Method for detecting fumonisin B1 based on fluorescence immunoassay quantitative test strip
A fluorescent immunity and fumonisin technology, applied in measurement devices, biological tests, material inspection products, etc., can solve the problems of poor particle size uniformity of colloidal gold particles, inability to meet rapid quantitative detection, and unstable immune markers. The effect of stable and controllable surface functional groups, elimination of non-specific fluorescence interference, and easy promotion and use
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Embodiment 1
[0049] Example 1 Eu 3+ - Preparation of fluorescent microsphere-labeled fumonisin B1 monoclonal antibody (fluorescent probe)
[0050] Preparation of relevant solutions:
[0051] Activation buffer: pH 6.0 0.05mol / L 2-(N-morpholine)ethanesulfonic acid (MES,C 6 h 13 NO 4 S·H 2 O);
[0052] Coupling buffer: pH 7.0 0.01mol / L phosphate buffer (PBS) (avoid using solvents with free amines);
[0053] Activator: 2mg / mL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, C 8 h 17 N 3 HCl) solution and 2 mg / mL N-hydroxysuccinimide (NHS, C 4 h 5 NO 3 ) solution;
[0054] Blocking buffer: phosphate buffer (PBS) containing 1.0% BSA (mass concentration), 0.05% Tween-20 (mass concentration) pH7.0;
[0055] Labeling buffer: Tris-HCl solution with a concentration of 0.05M at a pH of 7.0 containing 0.3% Tween-20;
[0056] Microsphere complex solution: a Tris-HCl solution with a pH of 7.0 and a concentration of 0.05mol / L containing 1.0% bovine serum albumin and 0.5% ...
Embodiment 2
[0067] The preparation method of fluorescence immunoquantitative test paper strip comprises the steps:
[0068] Spray 0.1 mg / mL fumonisin B1 complete antigen solution onto the nitrocellulose membrane (NC membrane) as the detection line T, and spray 1.0 mg / mL goat anti-mouse IgG solution onto the nitrocellulose membrane (NC membrane) as the detection line T. Quality control line C, the spraying volume is 1μL / cm (instrument parameters, refers to spraying 1μL of the corresponding solution on the NC membrane every time the nozzle moves 1cm), and dried at 37°C for 3h; the distance between the detection line T and the quality control line C The width of the test line T and the quality control line C is 4.0mm; the spraying adopts the XYZ3050 three-dimensional spraying platform of the American BioDot company;
[0069] After that, the sample pad, the nitrocellulose membrane containing the detection line and the quality control line, and the absorbent paper are pasted on the PVC bottom ...
Embodiment 3
[0071] The construction of embodiment 3 standard curve
[0072] The construction of the standard curve for detecting fumonisins B1 based on fluorescent immunoquantitative test strips comprises the following steps:
[0073] Take fumonisin B1 standard substance and use PBS buffer solution (concentration 0.01mol / L, pH7.4) to configure standard solutions with concentrations of 0, 0.75, 1.0, 1.5, 2.0, 5.0, 10.0ng / mL for immunofluorescence assay Detection of paper strips.
[0074] The Eu described in embodiment 1 3+ - Fumonisin B1 monoclonal antibody (Eu-FB1-mAb) labeled with fluorescent microspheres is used as a fluorescent probe, mix 60 μL of standard solution and 10 μL of fluorescent probe in a 1.5 mL centrifuge tube, shake at 37 ° C and 500 rpm Incubate for 10 minutes to obtain the solution to be tested;
[0075] Then use a pipette to slowly drop 70 μL of the solution to be tested into the sample pad of the fluorescent immunoquantitative test strip described in Example 2, and...
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