Magnetic immunochemiluminescence detection kit for apramycin, and applications thereof
A chemiluminescent detection and apramycin technology, which is applied in the field of immunological detection, can solve the problems of low sensitivity, high false negative rate and false positive rate, and achieve high sensitivity and specificity, short detection time, and fully automated Effect
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Embodiment 1
[0030] Example 1: Preparation of Specific Components of Apramycin Magnetic Immunochemiluminescent Detection Kit
[0031] 1. Synthesis of Apramycin hapten (see attached for synthetic route figure 1 )
[0032] A. Take 1.0g of apramycin, add 50mL of pyridine to dissolve, add 0.83g of pyridinium dichromate (PDC), stir at 80°C for 3h, stop the reaction, evaporate to dryness, add toluene, evaporate to dryness, and get black The oil was put on a silica gel column and eluted with dichloromethane / methanol (V / V, 5 / 1) to obtain 0.89 g of aldehyde apramycin, with a yield of 89.35%;
[0033] B. Take 0.89g of alpramycin, add 80mL of methanol to dissolve, add 0.32g of adipic acid 1-hydrazide, add 0.3g of sodium carbonate, stir at room temperature for 2h, stop the reaction, rotary steam, remove methanol, add water, ethyl acetate The ester was extracted and shaken, evaporated to dryness by rotary evaporation, and recrystallized from dichloromethane / methanol (V / V, 1:1) to obtain 0.71 g of apr...
Embodiment 2
[0054] Example 2: Construction of Apramycin Magnetic Immunochemiluminescence Detection Kit
[0055] The Apramycin Magnetic Immunochemiluminescence Detection Kit was assembled to include the following components:
[0056] (1) horseradish peroxidase-labeled apramycin hapten;
[0057] (2) The enzyme-labeled antigen diluent is pH 7.2-7.6, Na 3 PO 4 A buffer solution with a concentration of 0.01mol / L and a NaCl concentration of 0.25mol / L;
[0058] (3) Apramycin monoclonal antibody labeled with immunomagnetic beads;
[0059] (4) Magnetic label antibody diluent is pH 7.2-7.6, Na 3 PO 4 A buffer solution with a concentration of 0.01mol / L and a NaCl concentration of 0.25mol / L;
[0060] (5) Apramycin series standard solution concentrations are 0μg / L, 0.3μg / L, 0.9μg / L, 2.7μg / L, 5.4μg / L, 10.8μg / L;
[0061] (6) The chemiluminescence substrate A solution is trishydroxymethylaminomethane solution containing luminol and p-cresol, and the chemiluminescence substrate B solution contains ...
Embodiment 3
[0064] Embodiment 3: the detection of apramycin residual amount in the sample
[0065] 1. Sample pretreatment
[0066] Add 30 μL milk sample to 870 μL complex solution and vortex for 1 min for detection.
[0067] 2. Detection with kit
[0068] Dilute the enzyme-labeled antigen and the enzyme-labeled antigen diluent at a volume ratio of 1:10 to 1:20, and put it into the enzyme-labeled antigen working solution container of the chemiluminescent detector; dilute the magnetic-labeled antibody and the magnetic-labeled antibody diluent at 1:1 : Dilute at a volume ratio of 10 to 1:20, and put it into the magnetic label antibody working solution container of the chemiluminescence detector; set the position on the sample rack for each sample / standard product, enter the sample information and the name of the test item to be tested ;Put the sample tube / standard tube into the set sample rack, and the chemiluminescence detector draws 50 μL enzyme-labeled antigen working solution, 50 μL sa...
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