Application of allergen Blo t 24 in dust mite allergen component detection

Abstract

The invention discloses an application of an allergen Blo t 24 in detection of dust mite allergen components. The invention finds that the positive rate of the allergen Blo t 24 is equivalent to that of the allergen Blo t 5 in newly identified allergens, so that the Blo t 24 can be used for detecting allergen components, and the positive rate is as high as 91.36% when a combination formed by the allergens Blo 5, Blo t 21, Blo t 24 and Blo t 2 is used for detecting the allergen components.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease detection drug development, and in particular relates to an application of an allergen Blot 24 in the detection of dust mite allergen components. Background Art

[0002] Dust mite allergy affects a large number of people worldwide. It is estimated that more than 20% of people have experienced related symptoms, and more than 30% are in an asymptomatic sensitized state. The prevalence of dust mite allergy continues to rise, becoming a significant global public health burden. However, current allergy diagnosis and specific immunotherapy rely primarily on natural dust mite extracts, which have certain limitations. Dust mite extracts are mixtures of allergens and non-allergen components, making it difficult to accurately identify specific allergenic components. In addition, differences in extraction methods and storage conditions can lead to unstable components. This can lead to false-negative results in some patients, thus affecting the accuracy of diagnosis and the efficacy of immunotherapy.

[0003] To overcome these issues, there has been increasing interest in recent years in using single or combined allergen components for the precise diagnosis and treatment of allergies. This strategy relies on an in-depth analysis of various mite allergens to ensure consistent and effective diagnosis and treatment. However, the success of this approach relies on the precise identification of mite allergens, as each mite species typically contains approximately 40 different allergens, and in some species, a large number of allergens remain undiscovered.

[0004] Tropical Anonymous Mite ( Blomia tropicalis ) belongs to the family Echimyopodidae and is a common house dust mite found in tropical and subtropical regions, including the southern states of the United States. B. tropicalis Among them, allergens of types 5, 7, and 21 were identified as the main sensitizing components. Among them, the sensitization rate of type 5 allergen (Blo t 5) was particularly high, reaching 92.8% in the Brazilian population and even 96.7% in Singaporean asthma patients. Although B. tropicalis is less common in temperate regions, it can still be detected at a certain rate. For example, a study in northern China showed that in patients with asthma and / or allergic rhinitis, the sensitization rate to B. tropicalis was higher than that in patients with asthma and / or allergic rhinitis. B. tropicalis The proportion of positive skin prick tests was 28.5%.

[0005] IgE antibodies specific for a particular allergen are the primary effector molecules in type I hypersensitivity reactions. They bind to Fc receptors (FcRⅠ) on the surfaces of effector cells such as mast cells and basophils through their Fc domains. Inflammatory mediators such as histamine, prostaglandins, and leukotrienes released by these cells are the direct triggers of asthma, rhinitis, and dermatitis. IgE antibody levels are very low in normal human serum, but are significantly elevated in patients with type I hypersensitivity reactions. Following the discovery of IgE antibodies in the 1990s, the radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA), and UniCap assays were developed. In vitro detection of serum IgE antibodies has become the primary diagnostic method for type I hypersensitivity reactions. However, to date, both in vitro and in vivo diagnostic methods for type I hypersensitivity reactions have relied on mixtures of crude allergen extracts. Using this crude extract, it is possible to identify a patient's sensitization to a specific allergen, but it is not possible to pinpoint the specific allergen molecule responsible. Using this crude extract also fails to clearly identify whether a patient is allergic to multiple allergens simultaneously or to a single allergen with a cross-reactive effect. Therefore, determining which allergen component triggers elevated IgE is the primary concern in diagnosing type I hypersensitivity reactions. Only by identifying the allergen component that triggers the sensitization can immunotherapy be performed using the appropriate allergen component.

[0006] The use of modern immunological technology to detect the allergen components of sensitized organisms is called component-resolved diagnosis (CRD), which can clearly identify the allergen components that cause the disease in the organism, clarify the specific IgE reaction titer of each patient to different allergens in the crude extract mixture, and monitor the effect of immunotherapy in order to adjust the dosage and type of allergens in a timely manner. The prerequisite for establishing a component-resolved diagnosis method is to obtain pure allergens that bind to the body's specific IgE. In the past, biochemical methods were used to purify allergens, which required the breeding of mites to a certain density, which was time-consuming, cumbersome, and costly, and could not fundamentally improve the purity of allergens. In recent years, with the rapid development of molecular biology technology, a variety of recombinant allergens have emerged, and it has been proven that they are stable in nature, have no genetic or biological variations, and have high yields. They can completely replace traditional natural allergens for the clinical diagnosis and treatment of type I hypersensitivity diseases. Component-resolved diagnosis methods have therefore developed rapidly. Component-resolved diagnosis, also known as allergen molecular diagnosis (Molecular Diagnosis), https: / / www.thermofisher.com / phadia / wo / en / resources / allergen-encyclopedia.html?type=Component&search=blomia Only one component, Acyrthosaurus tropicalis, is listed in the FDA's ... Summary of the Invention

[0007] The purpose of the present invention is to provide an application of the allergen Blot 24 in the detection of dust mite allergen components, aiming to solve the problem of tropical dung mite allergy diagnosis.

[0008] The present invention is achieved by using the allergen Blot 24 in the preparation of dust mite allergen components or allergic disease detection reagents.

[0009] Preferably, the combination of the allergen Blo t 24, allergen Blo t 5, allergen Blo t 21 and allergen Blo t 2 is used in the preparation of a detection reagent for dust mite allergic diseases.

[0010] Preferably, the dust mite comprises Acyrthodes tropicalis.

[0011] The present invention further discloses a detection reagent for dust mite allergens, wherein the reagent comprises a coating antigen consisting of the allergen Blo t24.

[0012] Preferably, the coating antigens further include allergens Blo t 5, Blo t 21 and Blo t 2.

[0013] The present invention further discloses a method for detecting dust mite allergens, which comprises: using Blo t 24 as a coating antigen and establishing an indirect ELISA method to detect specific IgE in serum.

[0014] Preferably, the coating antigens further include allergens Blo t 5, Blo t 21 and Blo t 2.

[0015] The present invention overcomes the shortcomings of the existing technology and provides an application in the detection of dust mite allergen components. Chromosome-level genome assembly technology, combined with advanced allergen discovery and verification methods, has been proven to be a powerful strategy that helps to analyze allergens more comprehensively and accurately. The present invention further expands the application of this strategy and constructs B. tropicalis A chromosome-level genome assembly of the 1997 Allergens (BioProject ID PRJNA911905) was constructed and 39 genes encoding allergens were predicted, including nine newly identified allergens. These allergens have been clinically validated using recombinant proteins and officially accepted by the World Health Organization / International Union of Immunological Societies (WHO / IUIS) Allergen Nomenclature Panel. Among these newly identified allergens, the positive rate for allergen Blo t 24 is comparable to that for allergen Blo t 5. Therefore, Blo t 24 can be used for the detection of allergen components. Furthermore, a combination of allergens Blo t 5, Blo t 21, Blo t 24, and Blo t 2 has a positive rate of 91.36%. In the present invention, the allergens Blot 24, Blot 5, Blot 21 and Blot 2 can be recombinant proteins produced by genetic engineering methods, or can be natural proteins extracted from mites by biochemical methods.

[0016] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following advantages: Blo t 24 of the present invention can be used to detect allergen components with a high positive rate. In addition, a combination of allergens Blo 5, Blo t 21, Blo t 24, and Blo t 2 can be used to detect allergen components with a positive rate of up to 91.36%. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the agarose gel electrophoresis diagram of the total RNA extracted from Acyrthosaurus tropicalis; Lane M: DL2.000 DNA Marker; Lane 1: Total RNA.

[0018] Figure 2 pET28a(+)-plasmid used Nde I / Eco Agarose gel electrophoresis of double enzyme digestion reaction with RI; Lane M: l -Hin d Ⅲ digest Marker, Lane 1: pET28a(+)- Nde I / Eco RI.

[0019] Figure 3SDS-PAGE identification of pET28a(+)-Blo t 24 expressed in Rosetta2(DE3)pLysS; Lane 1: pET28a(+) empty vector control induced by IPTG, Lane 2: pET28a(+)-Blo t 24 transformed into Rosetta2(DE3)pLysS and clone 1 induced by IPTG, Lane 3: pET28a(+)-Blo t 24 transformed into Rosetta2(DE3)pLysS and clone 2 induced by IPTG, Lane 4: pET28a(+)-Blo t 24 clone 3 transformed into Rosetta2(DE3)pLysS and induced by IPTG. Lane M is a protein molecular weight marker.

[0020] Figure 4 This is a soluble analysis of pET28a(+)-Blo t 24 expressed in Rosetta2(DE3)pLysS; Lane 1: supernatant of pET28a(+)-Blo t 24 expressed in Rosetta2(DE3)pLysS, Lane 2: precipitate of pET28a(+)-Blo t 24 expressed in Rosetta2(DE3)pLysS.

[0021] Figure 5 pET28a(+)-Blot 24 was expressed in Rosetta2(DE3)pLysS E. coli Denaturing purification electrophoresis diagram; Lane M: Protein MW marker (Broad), Lane 1: elution, Lane 2: 20-1 (imidazole), Lane 3: 20-2 (imidazole), Lane 4: 50-1 (imidazole), Lane 5: 50-2 (imidazole), Lane 6: 150-1 (imidazole), Lane 7: 150-2 (imidazole), Lane 8: 250-1 (imidazole), Lane 9: 250-2 (imidazole), Lane 10: column material.

[0022] Figure 6 The recombinant protein rBlo t 24 was obtained by SDS-PAGE identification and purification.

[0023] Figure 7 The purified recombinant protein rBlo t 24 was identified by Western blotting.

[0024] Figure 8 This is the endotoxin test report of recombinant protein rBlo t 24.

[0025] Figure 9 This represents the positive rate of recombinant allergen Blo t 24 detected by IgE-Western blotting. Lane M is a protein molecular weight marker, lanes 1-10 represent sera from patients who tested positive for A. tropicalis crude extract, and lanes 11-13 represent sera from patients who tested negative for A. tropicalis crude extract. IgE-Western blotting revealed that lanes 1-7 represent sera from patients who tested positive for rBlo t 24, demonstrating that Blo t 24 binds to human serum.

[0026] Figure 10 These are the results of indirect ELISA detection of tropical anonymus mite allergen components Blo t 1, Blo t 2, Blo t 5, Blo t17, Blo t 21, and Blo t 24. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] 1. Cloning of the Blo t 24 gene, the tropical mite allergen 1. Total RNA extraction Total RNA from A. tropicalis was extracted using the TaKaRa MiniBEST Universal RNA Extraction Kit (Cat. No. 9767). 5 ml of the sample was subjected to agarose gel electrophoresis. The results were as follows: Figure 1 shown.

[0029] 2. Obtaining target genes 2.1. The synthetic primers are shown in Table 1 below.

[0030] Table 1. Synthetic primers

[0031] 2.2 Reverse transcription cDNA was synthesized using the TakaRa PrimeScript™ RT-PCR Kit (Cat. No. RR014A). The reaction mixture consisted of 1 mL of total RNA, 1 mL of 20 mM Random Six Mers, 1 mL of 2.5 mM Oligo dT Primer, and 1 mL of 10 mM dNTP Mixture. RNase-free dHO was then added to the final volume (10 mL). The reaction was incubated at 65°C for 5 minutes, followed by 2 minutes of incubation on ice.

[0032] Then, add the following components: 4 mL 5´ PrimeScript RT Buffer, 0.5 mL RNase Inhibitor (40 U / μl), 0.5 mL PrimeScript RTase (for 2 Steps), and RNase-free dH2O to 20 mL. Reaction conditions: 30°C for 10 min, 42°C for 30 min, 95°C for 5 min, and 70°C for 15 min.

[0033] 2.3 PCR amplification PCR amplification was performed using TaKaRa Tks Gflex DNA Polymerase (Cat. No. R060A). The reaction system consisted of 1 mL of the aforementioned reverse transcription cDNA reaction solution, 25 mL of 2× Gflex PCR Buffer (Mg2+, dNTPs plus), 1 mL of Tks Gflex DNA Polymerase (1.25 units / mL), 1 mL of F0 primer (20 pmol / μl), 1 mL of R0 primer (20 pmol / mL), and 21 mL of dH2O, for a total reaction volume of 50 mL. Reaction conditions: 94°C for 1 minute, followed by 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 68°C for 3 minutes. A 5 mL aliquot was analyzed by agarose gel electrophoresis.

[0034] The DNA was recovered from the gel using Takara MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Code No.9762), and F0 and R0 were used for sequencing.

[0035] 3. Target gene amplification (without signal peptide) 3.1. Primer synthesis is shown in Table 2 below.

[0036] Table 2 Synthetic primers

[0037] 3.2 PCR amplification TaKaRa Tks Gflex DNA Polymerase (Cat. No. R060A) was used for PCR amplification. Reaction system: 1 mL of the above reverse transcription cDNA reaction solution, 25 mL of 2× Gflex PCR Buffer (MgCl2 2+ , dNTP plus), 1 mL TksGflex DNA Polymerase (1.25 units / mL), 1 mL INF primer (20 pmol / mL), 1 mL INR primer (20 pmol / mL), and 21 mL dH2O for a total reaction volume of 50 mL. Reaction conditions: 94°C for 1 minute, followed by 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 68°C for 3 minutes. 5 mL of PCR product was analyzed by 1% agarose gel electrophoresis.

[0038] The above product was purified by gel excision using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Code No.9762).

[0039] 4. Vector DNA Preparation 4.1. Vector digestion Use for pET28a(+) empty plasmid Nde I / Eco RI double enzyme digestion reaction, the reaction system and conditions are as follows: 6 mgpET28a (+) plasmid, 10 mL 10 × H Buffer, 5 mL Nde I (10 U / mL), 5 mL Eco RI (10 U / mL) was added to 100 mL with dH2O. Reaction conditions: 37°C for 4 hours.

[0040] 4.2 Vector Purification The TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Code No.9762) was used to extract and purify the approximately 5.4 kbp enzyme digestion product. 1 mL of the product was subjected to 1% agarose gel electrophoresis. The results were as follows: Figure 2 shown.

[0041] 5. In-Fusion, transformation, positive clone screening and plasmid sequencing Using the In-Fusion HD Cloning Kit (Clontech, Code No. 639650), the purified PCR product was reacted with the digested pET28a(+) empty plasmid in an In-Fusion reaction. The reaction system consisted of 1 mL of pET28a(+) empty plasmid digested product (approximately 50 ng / mL), 2 mL of purified PCR product (approximately 20 ng / mL), and 2 mL of 5´ In-Fusion HD Enzyme Premix, for a total of 5 mL. Reaction conditions: 50°C for 15 min.

[0042] Take 2.5mL of the above In-Fusion reaction solution and heat-convert it to E. coli Spread the plate with HST08 Premium CompetentCells (Cat. No. 9128) and incubate overnight at 37°C. Select positive colonies, culture them, extract the plasmids, and sequence them using T7 sequencing.

[0043] 2. Expression of Tropical Anonymous Mite Allergen Blo t 24 Protein 1. Conversion Transfer 1 mL of plasmid pET28a(+)-Blood 24 into Rosetta2(DE3)pLysS. Spread 40 mL of the transformation solution onto LB plates containing 100 mg / mL Kanamycin and 34 mg / mL Chloramphenicol. Incubate at 37°C O / N. Repeat the same procedure for pET28a(+).

[0044] 2. Cultivation and induction 2.1. Seed cultivation Pick four single colonies (pET28a (+) empty vector control, clone 1, clone 2, and clone 3) and add them to 3 ml of LB / Kanamycin (100 mg / mL) + Chloramphenicol (34 mg / ml) medium and culture at 37°C O / N.

[0045] 2.2 Small-amount induction Add 5 ml of LB / Kanamycin (100 mg / mL) + Chloramphenicol (34 mg / mL) medium to a glass tube and add 100 ml of the seed culture. Incubate at 37°C until the OD600 nm value reaches approximately 0.6. Induce the clones by adding 50 ml of 100 mM IPTG (final 1 mM IPTG) and incubate at 37°C for 4 hours.

[0046] 2.3 Bacterial electrophoresis After collecting the bacteria, take 100 mL of the induced bacterial solution and centrifuge to separate the bacteria and culture medium. Discard the supernatant, add 40 mL of 50 mM Tris, 500 mM NaCl, pH 8.0 to the bacteria, resuspend them, add 10 mL of 5×SDS Loading Buffer, heat at 100°C for 15 minutes, and perform SDS-PAGE electrophoresis. Centrifuge the bacterial cell suspension (12,000 ´ rpm, 5 minutes). Electrophoresis conditions: constant voltage 220V, about 35 minutes. Use gel: 8%-20% polyacrylamide gel. After electrophoresis, stain with CBB-R250 and decolorize with decolorizing solution. The SDS-PAGE / CBB staining results are as follows: Figure 3 shown.

[0047] 3. Purification of the tropical anonymite allergen Blo t 24 recombinant protein 1. 750 ml × 2 bacterial culture experiment 1.1、Seed cultivation Take 2 mL of pET28a(+)-Blo t 24 glycerol culture and inoculate them into 10 ml of LB / Kanamycin (100 mg / mL) + Chloramphenicol (34 mg / ml) medium and culture at 37℃ O / N.

[0048] 1.2 Main culture and induction 5 mL of seed culture was transferred to two 750 mL LB / 2L flasks and cultured at 37°C and 200 rpm until the OD600 reached approximately 0.6. Induction was then performed by adding 750 mL of 1M IPTG (final 1mM IPTG) and continued for 4 hours. After incubation, the cells were harvested by centrifugation (7000 rpm / min for 5 minutes). The cells were washed with 50 mM Tris, 500 mM NaCl, pH 8.0, and centrifuged. The wet weight of the cells was then measured. The absorbance values ​​are shown in Table 3 below.

[0049] Table 3 Absorbance measurement values

[0050] 1.3 Protein solubility analysis Take a small amount of collected bacteria and resuspend them in 1g / 15mL disruption buffer (50 mM Tris, 500 mM NaCl, pH 8.0). Then add nuclease and lysozyme, and lyse the bacteria by freeze-thawing. After lysis, take 40mL and dilute it with the disruption buffer at a ratio of 1:1. After centrifugation, obtain the supernatant and precipitate (the precipitate is still resuspended in the disruption buffer). The prepared disruption buffer, supernatant, and precipitate samples are subjected to SDS-PAGE for solubility analysis. The results are as follows: Figure 4 shown.

[0051] 2. Protein purification experiment 2.1. Buffer preparation Buffer A: 50 mM Tris, 500 mM NaCl, pH8.0; Buffer B: 50 mM Tris, 500 mM NaCl, 7M GuHCl, pH8.0; Buffer C: 50 mM Tris, 500 mM NaCl, 8M Urea, pH8.0; Buffer D: 50 mM Tris, 500 mM NaCl, 8M Urea, 5M Imidazole, pH8.0; Buffer E: 50 mM Tris, 500 mM NaCl, 4 M Urea, 10% glycerol, pH 8.0; Buffer F: 50 mM Tris, 500 mM NaCl, 2 M Urea, 10% glycerol, pH 8.0; Buffer G: 50 mM Tris, 500 mM NaCl, 1 M Urea, 10% glycerol, pH 8.0; Buffer G: 50 mM Tris, 500 mM NaCl, 0.5 M Urea, 10% glycerol, pH 8.0; Buffer H: 50 mM Tris, 500 mM NaCl, 10% glycerol, pH 8.0; Buffer I: PBS, 10% glycerol, pH 7.4; Buffer J: PBS, pH 7.4.

[0052] 2.2. Bacteria disruption (1) Weigh the cells after culture.

[0053] (2) Add Buffer A at 1g / 15ml to the collected cells (3) Fully suspend at 4℃ and ultrasonically crush in ice water for 20 minutes (4) Centrifuge at 12000 rpm / min for 20 min at 4°C to obtain the precipitate, and dissolve it in Buffer B overnight. (5) Centrifuge the overnight dissolved precipitate at 12000 rpm / min for 20 min at 4°C to obtain the supernatant. 2.3 Ni column chromatography purification 2.3.1. Take 3 mL of GE Chelating Sepharose Fast Flow Ni-chelated column material and equilibrate the column material with 10 column volumes of sterile MilliQ H2O and 10 column volumes of Buffer B.

[0054] 2.3.2. Mix the equilibrated column material with the supernatant and incubate at 4°C for 2 h before purification.

[0055] 2.3.3. After retaining the column material, wash the column with Buffer B for 10 column volumes, then with Buffer B + 1% Triton X-114 (v / v) for 10 column volumes to remove endotoxins, and then wash the column with Buffer B for 10 column volumes to remove residual Triton X-114. Then, elute the column with buffers with imidazole concentrations of 20 mM, 50 mM, 150 mM, and 250 mM (prepared with Buffer C and Buffer D).

[0056] 2.3.4. Take small samples of each component after purification and perform SDS-PAGE detection. The results are as follows: Figure 5 shown.

[0057] 2.4 Dialysis 2.4.1. Select a higher purity fraction and add 1% N-lauroylsarcosine sodium salt (w / v). Dialyze the protein at 4°C for 3 h into Buffer E at a replacement ratio of 1:200. Continue to dialyze the protein at 4°C for 16 h into Buffer E. Then, dialyze the protein sequentially at 4°C into Buffer F, Buffer G, Buffer H, and Buffer I for 3 h at a replacement ratio of 1:200. Finally, dialyze the protein twice at 4°C into Buffer J at a replacement ratio of 1:1000 (dialysis membrane: Viskase Dialysis Tubing 14000 MWCO, MD34-14-2). 2.4.2. Take the dialyzed sample and perform SDS-PAGE electrophoresis (electrophoresis gel: 8%~20% SDS PAGE / Coomassie Brilliant Blue staining solution). The final purified rBlo t 24 protein is as follows: Figure 6 shown.

[0058] 2.5 Western Blotting Cut the PVDF membrane and filter paper into the same size as the gel. After treating with transfer buffer, place the filter paper, PVDF membrane, gel, and filter paper between the electrode plates of the transfer apparatus in that order. Start transfer. Set the transfer apparatus parameters as shown in Table 4 below.

[0059] Table 4 Transfer apparatus parameters

[0060] Place the PVDF membrane in 10 ml Blocking buffer containing 5% skim milk and incubate at 37°C for 1 hour. Use 10 ml of diluted Anti-His (1:500) solution and incubate for 1 hour. Wash 5 times with TBST buffer (20 ml), 10 minutes each time. Use 10 ml of diluted HRP-Rabbit Anti-Mouse IgG antibody (1:5000) solution to perform secondary antibody reaction for 0.5 hours. Wash 3 times with TBST buffer (20 ml), 10 minutes each time. After washing, immerse the PVDF membrane in 1 ml of luminescent solution (a mixture of luminol and hydrogen peroxide in a volume ratio of 1:1) and use the gel imager for automatic chemiluminescence exposure. The exposure image is shown as follows Figure 7 shown.

[0061] 3. Determination of protein concentration by BCA method 3.1. Take the final protein sample to determine the protein concentration 3.2. BCA Protein Assay Kit (Pierce BCA Protein Assay Kit; Prod #23227; Lot #MC155209) 3.3. The BCA concentration determination method can refer to SOP.

[0062] 4. Endotoxin determination 4.1. Preparation of bacterial endotoxin standard solution: Take one bacterial endotoxin working standard and prepare 0.5 EU / mL and 0.25 EU / mL endotoxin standard solutions according to the instructions for use of bacterial endotoxin working standard (the sensitivity of the limulus amebocyte lysate is 0.125 EU / mL). 4.2. Prepare negative control solution: water for bacterial endotoxin test.

[0063] 4.3. Prepare the positive control solution: an endotoxin standard solution with a concentration of 0.25 EU / mL.

[0064] 4.4. Prepare the positive control solution of the test sample: Add the test sample solution with an endotoxin standard concentration of 0.25EU / mL 4.5 Dissolving the Limulus Amebocyte Lysate: Add 100 mL of bacterial endotoxin test water to the Limulus Amebocyte Lysate and shake gently to completely dissolve the reagent. Be careful not to cause bubbles. The dissolved reagent should be used within 10 minutes (ready to use).

[0065] 4.6. Add 0.1 mL of negative control solution, positive control solution, test sample, and test sample-positive control solution to each reaction tube. Seal the tube, shake gently, and incubate in a 37°C incubator for 60 min ± 10 min. Avoid vibration during incubation.

[0066] 4.7. Gently remove the reaction tube from the incubator and slowly invert it 180°. If the contents of the tube form a solid gel that does not deform or slide off the tube wall, the test is positive and recorded as (+). If there is no gel, or if a gel is formed but cannot remain intact and slides off the tube wall, the test is negative and recorded as (-).

[0067] 4.8. The test is valid only when the results of the negative tubes are all negative and the results of the positive control tube and the test sample positive control tube are all positive. Otherwise, it is invalid.

[0068] Endotoxin test report Figure 8 shown.

[0069] IV. Application of the tropical mite allergen recombinant protein rBlo t 24 1. IgE-ELISA detection of recombinant allergen rBlo t 24 The recombinant allergen rBlo t 24 was used in precision medicine to diagnose patients with allergic diseases associated with tropical mites. For example, an indirect ELISA assay was developed based on the classic ELISA principle, using the recombinant allergen as the coating antigen to detect specific IgE in patient serum. A case study was conducted using rBlo t 24 as the coating antigen in sera from asthma patients. The ELISA assay procedure is as follows: ① Prepare the antigen coating solution: Dissolve the antigen (rBlo t 24) in 0.1M PBS buffer to a final concentration of 1 μg / mL. ② Antigen coating: Apply 100 μL per well at 2-8°C overnight for at least 15 hours. ③ Prepare PBST wash solution and wash the plate twice, adding 300 μL per well, then pat dry. ④ Prepare blocking solution and block the plate: Add 300 μL of blocking solution to each well at 37°C for 2 hours. Wash the plate five times and pat dry. ⑤ Dilute serum at a ratio of 1:4 (sample diluent), add 100 μL / well, and incubate at 4°C overnight. ⑥ Wash 5 times with PBST and pat dry. ⑦ Mouse anti-human IgE-HRP (secondary antibody): dilute 1:5000, add 100 μL per well, and incubate at 37°C for 1 hour. ⑧ Wash 5 times with PBST and pat dry. ⑨ Add substrate color development solution, 100 μL per well, and develop in the dark for 15 minutes. ⑩ Add stop solution, 50 μL / well, and measure OD with a microplate reader. 405 value.

[0070] There were 26 positive serum samples from asthma children, including 20 males and 6 females, with an average age of (7.2 3.1) years old, provided by Hainan Provincial Women and Children's Hospital, the selected tropical mite positive serum allergen-specific IgE averaged (18.8±22.7) kUA / L; allergen-specific IgE detection was performed using the UniCAP1000 detection system. Negative serum inclusion criteria: patients with a history of allergies, normal physical examination, serum total IgE <100 IU / mL, and concentration grade 0. 4 samples were screened and used as negative controls with tropical mite negative serum, 2 males and 2 females, aged (7.3) 1.5 years old, and all positive and negative sera for A. tropicalis were diagnosed with asthma / rhinitis. Negative sera were tested using the ELISA method described above, with the mean ± 3SD threshold used as the OD value. Sera above this threshold were considered positive, thus determining whether the patient had an allergic disease caused by Blo t 24.

[0071] Results: Among the 26 patients who tested positive for crude extracts of A. tropicalis, 19 tested positive for rBlo t 24, with a positive rate of 73%. See Table 5 for details.

[0072] Table 5 Results of IgE-ELISA detection of rBlo t 24

[0073]

[0074] Note: 1-26 are patients with tropical mite allergy, 28-30 are normal controls; P is positive, N is negative.

[0075] 2. Recombinant Allergen Blot 24 for IgE-Western Blotting ① Protein sample preparation: Mix recombinant allergen Blot 24 with 6' loading buffer at a ratio of 1:5 and heat at 100°C for 10 minutes. ② Sample loading: Add 5 mL to the Maker wells, with a protein loading volume of 5 mg depending on the concentration. ③ SDS-PAGE electrophoresis: Stacking gel at 80V for 30 minutes; Resolving gel at 120V for 60 minutes. ④ Transfer: Remove the gel, cut out the desired band, and prepare a PVDF membrane of the same size pre-soaked in methanol. Arrange the membrane in the order of black plate, fiber pad, filter paper, gel, PVDF membrane, filter paper, fiber pad, and white plate. Clamp the membrane and transfer to the membrane. ⑤ Blocking: Soak the PVDF membrane in TBST containing 5% skim milk and block for 2 hours on a shaker at room temperature. ⑥ Washing: Wash three times with PBST for 10 minutes each. ⑦ Primary antibody incubation: Serum diluted 1:10 (in sample diluent) is added to 2 mL / well and incubated overnight at 4°C. ⑧ Washing: Wash three times with PBST, 10 minutes each. ⑨ Secondary Antibody Incubation: Mouse anti-human IgE-HRP (secondary antibody): 1:2000 dilution, 2 ml per well, incubate at 37°C for 2 hours. ⑩ Color Development: Mix ECL Luminescent Solution A and Solution B in a 1:1 ratio. Place the PVDF on the imager and evenly add ECL working solution. Expel any bubbles and begin exposure.

[0076] There were 10 positive serum samples from asthma children, including 7 males and 3 females, with an average age of (8.3 3.2) years old, provided by Hainan Provincial Women and Children's Hospital, the selected tropical mite positive serum allergen-specific IgE averaged 10.5±12.4kUA / L; allergen-specific IgE was detected using the German UniCAP1000 allergen detection system. Negative serum inclusion criteria: patients with a history of allergies, normal physical examination, serum total IgE <100 IU / mL, and concentration grade 0. Three samples of tropical mite negative serum were screened and used as negative controls, including 2 males and 1 female, aged (9.7 6.8) years old. All positive and negative sera for A. tropicalis were diagnosed with asthma / rhinitis. IgE-Western blotting was used as described above (Table 6).

[0077] Results: Among the 10 patients who tested positive for the crude extract of Acyrthodes tropicalis, 7 showed specific bands for the prokaryotic expression product of Blo t 24, with a positive rate of 70.0%. Figure 9 shown.

[0078] Table 6 Information on positive / negative serum of crude extracts of tropical Acarus spp.

[0079] 5. Combined detection of single components of tropical anonymite allergens 48 A. tropicalis-positive sera and 8 A. tropicalis-negative sera were tested by IgE-ELISA (the same as the indirect ELISA method in the above “IgE-ELISA test of recombinant allergen Blo t 24”). The positive binding rates of allergens Blo t 1, Blo t 2, Blo t 5, Blo t 17, Blo t 21 and Blo t 24 were 31.25%, 72.9%, 60.42%, 20.8%, 60.42% and 68.8%, respectively. Figure 10 As shown in Tables 7 to 13.

[0080] The above 6 single components were considered positive if any one of them was positive. Through statistical analysis: the positive rate of Blo t 5 + Blo t 21 + Blo t 24 + Blo t 2 was as high as 91.36%.

[0081] Table 7 Basic information of 48 patients with tropical mite allergy and the control group

[0082]

[0083]

[0084] Note: M, male; F, female; HC, normal control group; AR, allergic rhinitis; AS, allergic asthma; Bt, crude extract of tropical Acarus truncatus allergen; Df, crude extract of dust mite allergen; Dp, crude extract of house dust mite allergen.

[0085] Table 8 Results of IgE-ELISA detection of recombinant allergen rBlo t 5 (29 / 48=60.42%)

[0086]

[0087]

[0088] Note: P, positive; N, negative.

[0089] Table 9 Results of IgE-ELISA detection of recombinant allergen rBlo t 24 (33 / 48 = 68.8%)

[0090]

[0091]

[0092] Note: P, positive; N, negative.

[0093] Table 10 Results of IgE-ELISA detection of recombinant allergen rBlo t 21 (29 / 48 = 60.42%)

[0094]

[0095]

[0096] Note: P, positive; N, negative.

[0097] Table 11 Results of IgE-ELISA detection of recombinant allergen rBlo t 2 (35 / 48 = 72.9%)

[0098]

[0099]

[0100] Note: P, positive; N, negative.

[0101] Table 12 Results of IgE-ELISA detection of recombinant allergen rBlo t 1 (15 / 48 = 31.25%)

[0102]

[0103]

[0104]

[0105] Note: P, positive; N, negative.

[0106] Table 13 Results of IgE-ELISA detection of recombinant allergen rBlo t 17 (10 / 48 = 20.8%)

[0107]

[0108]

[0109] Note: P, positive; N, negative.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of the allergen Blot 24 in the preparation of dust mite allergen components or allergic disease detection reagents.

2. The use according to claim 1, characterized in that Application of the combination of the allergen Blo t 24, allergen Blo 5, allergen Blo t 21 and allergen Blo t 2 in the preparation of a detection reagent for dust mite allergic diseases.

3. The use according to claim 1, characterized in that The dust mites include the tropical dust mite.

4. A detection reagent for dust mite allergens, characterized in that: The reagent includes a coating antigen consisting of the allergen Blo t 24.

5. The dust mite allergen detection reagent according to claim 4, characterized in that: The coating antigens also include allergens Blo t 5, Blo t 21 and Blo t 2.

6. A method for detecting dust mite allergens, characterized in that: The method is: using Blo t 24 as the coating antigen, an indirect ELISA method is established to detect specific IgE in serum.

7. The method according to claim 6, wherein The coating antigens also include allergens Blo t 5, Blo t 21 and Blo t 2.

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