Preparation method and application of dual-amplifying fluorescent immune labeling probe and method for preparing fluorescent immune chromatography reagent strip from probe
A fluorescence immunoassay and double amplification technology, applied in the field of fluorescent labeling, can solve the problems of inability to fully utilize the amplification mode of biotin and avidin, helplessness, and inability to detect, and achieve the effect of enhancing the performance of immunoassay reagents and increasing the intensity of fluorescent signals
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Embodiment 1
[0038] Example 1: Preparation of Double Amplified Fluorescent Microspheres
[0039] 1.1 Get 20mmol of purchased activated biotin, wherein 5mmol of activated biotin is mixed with 5mmol of analyte detection molecules for reaction (this embodiment takes cardiac troponin I (cTnI) antibody as an example), 15mmol of activated biotin is mixed with 15mmol of The bovine serum albumin was mixed and reacted, stirred at room temperature for 1-2 hours, and then dialyzed in a dialysis bag filled with PBS buffer (10mMpH7.4) for 48-72 hours to obtain biotinylated cTnI antibody and biotinylated bovine serum albumin.
[0040]1.2 Take 20 μL of biotinylated bovine serum albumin prepared in the above steps, adjust the pH to 8-9 with 10% NaHCO3, add a small-molecule fluorescent dye with the same fluorescent spectrum properties as the fluorescent microspheres, and gently stir at room temperature for 1-2 hours. After the reaction, use purified resin to remove unreacted fluorescent dye, then add fluor...
Embodiment 2
[0042] Example 2: Preparation of Fluorescence Immunochromatography Diagnostic Reagent Strips
[0043] Such as figure 2 As shown, a fluorescent immunochromatographic reagent strip for detecting cardiac troponin I includes a bottom plate 1, on which a sample pad 2, a binding pad 3, a nitrocellulose membrane 4 and a water-absorbing pad 5 are sequentially attached; wherein the sample Pad 2, binding pad 3, nitrocellulose membrane 4, and water-absorbing pad 5 are overlapped by 1 to 2 mm at the joints to ensure that the test sample passes from the sample area to the detection area through the binding pad. The preparation method of each component is as follows:
[0044] 2.1 Preparation of sample pad 2: Dissolve 0.5g of BSA protein with 100mM, pH7.2-7.4 PBS buffer solution 100mL, then add 0.01-0.05g of surfactant Tween20 to adjust the pH to 6-8; the sample pad is optional Glass fiber or polyester material, a 30cm long sample pad is placed in 2-5ml of the above-mentioned buffer soluti...
Embodiment 3
[0051] Example 3: Fluorescence Immunochromatography Diagnostic Reagent Detection
[0052] 3.1 Comparison of detection performance between traditional polystyrene-labeled fluorescence chromatography reagents and reagents of the present invention
[0053] Add different concentrations of cTnI antigen standards on the sample pad (take 10 different concentrations, respectively 0, 0.01, 0.02, 0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56, 5.12ng / mL, each sample concentration setting Repeat three times) respectively drop 120 μL into the sample wells of the two reagent strips, and read the signal through the fluorescence immunoquantitative analyzer Getein1100 of Jidan Biotechnology Co., Ltd. after 15 minutes. The experimental results are shown in image 3 :
[0054] Such as image 3 As shown, the lower limit of the detection range of the traditional polystyrene fluorescent microsphere system is 0.08ng / mL. After using the double-enhanced fluorescent microspheres of the present invention,...
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