Avian metapneumovirus antibody double-antigen sandwich method ELISA detection kit and application thereof
A double-antigen sandwich and avian metapneumovirus technology, which is applied in the field of avian metapneumovirus detection, can solve the problems that the avian metapneumovirus detection kit cannot detect poultry whole blood and cannot be applied to a variety of poultry, and achieves simple and fast sample processing , low cost, good sensitivity
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Embodiment 1A
[0056] Example 1 Preparation and purification of APMV / C-N protein
[0057] Preparation of reagents:
[0058] Buffer A: 1M Tris-HCl (pH 8.0) 50mL, 5M NaCl 100mL, glycerol 100mL, add water to make up to 1L, sterilize at 121°C for 20min, and store in a 4°C refrigerator.
[0059] Buffer B: imidazole 16.02g, 1M Tris-HCl (pH 8.0) 50mL, 5M NaCl 100mL, glycerol 100mL, add water to make up to 1L, sterilize at 121°C for 20min, and store in a refrigerator at 4°C.
[0060] Buffer C: imidazole 25.6g, 1M Tris-HCl (pH 8.0) 50mL, 5M NaCl 100mL, glycerol 100mL, add water to make up to 1L, sterilize at 121°C for 20min, and store in a refrigerator at 4°C.
[0061] Buffer D: imidazole 32.04g, 1M Tris-HCl (pH 8.0) 50mL, 5M NaCl 100mL, glycerol 100mL, add water to make up to 1L, sterilize at 121°C for 20min, and store in a refrigerator at 4°C.
[0062] Preparation process of AMPV / C-N protein:
[0063] (1) The pET-32a-AMPV / C-N prokaryotic expression vector plasmid constructed by BGI was introduce...
Embodiment 2
[0075] Example 2 HRP-AMPV / C-N horseradish peroxidase labeling
[0076] Take 10mg / mL horseradish peroxidase (HRP) solution 2mL, add 0.06M NaIO 4 2 mL of aqueous solution, oxidized at 4 °C for 30 min, added 2 mL of 0.4M ethylene glycol solution containing 22% NaCl, oxidized at 25 °C for 30 min, mixed with 24 mL of pre-cooled absolute ethanol, and centrifuged at 1500 rpm After 10 minutes, pour off the upper layer of liquid and place it on the test tube and filter paper. After the liquid is drained, add 8 mL of ultrapure water to dissolve the precipitate. After adding 4 mg of antigen solution (AMPV / C-N), adjust the pH to 9.0 with 0.5M CBS at pH 9.6. After standing at 4°C for 16-24h, add 10mg / mL KBH 4 Aqueous solution 0.2 mL, after standing for 2 h, add NaH 2 PO 4 Adjust pH to 7.0 to obtain HRP-AMPV / C-N with a protein concentration of 0.5 mg / mL.
Embodiment 3
[0077] Example 3 Establishment of avian metapneumovirus antibody double antigen sandwich ELISA detection kit
[0078] Preparation of ELISA plate:
[0079] 1. Coat AMPV / C-N protein on the ELISA reaction plate, the specific method is:
[0080] The AMPV / C-N protein was diluted to 0.6875mg / mL with 0.05M CBS pH 9.6, 100μL was added to each reaction well, coated at 4°C for 18h, and 120μL of blocking solution (10% sucrose solution) was added to each reaction well after washing the plate twice. , a mixed solution of 5% BSA and 5% fish gelatin, 1:1:1), closed at 37°C for 2 hours, then thrown off the blocking solution and dried the reaction plate in a 37°C oven to obtain a coated ELISA reaction plate.
[0081] Enzyme label preparation
[0082] 1. The HRP-AMPV / C-N obtained in Example 2 was diluted to 0.25 mg / L with 10% sucrose solution, and then 0.01% Royal blue (0.1 mL pigment per liter) was added to obtain the enzyme marker.
[0083] 3. Detection kit
[0084] Avian metapneumovirus an...
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