Hybridoma cell strain secreting clenbuterol resistance monoclonal antibody and application thereof
A hybridoma cell line and monoclonal antibody technology, applied in the biological field, can solve problems such as clenbuterol abuse
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Embodiment 1
[0018] Example 1 Preparation of Clenbuterol Kit Components
[0019] 1. Preparation of clenbuterol hapten
[0020] Take 1.0 g of 1-(4-amino-3,5-dichlorophenyl)ethanol, add 20 mL of pyridine to dissolve, clarify, add 0.61 g of succinic anhydride, and stir at 80°C for 5 h. The reaction was stopped, and the pyridine was removed by rotary evaporation to obtain a red oily substance, which was applied to a silica gel column, and eluted with ethyl acetate / n-hexane (v / v, 1 / 1) to obtain 1.3 g of succinic anhydride-clenbuterol analog, Yield 87.84%.
[0021] 2. Preparation of antigen
[0022] Immunogen preparation - Clenbuterol hapten was conjugated to bovine serum albumin (BSA) to obtain the immunogen.
[0023] Take 43 mg of succinic anhydride-clenbuterol analog hapten, add 7 ml of DMF to dissolve, add 39 mg of EDC, stir for 3 h, add 28 mg of NHS, and continue stirring for 2 h to obtain hapten activation solution A; take bovine serum albumin (BSA) 80mg, add 4ml 0.05mol / L PB buffer to...
Embodiment 2
[0040] Example 2 Construction of an enzyme-linked immunosorbent assay kit for detecting clenbuterol
[0041] Assemble an ELISA kit for the detection of clenbuterol, including the following components:
[0042] (1) ELISA plate coated with Clenbuterol-conjugated antigen;
[0043] (2) 6 bottles of Clenbuterol standard solution with concentrations of 0 μg / L, 0.1 μg / L, 0.3 μg / L, 0.9 μg / L, 2.7 μg / L, and 8.1 μg / L;
[0044] (3) Clenbuterol monoclonal antibody working solution;
[0045] (4) goat anti-mouse anti-antibody labeled with horseradish peroxidase;
[0046] (5) The substrate color developing solution is composed of A solution and B solution, A solution is carbamide peroxide, and B solution is tetramethylbenzidine;
[0047] (6) The stop solution is 2mol / L sulfuric acid;
[0048] (7) The washing solution is pH 7.4, containing 0.5%~1.0% Tween-20, 0.01‰~0.03‰ sodium azide preservative, 0.1~0.3mol / L phosphate buffer, and the percentage is weight volume percentage;
[0049](8) ...
Embodiment 3
[0050] Embodiment 3 Detection of clenbuterol in animal tissue and animal urine samples
[0051] 1. Sample pretreatment
[0052] (1) Animal tissue
[0053] Weigh 2.0±0.05g of the homogenized tissue sample into a 50ml polystyrene centrifuge tube; for liver sample: add 4ml of liver sample extract, shake until uniform, 3000g at room temperature (20-25℃ / 68-77℉ ) centrifugation for 5 minutes; muscle sample: add 4ml of muscle sample extract, shake it with a shaker until uniform, centrifuge at 3000g for 5 minutes at room temperature; take 1ml of supernatant, add 330μl of sample diluent and mix well, check whether the pH value is between 7-8 between (adjust pH with hydrochloric acid or sodium hydroxide solution); take 20 μl for analysis.
[0054] (2) animal urine
[0055] Take 20 μl of clear urine sample for direct determination (if the urine sample is cloudy, it must be filtered or centrifuged at 3000g at room temperature for 5 minutes until it is clear).
[0056] 2. Test with a k...
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