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Dicofol monoclonal antibody and application thereof
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A monoclonal antibody, dicofol technology, applied in material testing products, instruments, analytical materials, etc., can solve the problems of long detection time, intuitionistic detection results, and application limitations of dicofol, and achieve the effect of good detection sensitivity.
Pending Publication Date: 2021-04-09
SUZHOU KUAIJIEKANG BIOTECH CO LTD
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Problems solved by technology
ELISA (Enzyme Linked Immunosorbent Assay) method is a commonly used immunoassay method, but it has relatively many operating steps, a long detection time, the detection result is not intuitive enough, and it cannot be detected online.
Therefore, the application of ELISA in the rapid detection of dicofol is greatly limited
Method used
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[0026] Mix 2 g of dicofol, 4 g of succinic anhydride, and 1 mL of anhydrouspyridine, stir the reaction at 95°C for 4 h, cool the reactant to room temperature, distill off pyridine under reduced pressure, add 10 mL of ice water, then add chloroform for extraction, and separate the organic layer, repeated extraction once to obtain dicofol hapten succinate.
[0027] Immunization antigens were prepared by active ester method. Weigh 0.1 mmol hapten and 0.1 mmol NHS, respectively, and dissolve them in 600 μL DMF in the reaction device; weigh 0.1 mmol DCC, dissolve them in 400 μL DMF; slowly drop the DCC / DMF solution into the above reaction device. The reaction was sealed at room temperature for 7 hours under magnetic stirring. The final reaction solution was cooled in a refrigerator at 4 ℃ for more than 2 hours, centrifuged at 8000 rpm for 5 minutes, and the supernatant active ...
Embodiment 2
[0040] Example 2 Preparation of anti-dicofol monoclonal antibody
[0041] Take 8-10-week-old BALB / c mice, and inject 1 mL of sterile paraffin oil into each mouse; 7 days later, inject 1×10 6 For hybridoma cells, the ascites fluid was collected from the seventh day, and the antibody was purified from the ascites fluid by octanoic acid-saturated ammoniumsulfate method, and the obtained monoclonal antibody was stored at -20°C.
Embodiment 3
[0042] Example 3 Identification of Anti-dicofol MonoclonalAntibody Sensitivity
[0043] (1) Coating: Dilute the original coating with 0.05M pH9.6 carbonate buffer 3 times starting from 12ng / mL, 100μL / well, and react at 37°C for 2h;
[0044] (2) Washing: Pour off the solution in the plate, and wash with washing solution 3 times, each time for 3 minutes;
[0045] (3) Blocking: After patting dry, add 200 μL / well blocking solution, react at 37°C for 2 hours, wash and dry for later use;
[0046] (4) Adding samples: Dilute the antiserum starting from 1:1000, and add it to the coated wells of each dilution, 100 μL / well, and react at 37°C for 30 minutes; after fully washing, add 1:3000 diluted HRP - Goat anti-mouse IgG, 100 μL / well, react at 37°C
[0047] 30min.
[0048] (5) Color development: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react in the dark at 37°C
[0049] 15min;
[0050] Termination and measurement:...
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Abstract
The invention discloses a dicofolmonoclonalantibody and application thereof. The antibody comprises a light chain variable region and a heavy chain variable region; the CDR1 sequence of the light chain variable region is shown as SEQ ID NO:1; the CDR2 sequence of the light chain variable region is shown as SEQ ID NO:2; the CDR3 sequence of the light chain variable region is shown as SEQ ID NO:3; the CDR1 sequence of the heavy chain variable region is shown as SEQ ID NO:4; the CDR2 sequence of the heavy chain variable region is shown as SEQ ID NO:5; and the CDR3 sequence of the heavy chain variable region is shown as SEQ ID NO:6. The antibody has good detection sensitivity and addition recovery rate on the dicofol, can realize detection of the residual amount of the dicofol in agricultural products, and provides a raw material for immunodetection of the residual amount of the dicofol. The variable region gene sequence of the antibody is measured for the first time; and a sequence basis is provided for large-scale expression production when subsequent antibody reagents are applied to detection kits.
Description
technical field [0001] The invention belongs to the technical field of pesticide residue immune analysis, and in particular relates to a dicofol monoclonal antibody and an application thereof. Background technique [0002] Dicofol is an organochlorine insecticide, which is mainly used to control a variety of harmful mites in cotton, fruit trees and flowers. However, residues of dicofol have toxic and estrogenic effects on fish, reptiles, birds, mammals and humans, and are extremely toxic to aquatic organisms. [0003] At present, the detection methods for dicofol residues are mainly instrumental analysis methods, including gas chromatography (Gaschromatography, GC), gas chromatography-mass spectrometry (GC-MS) and so on. These methods have reliable results and high sensitivity, and relevant technical standards are available for reference. However, due to the need for expensive instruments, dedicated operators, and the complexity, high cost, and long time of sample pretreat...
Claims
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Application Information
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