Quantum dot labeled direct competitive fluorescence immunoassay method for detecting catalase
A technology of catalase and fluorescence immunity, which is applied in the direction of fluorescence/phosphorescence, measuring devices, and material analysis through optical means, which can solve the problems of narrow excitation wavelength range, low fluorescence efficiency, cumbersome operation, etc., and achieve fluorescence stabilization time Long, strong fluorescent intensity, easy to operate
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Embodiment 1
[0083] The method for the direct competitive fluorescent immunodetection catalase of quantum dot labeling of the present invention comprises the following steps:
[0084] (1) Activation of quantum dots
[0085] Measure 100 μL CdSe / ZnS quantum dots, add 100 μL 3mg / mL N-hydroxysuccinimide (NHS) phosphate solution, 100 μL 2mg / mL 1-ethyl-(3-dimethylaminopropyl)-carbodiethylene Dissolve in amine hydrochloride (EDC) phosphate solution and 700μL 25mM pH 6.0 phosphate buffer, after ultrasonic dispersion, react in a constant temperature incubator at 37°C and 250rmp for 30min, centrifuge at 10000rpm for 10min, remove the supernatant, and obtain the activated subsequent quantum dots.
[0086] (2) Coupling of activated quantum dots and catalase
[0087] Use 0.01M pH7.4 phosphate buffer as solvent to prepare 100μg / mL catalase phosphate solution, take 1mL catalase phosphate solution to redissolve the activated quantum dots, and ultrasonically disperse Mix evenly, and react in the dark fo...
Embodiment 2
[0101] Except for the following step (4), the same operation as in Example 1 was used to obtain the fluorescence intensity and prepare a standard curve. The linear correlation coefficient of the standard curve obtained in this study was 0.981.
[0102] (4) Antibody coating: use pH=7.4, 0.01M phosphate buffer to dilute the catalase polyclonal antibody at 1:500, add 100 μL of the obtained antibody dilution to each well of the microtiter plate, After overnight coating in a refrigerator at 4°C, take it out, wash three times with 0.01M pH7.4 phosphate buffer containing 0.5% Tween-20 at room temperature and spin dry to remove excess antibody, then add 300 μL to each well 1% BSA blocking solution was used to block the blank site at 37° C. for 1.5 h to obtain the coated catalase polyclonal antibody.
Embodiment 3
[0104] Except for the following step (5), the same operation as in Example 1 was used to obtain the fluorescence intensity and prepare a standard curve. The linear correlation coefficient of the standard curve obtained in this study was 0.927.
[0105] (5) Formation of luminescent immune complexes: Add 50 μL of standard solution containing catalase and 50 μL of QDs to each well of the blocked microtiter plate wells (containing the coated catalase polyclonal antibody) - CAT complex diluent. In this step, catalase will compete with QDs-CAT to bind to the solid-phase antibody coated on the microtiter plate, and the competition reaction lasts at 37°C for 0.5h. Wash three times with 0.01M pH7.4 phosphate buffer containing 0.5% Tween-20 to remove non-specific adsorption and form antibody-antigen luminescent immune complex.
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