Preparation and Application of Clenbuterol Monoclonal Antibody
A monoclonal antibody and antibody technology, used in enzyme-linked immunosorbent assay kits and colloidal gold test paper cards, the detection of clenbuterol drug residues in samples, and the preparation of clenbuterol monoclonal antibodies, which can solve the problem of kits. Low sensitivity, poor stability of detection products, cumbersome and time-consuming processing methods, etc.
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Embodiment 1
[0089] Example 1 Preparation of clenbuterol monoclonal antibody
[0090] 1. Preparation of clenbuterol antigen
[0091] 1. Synthesis of clenbuterol hapten (see Figures 1a-1d )
[0092](1) Add 1.0-10.0g clenbuterol hydrochloride to 20-100ml dimethylformamide (DMF), add 5-25ml triethylamine, 10-20% molar equivalent of 4-dimethylaminopyridine (DMAP ), add dropwise a solution of 1.1-1.5 molar equivalents of tert-butyldimethylsilyl chloride in 10-50ml DMF at 0°C, after the dropwise addition, continue to react at room temperature for 1-10 hours, evaporate most of the solvent, add water , extracted with ethyl acetate, washed with water, dried, and recrystallized in cyclohexane-ethyl acetate after removing the solvent to obtain a white solid, which is O-silylated clenbuterol; (the synthetic route is as follows Figure 1a )
[0093] (2) 11.0-5.0g of the product obtained in step (1), add 20-50ml DMF to dissolve, add 0.5-1.5g NaOH, add dropwise 1.1-1.5 molar equivalent of ethyl bromo...
Embodiment 2
[0130] Example 2 Detection of Clenbuterol ELISA Kit Preparation and Application
[0131] 1. Preparation of clenbuterol ELISA kit components
[0132] The present invention detects the ELISA kit of clenbuterol, which comprises:
[0133] (1) 96-well enzyme plate coated with clenbuterol antigen;
[0134] (2) Enzyme-labeled secondary antibody;
[0135] (3) Clenbuterol antibody working solution;
[0136] (4) clenbuterol standard solution;
[0137] (5) clenbuterol high concentration standard solution;
[0138] (6) Substrate solution A liquid;
[0139] (7) Substrate solution B solution;
[0140] (8) Termination solution;
[0141] (9) Concentrate the washing solution.
[0142] 1. Preparation of ELISA plate
[0143] Use 0.1 mol / L carbonate buffer solution with a pH of 9.6 to dilute the clenbuterol coating source in Example 1 to 0.1-0.5 μg / mL, and add 100 μl of the coating source dilution to the microwells of the microtiter plate. solution, incubated at 37°C for 2 hours, poured...
Embodiment 3
[0219] Example 3 Preparation and Application of Detecting Clenbuterol Colloidal Gold Test Paper Card
[0220] 1. Preparation of test strips
[0221] 1. Preparation of clenbuterol monoclonal antibody-colloidal gold marker
[0222] (1) Preparation of colloidal gold
[0223] Take 100ml of 0.01% chloroauric acid aqueous solution and place it in a conical flask, heat it to boiling with a constant temperature electromagnetic stirrer, add 2ml of 1% trisodium citrate aqueous solution under the condition of continuous stirring at high temperature, continue to stir and heat at a constant speed for 15min, the solution is Translucent red. Cool at room temperature, restore to the original volume with deionized water, and store at 4°C.
[0224] (2) Preparation of gold-labeled antibody
[0225] Adjust the colloidal gold solution to pH 7.2 with 0.1mol / L potassium carbonate, add the clenbuterol monoclonal antibody in Example 2 to the colloidal gold solution according to the standard of add...
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