Blocking method for activated quantum dots

A technology of quantum dots and mass ratio, which is applied in the closed field of activated quantum dots, can solve the problems of reducing reagent stability and sensitivity, inert protein shedding, and difficulty in completely covering the exposed areas of activated quantum dots, so as to improve sensitivity and stability, Effects of improving stability and increasing sensitivity

Active Publication Date: 2018-03-30
NANJING VAZYME MEDICAL TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, natural BSA is mainly composed of monomer BSA, and it is difficult to completely cover all exposed areas on the surface of activated quantum dots, so it is impossible to completely avoid non-specific reactions.
In addition, the connection between the inert protein and the quantum dots is physical adsorption. During the long-term storage of the quantum dot markers, the inert protein is prone to fall off, which reduces the stability and sensitivity of the reagent.

Method used

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  • Blocking method for activated quantum dots
  • Blocking method for activated quantum dots

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of Quantum Dot Antibody Complex and Test of Coupling Amount

[0028] 1. Activation and coupling of quantum dots (QDs)

[0029] Take 50 μl of QDs stock solution with a solid content of 10%, dilute to 500 μl with 50 mM boric acid-borax buffer (pH 9.0), mix well, add 50 μl EDC solution (5 mg / ml), 5 μl NHS solution (75 mg / ml), mix well Stir and react at room temperature (25°C) for 30 minutes, centrifuge and wash once after incubation, and resuspend with 500 μl of 20 mM HEPES buffer (pH 7.0), take 75 μg of L-1# antibody, add it to the QDs resuspension, mix After uniformity, stir at room temperature for 1 hour;

[0030] 2. Processing of BSA

[0031] A 20% BSA solution and a 2% EDC solution were prepared with blocking buffer (20mM HEPES buffer, pH 7.0), and the two solutions were mixed 1:1 and stirred at 4°C for 2 hours.

[0032] 3. Closed

[0033] Add 500 μl of poly-BSA solution to the system after the reaction in step 1 to resuspend, mix well, stir ...

Embodiment 2

[0034] Example 2 Preparation and Specific Detection of Lipoprotein Phospholipase A2 (Lp-PLA2) Quantum Dot Immunofluorescence Detection Kit

[0035] 1. Preparation of QDs-L-1# labeling complex

[0036]Prepare the QDs-L-1# labeling complex according to steps 1-4 in Example 1;

[0037] 2. Treatment of binding pads and immobilization of labeling complexes

[0038] Cut the binding pad into a width of 8mm and soak it in citric acid-sodium citrate buffer (pH8.0, 20mM) containing 5% trehalose and 1% Tween 20 for 5 minutes, and dry it at room temperature. The bonding pad is placed in a constant temperature blast drying oven, and dried at 37°C for 16 hours;

[0039] Take out the dried conjugation pad and fix the labeling complex prepared in step 1 on the conjugation pad with a gold sprayer at a spray volume of 5 μl / cm, place it in a constant temperature blast drying oven, and dry it at 37°C for 16 hours.

[0040] 3. Fixation of T-line and C-line on nitrocellulose membrane

[0041] D...

Embodiment 3

[0054] Example 3 Preparation, Sensitivity and Stability Detection of Heart-type Fatty Acid Binding Protein (H-FABP) Quantum Dot Immunofluorescence Detection Kit

[0055] 1. Preparation of QDs-FP1# labeling complex by prior art

[0056] Take 50 μl of QDs stock solution with a solid content of 10%, dilute to 500 μl with 50 mM boric acid-borax buffer (pH 9.0), mix well, add 5 μl EDC solution (50 mg / ml), 5 μl NHS solution (75 mg / ml), mix well Stir the reaction at room temperature (25°C) for 30 minutes, centrifuge and wash once after incubation, and resuspend with 500 μl of 20mM MOPS buffer (pH 6.5);

[0057] Take 75 μg of FP1# antibody, add it to the QDs resuspension in step (1), mix well, stir at room temperature for 1 hour, centrifuge and wash once after the reaction is completed, and add 500 μl of blocking solution (20% BSA) to resuspend After mixing, stir and react at room temperature for 1 hour. After the reaction is completed, centrifuge and wash once, resuspend with 500 μl...

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Abstract

The invention provides a blocking method for activated quantum dots. The activated quantum dots are blocked by adopting polymer inert protein formed by cross-linking reaction of 1-ethyl-3-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) and inert protein. The method disclosed by the invention has the advantages that effective blocking of the surfaces of the activated quantum dots can be ensured and protein linking in the blocking process is firmer, and thereby the sensitivity and the stability of a reagent are improved and basic performance of the quantum dot immunochromatography reagent is improved. The invention also aims at providing application of the method in an immunofluorescence assay kit for the quantum dots.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a sealing method for activating quantum dots. Background technique [0002] Immunochromatography technology is a rapid diagnostic technology that has emerged in the past ten years. Its principle is to use the capillary action of the liquid to allow the analyte to undergo an immune reaction on the detection line through this action, and the test can be obtained through the naked eye or corresponding instrument detection. result. Immunochromatographic reagents have the advantages of fast detection and simple operation, and do not require complicated detection equipment, which meets the needs of point-of-care detection (POCT). [0003] At present, the commonly used methodologies of immunochromatographic reagents mainly include colloidal gold method and fluorescence method. Among them, colloidal gold immunochromatography technology has been widely used in clinical testing. Its d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531
CPCG01N33/531
Inventor 曹林唐波朱婷婷许雯
Owner NANJING VAZYME MEDICAL TECH CO LTD
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