ELISA kit for detecting titer of novel coronavirus neutralizing antibody

A coronavirus and kit technology, applied in the field of immunoassays, can solve the problems of being unsuitable for the screening of neutralizing antibodies of the new coronavirus, and achieve the effects of small batch-to-batch differences, simple operation, and high sensitivity

Active Publication Date: 2020-10-16
ACROBIOSYSTEMS INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the complicated cell experiment process and limited by factors such as safety and reagent consumables, the exi

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  • ELISA kit for detecting titer of novel coronavirus neutralizing antibody
  • ELISA kit for detecting titer of novel coronavirus neutralizing antibody
  • ELISA kit for detecting titer of novel coronavirus neutralizing antibody

Examples

Experimental program
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Embodiment 1

[0037] Example 1 Construction of ELISA Kit for Detection of Neutralizing Antibody to New Coronavirus

[0038] The novel coronavirus neutralizing antibody titer detection ELISA kit provided in this embodiment includes the following components:

[0039] (1) ELISA plate pre-coated with biotin-streptavidin-labeled human ACE2 protein, the specification is 96-well plate;

[0040] (2) Horseradish peroxidase (HRP)-labeled novel coronavirus spike protein RBD, in the form of lyophilized powder, with a specification of 10 ug;

[0041] (3) The new coronavirus neutralizing antibody reference product was purchased from Shanghai Xiangyao Biotechnology Co., Ltd. in the form of freeze-dried powder with a specification of 20 ug;

[0042](4) Concentrated (10×) washing solution: 20mM Tris, 150mM NaCl, 0.5% v / v Tween-20, pH7.4, 60mL;

[0043] (6) TMB chromogenic solution: 50 mM Na 2 HPO 4 12H 2 O + 25 mM citric acid + 40 mg / mL TMB + 3% v / v H 2 o 2 , pH5.5, the specification is 12 mL;

[00...

Embodiment 2

[0045] Embodiment 2 Kit operation method

[0046] Unpack the vacuum packaging bag and take out the microtiter plate, and equilibrate at room temperature for 5 minutes for later use. Prepare 0 μg / mL, 0.16 μg / mL, 0.31 μg / mL, 0.63 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL of the new coronavirus neutralizing antibody standard solution, add 50 μL for processing Put the samples into each well, make 3 replicates of each standard sample and sample, add 50 μL of horseradish peroxidase (HRP)-labeled new crown spike protein RBD diluted to 0.12ug / mL, and incubate at 37°C for 60 minutes; pour out Wash the liquid in the well three times with the diluted lotion solution, invert the ELISA plate on absorbent paper and pat dry; take the single-component TMB development solution, add 100 μL to each well, and develop the color in the dark for 10-15 minutes Add 50 μl of stop solution to each well to stop the reaction, and measure the OD value of each well at a wavelength of 450 nm on a micr...

Embodiment 3

[0049] Embodiment 3 kit accelerated experiment

[0050] The test kit of embodiment 2 is placed in 37 0 Save in C, take the kits that have been placed for 0, 7, 14, 21, and 28 days respectively, and use the new coronavirus neutralizing antibody reference product as the fitting curve to calculate the IC of the curves at different times 50 value. The measurement results at different times are shown in Table 1:

[0051]

[0052] From the above results, it can be seen that the IC 50 The value does not change much, and the kit can be stored at -20°C for at least 12 months.

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Abstract

The invention provides an ELISA kit for detecting titer of novel coronavirus neutralizing antibody. The kit comprises an elisa plate coated with biotin-streptavidin labeled human ACE2 protein, horseradish peroxidase labeled novel coronavirus RBD protein, a novel coronavirus neutralizing antibody positive control, a coating solution, a washing solution, a diluent, a confining solution, a developingsolution, a stop solution and the like. The kit has the characteristics of low cost, simple operation, high sensitivity, high specificity and high accuracy, and can be used for batch and rapid detection of novel coronavirus neutralizing antibodies.

Description

technical field [0001] The invention relates to the technical field of immunoassay, in particular to an ELISA kit for detecting the titer of a novel coronavirus neutralizing antibody. Background technique [0002] The novel coronavirus SARS-CoV-2 (or 2019-nCoV) is a virus newly discovered in 2019 that can cause viral pneumonia or lung infection in humans. The coronavirus genome is sequentially encoded as spike protein, envelope protein, membrane protein and nucleocapsid protein. Among them, the spike protein is the most important surface membrane protein of coronavirus, which contains two subunits, S1 subunit and S2 subunit. Among them, S1 mainly contains the receptor binding domain (RBD), which is responsible for recognizing the receptors of cells. The spike protein of SARS-CoV-2 interacts with human angiotensin-converting enzyme 2 (ACE2) protein to infect human respiratory epithelial cells. The spike protein is responsible for the binding of the new coronavirus to the h...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/535
CPCG01N33/535G01N33/54306G01N33/56983G01N2333/165G01N2469/20
Inventor 葛平菊苗景赟张兴琳
Owner ACROBIOSYSTEMS INC
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