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Method for detecting novel coronavirus neutralizing antibody by using BLI technology

A coronavirus and technical detection technology, applied in the biological field, can solve the problems of inability to achieve high-throughput detection, many steps, and long time, and achieve the effect of qualitative and quantitative detection and simple operation

Active Publication Date: 2021-01-22
ACROBIOSYSTEMS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ELISA detection has many steps and takes a long time. It needs to wash the plate, add samples, and develop color many times. It cannot achieve high-throughput detection, and the coating protein may cover some binding sites, resulting in false negative results.

Method used

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  • Method for detecting novel coronavirus neutralizing antibody by using BLI technology
  • Method for detecting novel coronavirus neutralizing antibody by using BLI technology
  • Method for detecting novel coronavirus neutralizing antibody by using BLI technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Binding experiment of novel coronavirus spike protein RBD and human ACE2

[0041] 1. Protein preparation: Dilute human ACE2 protein to 10 μg / mL, add 200 μL per well into a black 96-well plate, dilute the new crown spike protein RBD from 100 nM to 2-fold concentration gradient, and add 200 μL per well to the analysis sample column .

[0042] 2. Set up the program for detection: first capture the same concentration of human ACE2 on the sensor, and then combine with different concentrations of the new crown spike protein RBD.

[0043] The binding curve of novel coronavirus spike protein RBD and human ACE2 is shown in figure 1 .

Embodiment 2

[0044] Example 2 New crown neutralizing antibody blocking experiment

[0045] This embodiment provides a short time-consuming, high-throughput, and highly sensitive method for screening novel coronavirus neutralizing antibodies using BLI technology. This method can realize the screening of new crown neutralizing antibodies within 15 minutes, and the operation is simple and does not require Wash the plate several times. The method includes:

[0046] 1. Prepare protein and antibody: Dilute human ACE2 protein to 10 μg / mL, add 200 μL per well to the Load column, dilute the new crown spike protein RBD to 50 nM, and dilute the new crown neutralizing antibody 1.5 times from 54 nM , Mix the diluted new crown spike protein RBD and new crown neutralizing antibody in equal volumes, and add 200 μL of the mixed new crown spike protein RBD and new crown neutralizing antibody to the analysis sample column per well.

[0047] 2. Set up the program for detection: first capture the same concen...

Embodiment 3

[0053] Example 3 Optimization of the RBD concentration of the new crown spike protein

[0054] Taking the new crown neutralizing antibody antibody2 as an example, by optimizing the concentration of the new crown spike protein RBD (100nM, 50nM, 25nM, 12.5nM), the IC was determined 50 Value and detection range, and compare the detection sensitivity under different conditions. The results are shown in Table 1:

[0055]

[0056] From the above experimental results, it can be seen that as the concentration of the new crown spike protein RBD decreases, the detection range becomes wider, and the IC 50 The lower the value, the higher the sensitivity, but the concentration of the new crown spike protein RBD should not be too low, otherwise the detection signal will decrease accordingly, so the final concentration of the new crown spike protein RBD of 25nM is the best.

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Abstract

The invention provides a method for detecting a novel coronavirus neutralizing antibody by using a BLI technology. The method comprises the following steps: capturing human ACE2 protein with the sameconcentration on the surface of a biosensor, and premixing novel coronavirus spinous process protein RBD with neutralizing antibodies to be detected with different concentrations respectively, respectively contacting each mixed solution with the human ACE2 protein captured on the surface of the biosensor, calculating the inhibition rate according to the change of the relative displacement intensity of the interference spectrum detected by a molecular interaction instrument based on the BLI technology, drawing an inhibition curve, and calculating IC50. The method is simple to operate, quick andefficient, coating, repeated sample adding and plate washing are not needed in the whole detection process, and experimental results can be obtained within 15 min. The detection reaction is carried out in a black pore plate, the detection of novel coronavirus neutralizing antibodies of a large batch of samples can be realized, and different from the traditional qualitative detection, the method has the advantages that the inhibition capability of different new crown neutralizing antibodies can be quickly compared by calculating the IC50 value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting novel coronavirus neutralizing antibodies using BLI technology. Background technique [0002] The new coronavirus discovered in 2019, namely SARS-CoV-2 (2019-nCoV), belongs to the genus β of the Coronaviridae family with the SARS coronavirus reported in 2002 and the MERS coronavirus reported in 2012, and is currently known The seventh coronavirus that can infect humans. The new coronavirus is composed of four glycoproteins (spike protein, envelope protein, membrane protein and nucleoprotein) and RNA nucleic acid chains on the surface of the membrane, among which the spike protein is the most important surface membrane protein of the coronavirus. Contains two subunits, S1 and S2. 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-co...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569G01N21/45G01B11/06
CPCG01B11/0675G01N21/45G01N33/56983G01N33/6854G01N2021/458G01N2333/165G01N2469/20
Inventor 高文静苗景赟张晓慧
Owner ACROBIOSYSTEMS INC
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