Colloidal gold test paper for rapidly detecting antibody of porcine reproductive and respiratory syndrome virus
A technology for respiratory syndrome and respiratory syndrome, applied in the field of colloidal gold rapid diagnostic test strips for porcine reproductive and respiratory syndrome virus antibodies, to achieve the effects of improved sensitivity and specificity, convenient storage, and simple operation
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Embodiment 1
[0020] Example 1 Preparation of expressing porcine reproductive and respiratory syndrome virus Nsp9 protein
[0021] 1. Amplification of 7817-8657nt of Nsp9 gene: According to the sequence of PRRSV BJ-4, a pair of primers were designed and synthesized to amplify a partial fragment of Nsp9 gene (7817-8657nt).
[0022]
[0023] The underlined sequence of the upstream primer is HindIII, and the underlined sequence of the downstream primer is XhoI; the PCR reaction conditions are hot start at 94°C for 5 minutes, denaturation at 94°C for 60 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, and final extension at 72°C for 10 minutes , the amplified product is 840bp, electrophoresed by 1% agarose; 1.2 Cloning of the Nsp9 gene fragment in pMD-18T: the amplified product was recovered with a gel recovery kit, connected with the pMD-18T vector using the T-A principle, and transformed into DH5α Competent Escherichia coli was spread on LB plates with ampicil...
Embodiment 2
[0025] Example 2 Preparation of anti-PRRSV Nsp9 protein monoclonal antibody:
[0026] 1. For animal immunization, select healthy Balb / C mice aged 6-8 weeks to emulsify the purified PRRSV Nsp9 protein with complete Freund's adjuvant, and inject about 100 μg intraperitoneally into each mouse, and emulsify the protein intraperitoneally with incomplete Freund's adjuvant 14 days later. 100μg was injected, and at the last booster immunization, 100μg purified protein was directly injected intraperitoneally, and 50μg purified protein was injected into tail vein 3-4 days before fusion.
[0027]2. Cell fusion Take splenocytes from immunized mice and mix them with SP2 / 0 in a fusion tube, centrifuge at 300g for 10 min, discard the supernatant, shake the cells to mix the two cells as evenly as possible, and then slowly drop and preheat within 60 seconds PEG-4000 solution, then slowly add serum-free 1640 medium to terminate the fusion, let it stand still and then centrifuge at 1000 r / min ...
Embodiment 3
[0030] Example 3 Preparation of gold-labeled antigen
[0031] 1. Preparation of colloidal gold particles Prepare gold chloride into 0.01% aqueous solution, take 100ml and boil for 2 minutes, add 2ml of 1% trisodium citrate while stirring, boil until the color of the solution turns wine red, continue to boil to an appropriate concentration of OD535 =0.9312, after cooling, add double distilled water to restore to the original volume, store at 4°C;
[0032] 2. Dilute the PRRSV Nsp9 protein to be labeled in a ratio, take 100μl and add it to 1ml colloidal gold, add 100μL of 10% NaCl after 10min, and stand at 4°C for 1h. Take the highest dilution factor with no change in the color of colloidal gold as the standard, add the purified PRRSV Nsp9 protein to the colloidal gold solution at pH 8.0 according to the ratio of the measured minimum protein concentration, stir while adding, and let stand at room temperature for 30 min; Add 10% bovine serum albumin (BSA) to make the final conc...
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