Dermatophagoides pteronyssinus (Der p) allergen diagnostic reagent and preparation method thereof
A technology of diagnostic reagents and allergens, which is applied in the biological field to achieve the effects of strong biological activity, high titer, and avoidance of secondary sensitization
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Embodiment 1
[0044] Example 1 Preparation of pure mite body allergen raw material of house dust mite
[0045] 1) Mite collection: Dust on the bed, pillows, quilts, sofas, and carpets in the living room.
[0046] 2) Separation and identification: Pass the collected dust through the No. 2 sampling sieve to remove impurities, then pass through the No. 4 sampling sieve, spread the remaining dust in a petri dish, smooth the surface with a cover glass, and place it in the dark. 4 hours in a cool and humid place, observed every half hour. Use an inoculation needle to pick up a little dust at the raised part, make a film, and carry out morphological identification of dust mites. After confirming that the dust mites in the dust are house dust mites, the house dust mites are separated under a microscope and used for strain cultivation.
[0047] 3) Strain cultivation: Take 250ml sterile cell culture bottles, insert 4 medicine spoons (about 3-7 grams) of culture medium into each culture bottle asepti...
Embodiment 2
[0053] The preparation of embodiment 2 house dust mite allergen pricking liquid
[0054] 1) Extraction: raw materials of pure mite allergens of Dust mite and phosphate buffer extract (sodium chloride 8.0-11.0g, potassium dihydrogen phosphate 0.6-0.8g, disodium hydrogen phosphate 12.0-16.0g, phenol 6.0-9.0 g, add water for injection to 1000ml) and mix at a ratio of 1:100 (W / V), and extract with continuous shaking for 24 hours at a temperature of 2-8°C.
[0055] 2) Coarse filtration: use filter paper to remove slag.
[0056] 3) Sterilizing filtration: 0.22 μm membrane ultrafiltration membrane positive pressure filtration to obtain allergen extract.
[0057] 4) Mix the allergen extraction solution obtained above with an equal volume of glycerin under aseptic conditions to obtain D. dust mite puncture solution.
Embodiment 3
[0058] Example 3 Determination of protein content in prick fluid of house dust mite allergens (Bradford method)
[0059] 1) Preparation of dye working solution: dilute 5×Coomassie concentrated solution with distilled water to 400ml 1×dye working solution. The diluted 1× dye working solution was filtered with ordinary filter paper to remove the precipitate.
[0060] 2) Preparation of standard protein solution: Dilute standard bovine serum albumin (BSA, 4mg / ml, -20°C) with solvent. Dilute the BSA standard solution to 500, 400, 300, 200, 100, 50, 10, 5μg / ml each dilution, mix well, and stand for 2min.
[0061] 3) Standard curve preparation: take 15 μl of the standard BSA solution of each dilution mentioned above, and mix it with 285 μl of the dye working solution. The final volume of the reaction was 300 μl. Use a 96-well microplate, set it in a microplate reader, and measure the absorbance value OD595 at 595 nm. Take the standard protein concentration (μg / ml) as the abscissa...
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