Immunofluorescent rapid test strip of zearalenone and preparation method and use thereof
A technology for detecting zearalenone and test paper, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve problems such as large impact, difficult removal, and harm to human health, and achieves a simple preparation method, convenient use, and low cost. Effect
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Embodiment 1
[0039] Example 1 Preparation of Zearalenone Monoclonal Antibody
[0040] 1. Use the active ester method to couple ZEN with OVA to prepare the immune antigen (ZEN-OVA), couple ZEN with BSA to prepare the detection antigen (ZEN-BSA), immunize BALB / c mice, cell fusion, and prepare ZEN monoclonal antibody.
[0041] Three healthy BALB / c mice aged 6-8 weeks (purchased from the Institute of Animals, Southern Medical University) were taken and numbered respectively. Each mouse was immunized with a dose of 100 μg each time. The immunogen ZEN-OVA was mixed with an equal volume of Freund’s complete Adjuvant (prime immunization) or incomplete adjuvant (boost) emulsification (0.2 mL), subcutaneous injection at multiple points on the back, immunization cycle of 14 days, 7 days after each booster immunization, blood was collected from the tail to obtain serum, and stored in Store at -20°C for later use. ZEN-BSA was used as the coating agent, the titer of antiserum was determined by indire...
Embodiment 2
[0044] Example 2 Preparation of anti-ZEN monoclonal antibody-fluorescent microsphere probe
[0045] Carboxylation of fluorescent microspheres: Dissolve 100 μl fluorescent microspheres in 400 μl triple-distilled water, then add 100 μl 100 mg / ml EDC and 60 mg / ml NHS respectively, and incubate at room temperature for 30 min. The reaction solution was centrifuged at 4°C and 9000 rpm / min for 2 min, the supernatant was discarded, and this step was repeated 3 times to remove excess EDC and NHS. The pellet was resuspended in 500 μl PH7.2 PBS, and sonicated for 3 minutes.
[0046] Preparation of anti-ZEN monoclonal antibody-fluorescent microsphere probe: Mix 500 μl of the carboxylated fluorescent microsphere solution with 100 μl of anti-ZEN monoclonal antibody (45 μg / ml) dissolved in triple distilled water, and react at room temperature for 2 h. Then 2 μl of 10% BSA was added to block the remaining carboxyl groups of the microspheres and incubated for 1 h. Centrifuge at 4°C, 90...
Embodiment 3
[0047] Example 3 Coating of antigens and antibodies
[0048] Spray C and T lines on the nitrocellulose membrane (NC membrane) using an automatic film sprayer, C line is the quality control line, coated with goat anti-mouse IgG, the concentration of goat anti-mouse IgG is 0.8~1.2mg / ml, preferably 1mg / ml, the spray volume is 1μl / cm; the T line is the detection line, the complete antigen ZEN-BSA coated with ZEN, the concentration is 1.2~1.8mg / ml, preferably 1.5mg / ml, the spray volume is 1μl / cm; C , T line spacing is 5mm. Dry the NC film sprayed with C and T lines at 37°C for 6-12 hours.
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