Double-antibody biotin-Avidin ELISA (enzyme-linked immuno sorbent assay) detection kit for cattle viral diarrhea virus and application method thereof
A bovine viral diarrhea and detection kit technology, which is applied to measuring devices, instruments, scientific instruments, etc., can solve the problems of expensive kits, large-scale application detection limitations, etc., and achieve high specificity
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Embodiment 1
[0042] Embodiment 1: bovine viral diarrhea virus monoclonal antibody ( McAb ) 3D8 development
[0043] 1 Experimental method
[0044] 1.1 Preparation of immunogen
[0045] The pEC143 plasmid was used to transform the competent host strain JM109, and a single colony was picked to expand culture and harvest the recombinant bacteria. A large amount of plasmid pEC143 was extracted by alkaline lysis, and the plasmid DNA was purified by polyethylene glycol precipitation. After double enzyme digestion and identification with XbaI and BglII, the DNA was quantified and the mice were immunized.
[0046] 1.2 Immunization of mice
[0047] Immunize 6-week-old BALB / c mice with the recombinant pEC143 plasmid by thigh intramuscular injection, inject 100 μL of 25% sucrose solution to each mouse 24 hours before injecting the plasmid, and inject 100 μg of the plasmid at multiple points each time, and immunize once every two weeks , a total of 6 times of immunization, the indirect ELISA an...
Embodiment 2
[0085] Implementation Example 2: Development of Bovine Viral Diarrhea Virus Monoclonal Antibody 3F9
[0086] 1 Test method
[0087] 1.1 Concentration and purification of virus
[0088]The harvested 890 virus-infected cell liquid was frozen and thawed repeatedly 2-3 times, and centrifuged at 8000rpm / min at 4°C for 40 minutes to remove cell debris; take the virus supernatant, and slowly add 40% PEG-6000 and solid under stirring with a magnetic stirrer NaCl, to the final concentrations of 10% and 2.3%, respectively; magnetic stirring overnight at 4°C; the next day, centrifuge at 12000rpm / min at 4°C for 50min, remove the supernatant, and use TNE buffer (0.01mol / L Tris, 0.1 mol / L NaCl, 0.01 mol / L LEDTA sodium salt, pH 7.4) Suspended and precipitated at 1% of the volume of the original culture solution, this is the PEG concentrated virus.
[0089] The virus was purified by centrifugation with 20% sucrose at the bottom. Use a 20ml centrifuge tube, use 20% sucrose solution as the...
Embodiment 3
[0118] Example 3: Establishment of double-antibody sandwich biotin-avidin ELISA method
[0119] 1 Experimental method
[0120] 1.1 Establishment of double-antibody sandwich ELISA method
[0121] The specific operation steps are as follows: Dilute the purified ascitic fluid 3D8 to 1-20ug / ml with the coating solution, add 100ul / well into the wells of the microplate plate, act at 37°C for 2 hours, then place it at 4°C overnight, discard the liquid in the wells, and wash with PBST 3 times, 5min each time, pat dry; add 150ul of blocking solution to each well for blocking at 37°C for 2h, wash with PBST as before, at this time, the coated plate can be stored at -20°C for later use; add 100ul of the sample to be tested (E2 protein) to each well, Set up negative control and blank control at the same time, incubate at 37°C for 1h, wash and pat dry; add biotin-3F9, 100ul / well, incubate at 37°C for 1h, wash and pat dry; add Streptavidin-HRP, 100ul / well, act at 37°C for 10-20min , ...
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