Enzyme linked immunosorbent assay kit for detecting sever fever with thrombocytopenia syndrome virus antigen
A technology of enzyme-linked immunosorbent reagents and virus antigens, which is applied in the field of disease detection to achieve the effects of improving detection efficiency, high sensitivity, and strong specificity
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Embodiment 1
[0031] The ELISA coated plate coated with the capture antibody was made by the following method: the rabbit polyclonal antibody (Jingtiancheng Biotechnology Co., Ltd.) against the NP antigen of SFTSV was mixed with 0.05M sodium carbonate buffer solution of pH=9.6 Dilute to 40 μg / mL, add to 96-well ELISA plate, 100 μL per well, stand at 4°C for 16 hours and wash with washing buffer; add 5% skimmed milk of final mass, block at 37°C for 2 hours, wash with washing buffer, Pat dry on clean absorbent paper, put in a sealed bag, vacuumize and store at 4°C; SFTSV is the abbreviation for fever with thrombocytopenia syndrome virus, and NP is the abbreviation for nucleoprotein.
[0032] Wash buffer was 0.01M PBS buffer pH 7.4.
Embodiment 2
[0034] The antigen NP solution with a concentration of 15.6ng / mL was prepared by the following method:
[0035]The nucleotide sequence of the NP of the fever with thrombocytopenia syndrome virus of GENBANK: MF574213.1 was artificially synthesized, and cloned into the pET30a expression vector (commercial product), transferred into BL21 (DE3) competent cells (commercial product), and spread evenly Spread in solid LB medium containing 50 μg / mL kanamycin resistance, culture overnight at 37°C, pick out the correct clones and inoculate them into 2×YT medium containing 50 μg / mL kanamycin resistance, at 37°C Cultivate until the OD value reaches 0.6, then add IPTG to a final concentration of 1 mM, culture overnight at 30°C at 200 rpm; centrifuge at 4°C, 8000 rpm for 20 min, discard the culture supernatant, resuspend the bacteria in PBS, centrifuge, discard the supernatant; Suspension liquid, sonicate in ice bath, power 800W, sonicate for 5s, stop for 5s, repeat 200 times; centrifuge, 4...
Embodiment 3
[0037] AuNP-Ab at a gold nanoparticle concentration of 100 μg / mL 2 -HRP Probe Concentrate was prepared as follows:
[0038] The newly prepared 50 μg / mL aqueous solution of gold nanoparticles (Gold nanoparticles, AuNPs) was adjusted to a pH of 7.0 (or any value between 7.0 and 8.0) with 0.2 M potassium carbonate aqueous solution, and the final concentration was 15 μg / mL. mL horseradish peroxidase-labeled antibody, mixed and stirred at 4°C for 12h, added PEG20000 with a final mass concentration of 0.2%, stirred at room temperature for 30min, centrifuged at 10000rpm for 40min and discarded the supernatant to obtain AuNP-Ab 2 -HRP probe, washed twice with double distilled water with pH 7.0 (or any value between 7.0 and 8.0), redissolved in storage buffer, and prepared AuNP with a gold nanoparticle concentration of 100 μg / mL -Ab 2 -HRP probe concentrate, store at 4°C for later use;
[0039] The antibody labeled with horseradish peroxidase is a monoclonal antibody (1H10) against ...
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