Monoclonal antibody and its application, kit, dot-elisa method for detecting water-soluble protein content of Ginkgo biloba
A water-soluble protein, monoclonal antibody technology, applied in anti-plant immunoglobulins, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of time-consuming and other problems, and achieve the effect of low detection limit and short operation time
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Embodiment 1
[0037] Example 1 Preparation of ginkgo leaf water-soluble whole protein (antigen)
[0038] Pick fresh ginkgo leaves and store them at -70°C; cut them into pieces, add an appropriate amount of liquid nitrogen to grind them into powder; take 0.1 g of ground ginkgo leaf powder, add 1.5 mL of 10% trichloroacetic acid (TCA)-acetone, -20 Centrifuge at 12,000rpm / min for 1h at 4°C, discard the supernatant, add 1.5mL ice-bathed acetone, and keep at -20°C for 2h; repeat the previous step until the supernatant becomes clear, centrifuge at 4°C and discard the supernatant; precipitate Air-dry in a fume hood to obtain a precipitated powder containing ginkgo biloba whole protein, and store at -70°C.
[0039] Take 0.15g of Ginkgo biloba whole protein precipitation powder, lyse it with 1mL pH7.4 phosphate buffered saline (PBS), centrifuge at 12000rpm / min for 0.5h, take the supernatant, and get Ginkgo biloba water-soluble whole protein solution, in order to prevent protein degradation , add pr...
Embodiment 2
[0040] The preparation of embodiment 2 monoclonal antibody
[0041] Select 6-week-old female BALB / C mice, and use the Ginkgo biloba water-soluble whole protein solution provided in Example 1 for intraperitoneal immunization injection, once every 14 days, and immunize three times in total. 200μg / piece. After the third immunization, a booster immunization was carried out, and the dose of booster immunization was 300 μg / bird. It was mixed with complete Freund's adjuvant (1:1.1) for the first immunization, mixed with incomplete Freund's adjuvant (1:1.5) for the second immunization, and no adjuvant was added for subsequent immunizations.
[0042] Take the mice 72-96 hours after the booster immunization, collect blood through the eyes, dislocate the neck after anesthesia to kill the mice, soak them in 75% alcohol prepared in advance, fix them on their backs with needles, and place them on the ultra-clean bench after disinfection and sterilization. Aseptically cut the abdominal ski...
Embodiment 3
[0055] Embodiment 3 detects the establishment of the Dot-ELISA method of Ginkgo biloba water-soluble protein
[0056] Draw a 2×5cm square on the smooth surface of the nitrocellulose membrane (the size can be determined according to actual needs), mark it as a 1cm×1cm / grid membrane, and mark it as negative control area, positive control area 1, and positive control area. 2. Positive control area 3, positive control area 4, positive control area 5, and positive control area 6, after soaking (saturated) in 0.05mol / L, pH7.4 PBS buffer for 1 hour, take it out and put it in a ventilated room at room temperature or 37°C. Kitchen dry.
[0057] Dilute the Ginkgo biloba water-soluble total protein solution provided in Example 1 with 0.05mol / L, pH7.4 PBS buffer solution, the concentrations are 0.156 μg / μL, 0.078 μg / μL, 0.039 μg / μL, 0.0195 μg / μL, 0.00975μg / μL, 0.004875μg / μL. PBS was used as a negative control point in the negative control area, and the water-soluble The whole protein s...
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