25-hydroxyvitamin D kit for detecting peripheral blood, and preparation method and application thereof
A hydroxyvitamin and kit technology, which can be applied to measurement devices, instruments, scientific instruments, etc., can solve the problems of large demand for blood samples, difficult to repeatedly draw, and complicated operations, and achieves short sample processing time, small sample consumption, and preparation. simple effect
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Embodiment 1
[0040] Embodiment 1: Preparation of 25-hydroxyvitamin D (25-OH-D) kit
[0041] 1.1 Preparation of 25-hydroxyvitamin D (25-OH-D) immunochromatographic detection card
[0042] 1) Goat anti-25-hydroxyvitamin D (25-OH-D) monoclonal antibody labeled latex fluorescent microspheres
[0043] Take 1 mL of latex fluorescent microspheres (purchased from Merck Millipore) and add 1 mg of carbodiimide (EDC), 1 mg of N-hydroxysuccinimide (NHS) and stir at room temperature for 3 h, then add 0.1 mg of goat anti-25-hydroxyvitamin D monoclonal Antibody, stirred at room temperature for 1 h, then added 10 mg of BSA blocking solution, and continued to stir for 1 h. Centrifuge at 11000r / min for 30min at 2-8°C, and remove the supernatant. Finally, take 1 mL of 0.2M phosphate buffer solution (pH=7.4) and add it to the solid precipitate obtained after centrifugation, then add 1 μL of Proclin300, shake on a 40 Hz ultrasonic oscillator for 1 min to mix well, and then incubate at 4 °C Save for later us...
Embodiment 2 Embodiment 1
[0059] The detection of the 25-hydroxyvitamin D kit of embodiment 2 embodiment 1
[0060] 2.1 Detection process (see image 3 )
[0061] 1) Dilute the collected blood sample 21 such as fingertip blood with the release agent 22 at a volume ratio of 3:10;
[0062] 2) Take 60 μL of the blood sample 23 diluted in step 1) and drop it into the sample hole (corresponding to the sample pad 9 of the test strip) of the 25-hydroxyvitamin D test card 24, and let it stand at room temperature for 15 minutes to perform the immune layer. The analysis reaction is performed, and then the 25-hydroxyvitamin D detection card 24 is inserted into the fluorescent immunoassay analyzer 25 for detection, and the detection result can be obtained immediately.
[0063] 2.2. Establishment of standard curve
[0064] Prepare 25-hydroxyvitamin D standard substances (0, 5, 15, 30, 60, 120ng / mL) of different concentrations, and use the 25-hydroxyvitamin D kit prepared in Example 1 of the present invention to ...
Embodiment 3
[0067] Embodiment 3 comparison experiment
[0068] The 25-hydroxyvitamin D test kit prepared in Examples 1 and 2 was compared with the Siemens 25-hydroxyvitamin D detection kit. Detect by collecting 20 patients' fingertip peripheral blood and venous blood (serum, plasma, whole blood), to determine whether the 25-hydroxyvitamin D kit prepared in the application of the present invention is applicable to 25-hydroxyvitamin D in fingertip peripheral blood For the detection of D, the fingertip blood detection was repeated twice. The test results are shown in Table 2 below.
[0069] Table 2 Detection data
[0070]
[0071]
[0072] In the embodiment of the present invention 1, 25-OH-D kit detects fingertip peripheral blood and Siemens 25-OH-D kit detects the detection result of venous blood (serum) and carries out correlation analysis such as Figure 5 As shown, the regression equation is y=1.0113x+0.3081, R 2 =0.9982, n=20, showing that the test results of the 25-OH-D kit ...
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