Process for preparing allergoid sublingual buccal vaccine using chitosan as adjuvant
An allergen-like, sublingual technology, applied in the field of new vaccine inventions, can solve problems related to immune tolerance, achieve controlled-release gastrointestinal enzyme degradation, simple production process, and reduce hypersensitivity reactions.
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Embodiment 1
[0013] Example 1: Using biochemical extraction technology to finely extract dust mite allergens
[0014] ①. Sample preparation: Dust mite comes from the Key Laboratory of Microbiology and Genetic Engineering, School of Life Sciences, Shenzhen University. Take 1 gram of Dust mite sample and freeze it in a -70°C refrigerator for 10 minutes. Degrease with cold ether-acetone (1:2). Acetone degreases continuously for 48 hours, during which time the liquid is changed 5 times (every 8-10 hours), and placed under a fume hood to let it dry naturally. All the above operations were carried out on ice bath.
[0015] After degreasing, the samples were weighed and soaked in bicarbonate-saline extract (Coca’s) at a certain ratio. Add the extract at the ratio of 1g sample: 20ml Coca's solution (w / v). At the same time, the samples were fully ground and crushed with a plunger homogenizer. The crushed samples were extracted with Coca's solution for 48hr. During this period, the samples were...
Embodiment 2
[0056] Example 2: Preparation of dust mite allergens
[0057] At 4°C, 1% formaldehyde aqueous solution and 10% D. farinae allergen solution were mixed in equal volumes and stirred gently. Prepare dust mite allergen solution.
Embodiment 3
[0058] Example 3: Identification of dust mite allergens
[0059] Identify the molecular weight of the allergen by SDS-PAGE; detect the IgE reactivity (allergenicity) by competitive ELISA: coat the microwell plate with the allergen solution of D. (PBS; 100mmol / L, pH7.4) to wash the plate. Wells were blocked overnight with 200 [mu]l of PBS containing 10 g / L BSA (BSA / PBS). The five-point calibration curve was operated after serial dilution of the D. farinae allergen solution with BSA / PBS. Immunoglobulin (D. farinae specific IgE) and competitor (diluted calibrator or D. farinae allergen) diluted 1:1 in BSA / PBS were added to the tubes. Take 100 μl from each tube and add it to a microwell plate for 2 hours at 37°C, wash the plate 3 times, add 100 μl horseradish peroxidase-conjugated goat anti-mouse IgE BSA / PBS solution, wash the plate three times at 37°C for 2 hours, wash the plate with 3, 3, 5, 5, and tetramethylbenzidine were used as substrates to determine the binding peroxida...
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