Detection kit of doxycycline in food
A technology for detecting kits and doxycycline, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of long detection cycle, large error in results, difficulty in popularization, etc., and achieve high sensitivity and specificity
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Embodiment 1
[0027] Embodiment 1: Preparation of specific components of the kit
[0028] 1. Doxycycline hapten synthesis
[0029] A mixture of 0.58 g doxycycline and 0.07 g ethylenediamine in 50 ml methanol was reacted at room temperature for 3 to 4 hours, and the solvent was evaporated to obtain doxycycline hapten quantitatively.
[0030] 2. Preparation of enzyme-labeled antigen
[0031] Take 10~15mg doxycycline hapten, dissolve in 1~1.5ml N,N-dimethylformamide (DMF); take 27~32mg dichloroethane (EDC) and N-hydroxysuccinimide ( NHS) was fully dissolved in 0.1-0.3ml of water, then added to the hapten solution, and stirred at room temperature for 24 hours to obtain reaction solution A; weigh 30-50mg of horseradish peroxidase (HRP) to fully dissolve In the pH value of 7.2, 3.8ml of phosphate buffer solution, the reaction solution A was slowly added dropwise to the HRP solution, and stirred at room temperature for 24h; dialyzed with 0.01mol / L phosphate buffer solution at 4°C for 3 Change t...
Embodiment 2
[0044] Embodiment 2: the formation of kit
[0045] Set up the detection kit of doxycycline in food so that it contains the following components:
[0046] Horseradish peroxidase-labeled doxycycline hapten marker
[0047] Enzyme-labeled Antigen Diluent
[0048] Conjugate of doxycycline monoclonal antibody and magnetic beads
[0049] Magnetic Label Antibody Diluent
[0050] Doxycycline series standard solutions, concentrations are: 0μg / L, 0.25μg / L, 0.50μg / L, 1.00μg / L, 2.0μg / L, 4.0μg / L, 8.0μg / L, 16.0μg / L , the standard diluent is a phosphate buffer containing 0.04wt.% Tween-20, 0.04mol / L pH value 7.4.
[0051] Concentrated complex solution is 10 times concentrated complex solution, specifically containing 30-40g NaH per liter 2 PO 4 2H 2 O. 150~160g Na 2 HPO 4 12H 2 Aqueous solution of O.
[0052] The concentrated washing solution is a 10-fold concentrated washing solution, specifically a phosphate buffer solution with a pH value of 7.4-7.5 containing 0.25-0.30 wt.% Twe...
Embodiment 3
[0053] Embodiment 3: the detection of doxycycline residual amount in the sample
[0054] 1. Sample pretreatment method
[0055] (1) milk
[0056] Take 25 μL of fresh milk sample and add 950 μL of sample diluent (dilute the concentrated complex solution to 10 times the volume with deionized water), vortex and mix well, and take the solution for sample analysis.
[0057] (2) milk powder
[0058] Weigh 0.5g±0.05g milk powder sample, add 5ml sample diluent, vortex mix, take 200μL from it and add to 600μL sample diluent, vortex mix, take the solution for sample analysis.
[0059] 2. Detection with kit and analysis of results
[0060] Dilute the enzyme-labeled antigen and the enzyme-labeled antigen diluent at a volume ratio of 1:10 to obtain the enzyme-labeled antigen working solution; dilute the magnetic-labeled antibody and the magnetic-labeled antibody diluent at a volume ratio of 1:10 to obtain the magnetic-labeled antibody Antibody working solution: Take 20-25 μL of enzyme-...
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