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Human respiratory syncytial virus detection kit based on nanogold signal amplification

A detection kit and signal amplification technology, applied in the field of medical testing, can solve the problem of low sensitivity of the kit

Inactive Publication Date: 2013-11-20
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RSV ELISA detection kit is a common reagent in laboratory scientific research, but the existing kits have low sensitivity

Method used

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  • Human respiratory syncytial virus detection kit based on nanogold signal amplification
  • Human respiratory syncytial virus detection kit based on nanogold signal amplification
  • Human respiratory syncytial virus detection kit based on nanogold signal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preparation of embodiment 1 kit of the present invention

[0044] 1. RSV reference

[0045] (1) RSV virus infects Hep-2 cells in the 75mL culture flask;

[0046] (2) After 6 days, pour out the cell culture medium, add 2 mL of phosphate buffer (pH7.4) to the culture bottle, and freeze and thaw twice at -80°C and room temperature;

[0047] (3) 3000rpm, 4°C, centrifuge for 10min, collect the supernatant, freeze at -80°C, the concentration is 60pg / mL. When drawing the standard curve, dilute it to 0.5, 5, 10, 25, 50 pg / mL with the said diluent respectively.

[0048] 2. RSV polyclonal antibody (anti-RSV), purchased from abcam company, product number is ab20745.

[0049] 3. HRP-labeled RSV polyclonal antibody (anti-RSV-HRP), purchased from abcam company, product number is ab20686.

[0050] 4. Gold nanoparticles enzyme-labeled RSV antibody complex

[0051] (1) Synthesis of 13nm gold nanoparticles: Add 98mL of ultrapure water and 4mL of 1% chloroauric acid solution to a...

Embodiment 2

[0059] The determination of the amount of embodiment 2 enzyme-labeled RSV polyclonal antibody (anti-RSV-HRP)

[0060] (1) Dilute the RSV polyclonal antibody with an initial concentration of 20 μg / mL to 0.17 μg / mL, 0.33 μg / mL, 0.5 μg / mL, 0.67 μg / mL, 0.83 μg / mL, 1 μg / mL with phosphate buffer , 1.17μg / mL, 1.33μg / mL, 1.5μg / mL, 1.67μg / mL;

[0061] (2) Add 20 μL of antibody solutions of different concentrations to 200 μL of gold nanoparticle solution, mix well, and react at room temperature for 30 minutes;

[0062] (3) Add 20 μL of NaCl (2.5%, w / v) solution, mix well, and incubate at room temperature for another 15 minutes. Measure its spectrum and examine the amount of antibody required, the results are as follows figure 1 shown. The enzyme-labeled antibody concentration of 0.9 μg / mL was selected as the concentration used in this kit.

Embodiment 3

[0063] Embodiment 3 test kit stability investigation

[0064] (1) Store all the products provided by the kit of the present invention at a temperature of 4°C for 7 days, 14 days, and 21 days;

[0065] (2) Add the RSV reference substance with a concentration of 50 pg / mL to the microtiter plate, the amount added to each well is 100 μL, and incubate at 37°C for 1 hour;

[0066] (3) Rinse the enzyme-labeled plate with washing solution, add 100 μL of gold nanoparticle enzyme-labeled RSV antibody complex solution to each well of the enzyme-labeled plate, and incubate at 37°C for 1 hour after sealing the plate with a sealing film;

[0067] (4) Rinse the ELISA plate with washing solution, add 100 μL of substrate color development solution to each well, and develop color at 37°C in the dark for 30 minutes;

[0068] (5) Add 50 μL of stop solution to each well, and use a microplate reader to measure the optical density (OD) value, such as figure 2 As shown, the RSV highly sensitive de...

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Abstract

Belonging to the technical field of medical test, the invention in particular relates to a human respiratory syncytial virus (RSV) detection kit based on nanogold signal amplification. Technically, the invention aims to provide a new choice for human respiratory syncytial virus detection. According to a technical scheme adopted by the invention, the human respiratory syncytial virus detection kit based on nanogold signal amplification comprises an RSV reference, an RSV polyclonal antibody, and a gold nanoparticle enzyme labeled RSV antibody complex. The RSV reference is an RSV viral antigen, and the antigen concentration is 60-80pg / mL. The gold nanoparticle enzyme labeled RSV antibody complex is a horse radish peroxidase labeled RSV polyclonal antibody with gold nanoparticles as the carrier, and the antibody concentration is 0.83-1.67 microgram / mL. The kit provided in the invention provides a new choice for RSV detection.

Description

technical field [0001] The invention belongs to the technical field of medical testing, and in particular relates to a detection kit for human respiratory syncytial virus based on a nano-gold signal. Background technique [0002] Respiratory syncytial virus (RSV) belongs to the Pneumovirus genus of the Paramyxoviridae family and is a single-stranded negative-sense RNA virus. The virus is one of the most common and major pathogens causing lower respiratory tract infections in infants and young children in winter and spring. About 50% of infant pneumonia and 90% of infant bronchitis are caused by RSV infection; the latest research has found that adults with immunodeficiency People and the elderly are also susceptible to RSV. The virus is highly contagious, and there is no effective vaccine to prevent it, which can easily cause outbreaks of diseases. Therefore, the development of accurate and highly sensitive RSV detection technology and kits is not only of great significance...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/535
Inventor 黄承志詹蕾
Owner SOUTHWEST UNIVERSITY
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