A kind of enzyme-linked immunosorbent assay kit and application thereof for detecting Zhongbuling
An enzyme-linked immunosorbent reagent, the technology of Zhongbuling, which is applied in the field of enzyme-linked immunosorbent detection, can solve the problems of cumbersome processing, time-consuming, and inability to conduct large-scale on-site detection.
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Embodiment 1
[0028] Embodiment 1 Preparation of kit components
[0029] 1. Synthesis of Zhongbutaline hapten (see the appendix for the synthetic route figure 1 ) and identification
[0030] Take 1.00 g of 4-chloro-3,5-dinitrophenylacetic acid, add 50 mL of ethanol to dissolve, add 0.45 g of sodium carbonate, stir, add 0.31 g of butylamine, heat in an oil bath at 80°C, stir for 3 h, and detect the raw material The basic reaction is complete; stop the reaction, cool to room temperature, rotary evaporate, remove ethanol, add 80 mL of water, adjust the pH value to 4 with 1 mol / L hydrochloric acid, there is a lot of turbidity, add 80 mL of ethyl acetate to extract, wash with water, and apply silica gel The column was eluted and separated with dichloromethane / methanol with a volume ratio of 5:1 to obtain 1.05 g of the hapten product of sec-butyrin, with a yield of 92.11%.
[0031] NMR identification 1 H NMR (CDCl 3 , 300MHz) δ: 11.01 (1H, d, J=0.000), 8.281 (1H, d, J=0.000), 4.011 (1H, tq, J...
Embodiment 2
[0064] Example 2 The formation of an enzyme-linked immunosorbent assay kit for detecting Zhongbutaline
[0065] Set up the enzyme-linked immunosorbent assay kit that detects Zhongbulin, so that it includes the following components:
[0066] 1) A microtiter plate coated with secbutaline-coupled antigen;
[0067] 2) Zhongbuling standard solution, the concentrations are 0 μg / L, 10 μg / L, 30 μg / L, 90 μg / L, 270 μg / L, 810 μg / L;
[0068] 3) Zhongbuling monoclonal antibody working solution;
[0069] 4) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;
[0070] 5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A is carbamide peroxide, and the substrate chromogenic liquid B is tetramethylbenzidine;
[0071] 6) The stop solution is 2 mol / L sulfuric acid buffer;
[0072] 7) The washing solution is a 0.02 mol / L phosphate buffer solution with a pH value of 7.4, containing 0.05% Tween-20 and 0.01‰thimerosal preser...
Embodiment 3
[0074] Example 3 The detection of secbutaline residues in tobacco leaf samples
[0075] 1. Sample pretreatment
[0076] After crushing the tobacco leaf sample, weigh 1.0±0.05 g of the sample into a 50 mL polystyrene centrifuge tube, add 10 mL of acetonitrile, vortex for 2 min with a vortex mixer, and mix well; ) and centrifuge for 5 min, take 100 μL of the supernatant to a 2 mL polystyrene centrifuge tube, add 900 μL of reconstitution solution, and vortex for 20 s; take 50 μL for analysis.
[0077] 2. Detection with kit
[0078] Add 50 μL / well of the standard solution of secbutaline or the pretreated sample solution to the microwells of the microplate of the microplate coated with secbutaline-conjugated antigen, and then add 50 μL / well of secbutaline monoclonal antibody working solution Gently oscillate and mix well, cover the plate with a cover film, and place it in a dark environment at 25°C for 30 min; pour out the liquid in the well, add 250 μL of washing solution to eac...
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