the marker name you just edited, and a list of edited markers will pop up. Click the arrow corresponding to Type 1 on the left side of the list, and then click on the marker lane corresponding to the molecular weight in the image to generate the marker lane. Click the Match menu, select Standard Curve, and then click on the location of marker lane band 1. A dialog box will pop up; select Point To Point (semi-logarithmic curve). Select all options on the right side of the dialog box to create a standard curve for the marker. Next, click on the band in the lane where you want to measure the molecular weight to obtain the corresponding molecular weight for each band. Use the Gauss Model to obtain the area under the curve for each band to accurately quantify the band grayscale value. Then, use GraphPad Prism9 to plot a quantitative grayscale intensity matching map of all band grayscale results. See the detailed results below. Figure 2 The specific explanation is as follows:

[0060] The horizontal axis represents 12 extraction conditions (6hTris, 12hTris, 24hTris, 48hTris, 6hPBS, 12hPBS, 24hPBS, 48hPBS, 6hNH4, 12hNH4, 24hNH4, 48hNH4).

[0061] The vertical axis represents the band gray value (AUC, int x mm), and AUC is positively correlated with protein content.

[0062] Curves numbered 1-10: correspond to the 10 core feature bands in Figure 1 (sorted from largest to smallest molecular weight, with band 9 corresponding to an 11kDa sensitizing molecule).

[0063] PBS+24h group: Band 9 (11kDa) had the highest AUC (approximately 196 Int x mm), which was higher than other conditions (e.g., the AUC of band 9 in the NH4+24h group was approximately 183 Int x mm).

[0064] 48h extraction group: The AUC of band 9 extracted by PBS showed limited improvement (e.g., the AUC of band 9 in the PBS+48h group was ≈202 Int x mm, slightly higher than that in the 24h group of 196 Int x mm), while it decreased under other conditions, proving that excessively long extraction is meaningless.

[0065] The core band characteristics of "PBS+24h reference standard" were identified. The 11 kDa band of No. 9 is the core band of Humulus scandens. The characteristics of subsequent test samples must match those of the standard to determine that the "overall quality is qualified".

[0066] Example 2: Preparation of allergen molecular standards and their use as a quality control enzyme-linked immunosorbent assay (ELISA) for Humulus scandens pollen allergen extract.

[0067] Preparation of allergen molecular standards: First, the 11kDa protein obtained in Example 1 was separated by SDS-PAGE gel electrophoresis and stained with Coomassie Brilliant Blue R250. Protein bands were excised from the gel, digested with trypsin, and analyzed by mass spectrometry. The raw data was analyzed using pFind software to obtain six internal peptides. These peptides were matched with transcripts to obtain the theoretical cDNA sequence encoding the 11kDa protein, which was further confirmed by TA cloning and Sanger sequencing. Specifically, total RNA was extracted from Hops pollen and reverse transcribed to obtain cDNA. Based on the matched theoretical cDNA sequence, primers for PCR amplification were designed: the upstream primer was 5′-CACCATATTCATATCCAT-3′, and the downstream primer was 5′-TTTCAAGTCAGGTTTCG-3′. PCR conditions were as follows: 98℃ pre-denaturation / 5s (1 cycle), 98℃ denaturation / 10s, 55℃ annealing / 30s, and 72℃ extension / 1min (30 cycles), followed by 72℃ final extension / 10min (1 cycle). The PCR amplification product was recovered, purified, and ligated into the pCE2 TA / Blunt plasmid vector, which was then transformed into JM109 competent cells. Positive clones were screened on Luria-Bertani (LB) plates containing 100 µg / ml kanamycin, and confirmed by DNA sequencing. The gene is 336 bp in length, encoding 111 amino acids. Its nucleotide sequence is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2.

[0068] SEQ ID NO: 1:

[0069] caccatattc atatccatta atttatttgt aagaaatata gaacaaacaa aatggcgagc 60

[0070] aagatggttg tcgttttcgc cattttccta gtcgtttttg cagcactcaa cgtgagtgaa 120

[0071] tgcaatgctg acaactgttt tgaaaacggt atgaaagcat gcactagttt atatgataag 180

[0072] tattatcaaa actgtgtaat gaaactaccg ccaggggcat gtattgatag tgagaattat 240

[0073] cgaaagtgcc taacgaacca tattggtagc tgcgatattg acacctgctt tgaagacgtt 300

[0074] tcgatagcat gccgtagtat ttatccgagt aattatgcag aatgcgcaac aacacatcat 360

[0075] aatatctgtg gtgatttgca aggatgacga aaaacttaat atatcaaaaa ctgaagtgag 420

[0076] cagcgaaacc tgacttgaaa 440

[0077] SEQ ID NO: 2:

[0078] Met Ala Ser Lys Met Val Val Val Phe Ala Ile Phe Leu Val Val Phe 1 5 10 15

[0080] Ala Ala Leu Asn Val Ser Glu Cys Asn Ala Asp Asn Cys Phe Glu Asn 20 25 30

[0082] Gly Met Lys Ala Cys Thr Ser Leu Tyr Asp Lys Tyr Tyr Gln Asn Cys 35 40 45

[0084] Val Met Lys Leu Pro Pro Gly Ala Cys Ile Asp Ser Glu Asn Tyr Arg 50 55 60

[0086] Lys Cys Leu Thr Asn His Ile Gly Ser Cys Asp Ile Asp Thr Cys Phe 65 70 75 80

[0088] Glu Asp Val Ser Ile Ala Cys Arg Ser Ile Tyr Pro Ser Asn Tyr Ala 85 90 95

[0090] Glu Cys Ala Thr Thr His His Asn Ile Cys Gly Asp Leu Gln Gly 100 105 110

[0092] The nucleotide sequence of the 11kDa protein, after removing the coding region of the signal peptide, was cloned and ligated between the NcoⅠ and Xhol Ⅰ sites on the pET28a+ plasmid. The constructed recombinant allergen plasmid was heat-shock transformed into BL21(DE3) host bacteria for plate cloning. Single colonies were picked and cultured in liquid medium at 37°C and 200 rpm until the OD600 reached 0.5. IPTG inducer was added to a final concentration of 1 mM, and the culture was continued for 4 hours. SDS-PAGE electrophoresis results showed that the protein was mainly expressed in inclusion bodies after induction. Detailed detection results are shown below. Figure 4 The specific explanation is as follows:

[0093] Meaning of swimming lanes:

[0094] M: Molecular weight marker (labeled as 180, 130, 100, 70, 55, 40, 35, 25, 20, 15 kDa);

[0095] 1: Uninduced whole cell control (no 11kDa band);

[0096] 2: IPTG-induced whole-cell control (clear band appears at 11kDa);

[0097] 3: Supernatant after ultrasonic lysis of bacterial cells (very faint 11kDa band).

[0098] 4: Inclusion bodies after ultrasonic lysis of bacterial cells (11kDa band is clear, proving that the protein is mainly expressed in inclusion bodies).

[0099] The 11kDa recombinant protein is mainly expressed in inclusion bodies (four clear bands in lane 4), and needs to be dissolved in 8M urea for purification (Example 2 clearly states "dissolve inclusion bodies in 8M urea solution").

[0100] Bacterial cells were collected by centrifugation, resuspended in 8-10 ml of PBS buffer, and then lysed by sonication at 70% power for 30 minutes, pausing for 5 seconds every 9 seconds. The precipitate was collected and dissolved in 8M urea solution. After centrifugation, the supernatant was used for preliminary purification by nickel affinity chromatography. Elution was performed using a gradient of imidazoles at different concentrations. The purification effect was verified by SDS-PAGE gel electrophoresis. The eluates containing the target protein were combined and then subjected to a gradient dialysate containing 6M-4M-2M-0M urea for refolding. Finally, the protein was further purified by linear gradient elution with 20 mM Tris-1 M NaCl (pH 8.5) on an AKTA-go system using a HiTrapQ HP anion exchange chromatography column to obtain a high-purity 11 kDa protein as a standard. The detection results are shown in [link to results]. Figure 5 As shown below,

[0101] Figure 5A: Results of nickel column affinity chromatography purification (preliminary purification);

[0102] Figure 5B: HiTrap Q HP anion exchange chromatography purification results (fine purification);

[0103] Lane meaning: M = Molecular weight marker, 1 = Purified 11kDa recombinant protein (arrow indicates target band)

[0104] Figure 5A: After nickel column purification, a single main band appears at 11 kDa (with very few impurities);

[0105] Figure 5B: After anion exchange purification, only a single 11kDa band is retained, with a purity ≥95% (Example 2 clearly states "obtain a high-purity 11kDa protein as a standard").

[0106] Polyclonal antibodies against the 11kDa recombinant protein were prepared by immunizing New Zealand white rabbits, commissioned to Nanjing Zhongding Biotechnology Co., Ltd. The main preparation method is as follows: Male New Zealand white rabbits weighing approximately 2.5kg were selected. The initial immunization regimen involved subcutaneously immunizing the rabbits with an emulsion prepared by mixing 0.3-0.5mg of recombinant protein with an equal volume of Freund's complete adjuvant. Subsequent booster immunizations involved subcutaneously immunizing the rabbits with an emulsion prepared by mixing an equal volume of recombinant protein with Freund's incomplete adjuvant, once a week for three weeks. Serum from the immunized rabbits was collected within 10 days after the last injection; this serum contained as anti-recombinant protein IgG antibody was obtained from the serum.

[0107] Different buffer standards of *Humulus scandens* pollen allergen extract were diluted to 10 μg / ml using coating buffer (10 mM phosphate, pH 7.2). 100 μl of the solution was added to each well of a 96-well microplate, with two replicates per well. The plates were coated overnight with 11 kDa recombinant protein at concentrations of 0.15, 0.075, 0.0375, and 0.01875 μg / ml. The plates were then blocked with PBST (PBS containing 0.1% Tween 20) containing 1% bovine serum albumin (BSA) at 37°C for 2 h. After washing with PBST, anti-11 kDa recombinant protein antibody (1:10000 dilution) was added and incubated at 37°C for 2 h. Finally, the plates were incubated with 100 μL of goat anti-rabbit IgG-HRP (1:5000 dilution) at 37°C for 1 h. After washing, 100 μl of TMB chromogenic solution (Beyotime Biotechnology Co., Ltd., catalog number: P0209) was added and reacted for 5 min. The reaction was then terminated by adding 50 μl of 2M sulfuric acid solution. The absorbance of each well at 450 nm was read using a Multiskan microplate reader (Thermo Fisher Scientific). The TREND function in Excel was used to calculate the 11 kDa allergen molecule content in different buffers of the *Humulus scandens* pollen allergen extract. The results showed that the concentrations of 11 kDa protein in the allergen extract of *Humulus scandens* pollen in different buffers were 0.126 mg / ml (PBS), 0.144 mg / ml (Tris), and 0.152 mg / ml (NH4), respectively. See below for detailed results. Figure 3 The specific explanation is as follows:

[0108] Meaning of coordinate axes:

[0109] x-axis: 3 types of buffer solutions (PBS, Tris, NH4);

[0110] The vertical axis represents the absorbance value at 450 nm (OD value), which is positively correlated with the 11 kDa allergen concentration.

[0111] Column: Mean OD values ​​of different buffer groups (3 parallel experiments).

[0112] 11kDa allergen concentrations: PBS group 0.126mg / ml (OD value highest ≈0.15), Tris group 0.144mg / ml (OD value ≈0.13), NH4 group 0.152mg / ml (OD value ≈0.14);

[0113] Trend: Consistent with Figures 1 and 2, although the concentration of the PBS group was slightly lower, it may be due to differences in extraction batches. This also shows the effectiveness of quality control based on 11kDa molecules, and it was selected as the optimal buffer solution.

[0114] Establish a "11kDa acceptable concentration range for allergen molecules" (e.g., 0.12~0.13mg / ml for PBS group) to provide a quantitative basis for subsequent molecular-level quality control of test samples.

[0115] Example 3: Quality control kit for preparing hop pollen allergen extract and its practical application.

[0116] This invention provides a quality control kit for the preparation of allergen extracts from *Humulus scandens* pollen. The kit employs a multi-level quality control strategy, including reference standard comparison and standard quantification, to ensure the quality stability and effectiveness of the allergen extract. The kit includes a standardized crude *Humulus scandens* pollen extract reference standard. A stable SDS-PAGE gel electrophoresis pattern is established through optimized extraction process, allowing for direct comparison of band characteristics in the test samples. Precise quantification of the target component is achieved by using an 11kDa protein antibody to determine the content of key allergens at the 11kDa level in the test samples. The kit includes: *Humulus scandens* pollen extract reference standard, 11kDa recombinant protein standard, anti-11kDa polyclonal antibody, HRP-labeled goat anti-rabbit secondary antibody, ELISA plate, TMB chromogenic solution, ELISA stop solution, and an SDS-PAGE gel electrophoresis kit.

[0117] Operating steps:

[0118] I. Comparison of SDS-PAGE gel map features

[0119] 1. Sample preparation: Dilute the test sample and the Humulus scandens pollen extract reference standard with PBS buffer to the same concentration, add an equal volume of loading buffer at a ratio of 4:1, and boil in a metal bath at 100°C for 10 minutes.

[0120] 2. Electrophoresis conditions: Load 10µl per well, run at 70V for 30 minutes, then adjust to 110V until bromophenol blue reaches the bottom of the gel; after peeling off the electrophoresis gel, stain with Coomassie Brilliant Blue for 2 hours, then destain with pure water until the background is clear;

[0121] 3. Results Analysis: The gel obtained in the previous step was imaged using a chemiluminescence imaging system (Bio-rad ChemiDoc XRS+). The position of the bands was compared using Quantity One software, the gray value of the target band was calculated, and the difference between the test sample and the control was compared.

[0122] II. ELISA Quantitative Detection

[0123] 1. Coating: Coat the protein standard (serial dilution), control standard of the same concentration and the sample to be tested with PBS buffer and add to the microplate, 100 µl / well, and incubate overnight at 4°C;

[0124] 2. Blocking: After washing three times with 200µl / well of PBST, add 200µl / well of 1% BSA-PBST solution and block at 37℃ for 2 hours;

[0125] 3. Primary antibody incubation: After washing three times with 200µl / well of PBST, add 100µl / well of anti-11kDa protein antibody (1:10000 dilution) and incubate at 37°C for 2 hours;

[0126] 4. Secondary antibody incubation: After washing three times with 200µl / well of PBST, add 100µl / well of HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution) and incubate at 37°C for 1 hour;

[0127] 5. Color development and measurement: After washing three times with 200µl / well of PBST, add 100µl / well of TMB color development solution, react in the dark for 5 minutes, then add 50µl / well of stop solution, and measure the OD value at 450nm using a microplate reader.

[0128] 6. Results Analysis: A standard curve was plotted using the concentration of the standard, and the content of the standard protein in the test sample was calculated and compared with the control.

[0129] Example 4: Application of a quality control kit for preparing hop pollen allergen extract

[0130] 1. Comparison of SDS-PAGE gel chromatogram characteristics of Humulus scandens pollen allergen extract reference standard and different samples

[0131] The *Humulus scandens* pollen allergen extract prepared in Example 1 underwent extraction with ammonium bicarbonate buffer for 6 hours as a control. This control was diluted 2-fold to serve as Sample 1 for kit performance validation. Crude extracts of *Artemisia capillaris* pollen and *Ragnarökyi Nyctaginata* pollen were prepared under the same conditions as the control, and diluted 5-fold to serve as Samples 2 and 3 for specificity validation of non-*Humulus scandens* species in the kit. SDS-PAGE gel chromatography was constructed for Samples 1, 2, and 3 according to the method in Example 1. The characteristic bands were compared with those of the control. It was found that the characteristic band position of Sample 1 (the diluted control) matched that of the control. However, the area under the curve of the key peak at 11 kDa was 242 Int x mm for the control and 61.3 Int x mm for Sample 1. Since Sample 1 was significantly lower than the control, the quality control was deemed unsuccessful. The characteristic bands of Sample 2 (crude extract of Artemisia capillaris) and Sample 3 (crude extract of Ragnarok mongholicus) were not located in the same positions as the control, indicating that they were not Hops pollen samples and therefore failed quality control. See details below. Figure 6 In the electrophoresis diagram, M represents the standard molecular weight, the reference standard is the hop allergen extract standard, and samples 1-3 are diluted hop allergen standards and allergen extracts from unrelated species (Artemisia annua and Artemisia argyi pollen), respectively. The grayscale feature map is the band distribution feature map drawn after each sample is scanned by the swimming lanes.

[0132] 2. Comparison of 11kDa protein monomer content determination between crude extract of Hops pollen reference standard and test sample

[0133] The 11kDa protein content of the *Humulus scandens* pollen allergen extract reference standard prepared in Example 1, and samples 1, 2, and 3 (crude extracts of *Humulus scandens* pollen diluted 2-fold, crude extracts of *Artemisia capillaris* pollen, and crude extracts of *Ragnarok* pollen) was determined by ELISA. The results showed that the 11kDa protein content of the *Humulus scandens* pollen allergen extract reference standard was 0.067 mg / ml, that of sample 1 (crude extract of *Humulus scandens* pollen diluted 2-fold) was 0.038 mg / ml, and that of samples 2 (crude extract of *Artemisia capillaris* pollen) and 3 (crude extract of *Ragnarok* pollen) was 0 mg / ml. Since the 11kDa protein content of samples 1, 2, and 3 was lower than that of the reference standard, the quality control of these three samples failed. See [details omitted]. Figure 7 The x-axis represents the control standard (Humulus scandens pollen allergen extract), sample 1 (diluted Humulus scandens pollen allergen standard), and samples 2-3 (allergen extracts from unrelated species, Artemisia annua and Artemisia argyi pollen). The y-axis represents the concentration level of the 11 kDa core allergen from Humulus scandens detected using this kit.

[0134] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A quality control method for preparing hop pollen allergen extract, characterized in that: Includes the following steps: S1: Prepare a reference standard of hop pollen allergen extract. The reference standard is subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to establish a characteristic gel pattern. The molecular weight and gray value of the bands are recorded as the overall quality control benchmark. S2: Purify specific allergen molecules from hop pollen and prepare allergen molecule standards with gradient concentrations; S3: Using enzyme-linked immunosorbent assay (ELISA), the reference standard of S1 is reacted with the allergen molecular standard of S2 to quantitatively detect the content of the specific allergen molecule in the reference standard and establish a molecular-level quality control benchmark. S4: Perform SDS-PAGE gel mapping and ELISA quantitative detection on the extract to be tested. By comparing with the characteristic gel mapping of S1 and the molecular content benchmark of S3, determine whether the quality of the extract to be tested is qualified.

2. The quality control method for preparing a hop pollen allergen extract according to claim 1, characterized in that: The preparation method of the reference standard in S1 is as follows: mix hop pollen with buffer solution, add phenylmethylsulfonyl fluoride (PMSF), perform a rotational extraction gradient time, centrifuge to collect the supernatant and store it separately.

3. The quality control method for preparing a hop pollen allergen extract according to claim 1, characterized in that: The SDS-PAGE operation conditions in S1 are as follows: The reference standard was mixed with loading buffer, heated, and loaded onto a polyacrylamide gel. The mixture was run until bromophenol blue reached the bottom of the gel. After Coomassie brilliant blue staining and destaining, the Rolling Disk Size parameter was adjusted using Image Lab software to automatically identify the bands and calculate the molecular weight and AUC.

4. The quality control method for preparing a hop pollen allergen extract according to claim 1, characterized in that: The specific allergen molecule is an 11kDa allergen, and the coding gene sequence of the 11kDa allergen is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:

2.

5. The quality control method for preparing a hop pollen allergen extract according to claim 1, characterized in that: The specific allergen molecule is disease-associated protein 1.

6. The quality control method for preparing a hop pollen allergen extract according to claim 4, characterized in that: The preparation method of the allergen molecular standard in S2 is as follows: the 11kDa allergen gene is amplified by RT-PCR, cloned into the pET28a+ vector and transformed into BL21 competent cells. After expression is induced by 1mM IPTG, the molecular standard is obtained by sonication, dissolution of inclusion bodies with 8M urea, nickel column affinity chromatography, gradient dialysis refolding and purification by HiTrap Q HP anion exchange chromatography. The concentration range of the molecular standard is 1ng / ml to 10mg / ml, and it is attached to a polystyrene microplate or nitrocellulose membrane.

7. The quality control method for preparing a hop pollen allergen extract according to claim 4, characterized in that: The operation steps of the ELISA in S3 are as follows: (1) Coating: Dilute the control with phosphate coating solution and add 100 μl / well of allergen molecular standards of gradient concentration to the microplate and incubate overnight; (2) Blocking: Wash with PBST, add BSA-PBST blocking solution, and incubate; (3) Primary antibody incubation: Wash with PBST, add diluted anti-11kDa polyclonal antibody, and incubate; (4) Secondary antibody incubation: Wash with PBST, add diluted horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG, and incubate; (5) Color development and calculation: Add TMB color development solution, react in the dark, add sulfuric acid stop solution, read the absorbance value with an ELISA reader, fit the standard curve using the Excel TREND function, and calculate the content of specific allergen molecules in the control.

8. The quality control method for preparing a hop pollen allergen extract according to claim 4, characterized in that: The specific criteria for determining whether the quality of the extract to be tested is qualified in S4 are as follows: SDS-PAGE comparison requires that the characteristic band positions of the test sample and the control sample match and the difference in AUC of the key band is ≤±20%; ELISA quantification requires that the content of specific allergen molecules in the test sample is within ±15% of the corresponding molecule content of the control sample.

9. A reagent kit obtained by any one of the quality control methods described in claims 1 to 8, characterized in that, The kit includes: A reference standard for hop pollen allergen extract, wherein the reference standard is the extract prepared according to claim 2, and is accompanied by the corresponding SDS-PAGE characteristic gel pattern and band molecular weight and AUC data; Allergen molecular standards, wherein the standards are the disease-related protein 1 and 11kDa allergen prepared according to claim 4, which are attached to polystyrene microplates at gradient concentrations of 10ng / ml to 100μg / ml; Specific detection reagents include rabbit polyclonal antibodies against 11kDa allergens and HRP-labeled goat anti-rabbit IgG; Auxiliary reagents and SDS-PAGE electrophoresis reagent kit.

10. A reagent kit obtained by any one of the quality control methods described in claims 1 to 8, characterized in that, The specific detection reagent includes allergen-specific binding antibodies, including polyclonal antibodies, monoclonal monomers, single-domain antibodies, and nanobodies that can specifically bind to allergen proteins. The detection signal can be generated by directly or indirectly labeling fluorescent or enzyme-based substances that can produce the detected signal.

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