Monoclonal antibody blocking agent for novel crown antigen detection

A technology of blocking agent and kit, applied in the field of new coronavirus detection, can solve the problems of uncomfortable promotion and application, high detection cost, difficult operation, etc., and achieve good neutralization activity, improve accuracy, and reduce false positives.

Active Publication Date: 2022-07-15
厦门博昂生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection cost of pseudovirus neutralization test is high, and the operation is difficult,

Method used

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  • Monoclonal antibody blocking agent for novel crown antigen detection
  • Monoclonal antibody blocking agent for novel crown antigen detection
  • Monoclonal antibody blocking agent for novel crown antigen detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Preparation of monoclonal antibody blocking agent for new coronavirus detection

[0017] The monoclonal antibody blocking agent was prepared using the new coronavirus S protein RBD protein (Cat. No. CSB-DP703I) purchased from Wuhan Huamei Bioengineering Co., Ltd. as the antigen. Specific experimental methods: 50 μg of new coronavirus S protein RBD protein was used to immunize 6-week-old female BALB / c mice; the same method was used to boost the immunization once on the 14th day after the first immunization; the third immunization was performed on the 14th day after the second immunization Injection, the method is the same as the dose. On the 10th day after the third immunization, blood was collected from the tail of the mice, and the serum antibody titer was monitored by indirect ELISA. When the antibody level reached the requirement, on the 14th day after the third immunization, the immunized mouse with the highest titer was selected, and the immunization was...

Embodiment 2

[0019] Example 2 Affinity kinetic analysis of mAb blocker A3-4 with SARS-CoV-2 S protein RBD and its mutants (T478K, P681R and L452R)

[0020] GE Biacore T200 was used to detect the affinity kinetic constant of the monoclonal antibody blocker A3-4. The specific experimental operation is shown in the instrument manual. Experimental results: According to the test results, the antibody affinity data are as follows in Table 1:

[0021] Table 1. Antibody Affinity Kinetic Analysis

[0022]

[0023] Experimental conclusion: The monoclonal antibody blocker A3-4 obtained in the present invention has high affinity with the new coronavirus S protein RBD and its mutants (T478K, P681R, L452R), especially with the RBD (T478K, P681R, L452R) mutation The equilibrium dissociation constant is only 4.614×10 -11 .

Embodiment 3

[0024] Example 3 Competitive inhibitory effect of monoclonal antibody blocker A3-4 on the binding of new coronavirus S protein RBD and its mutants (T478K, P681R and L452R) to ACE2

[0025] Indirect ELISA was used to detect the competitive inhibition of monoclonal antibody blocker A3-4 on the binding of 2019-nCoV S protein RBD and its mutants (L452R, T478K) to ACE2. The test results are shown in the following table:

[0026] Table 2. Competitive inhibition of antibodies

[0027]

[0028] Experimental conclusion: The monoclonal antibody blocker A3-4 obtained in the present invention has obvious competitive inhibitory effect on the binding of the new coronavirus S protein RBD and its mutants (T478K, P681R, L452R) to ACE2.

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Abstract

The invention relates to a specific monoclonal antibody blocking agent aiming at a new coronavirus S protein RBD protein. The obtained monoclonal antibody blocking agent has high affinity, and can effectively and competitively inhibit the combination of the new coronavirus S protein RBD and ACE2. Pseudovirus neutralization experiments show that the antibody has good neutralization activity on new coronal pseudovirus and mutant strain (Detla) pseudovirus of the new coronal pseudovirus. The lower detection limit of the monoclonal antibody blocking agent is 3.9 ng/mL, the linear range is 1000 ng/mL to 7.8 ng/mL, the monoclonal antibody blocking agent does not have cross reaction with new coronavirus N protein, influenza virus, mycoplasma pneumoniae antigen, BSA, Vero cells and the like, and false positive of the reagent can be greatly reduced, so that the accuracy of the detection reagent is improved.

Description

technical field [0001] The present invention relates to the technical field of novel coronavirus detection, and more particularly, to a monoclonal antibody blocker for novel coronavirus detection. Background technique [0002] In order to control the further development of the epidemic, rapid and effective detection methods for the new coronavirus are urgently needed. At present, the diagnostic methods for 2019-nCoV infection mainly use RT-PCR on respiratory specimens, that is, nucleic acid detection as the gold standard. In addition, there are antibody-based serological detection, epidemiological diagnosis, and pseudovirus neutralization test detection. [0003] However, nucleic acid detection requires corresponding equipment and professional operators, and it takes a long time from sample collection to inspection, which greatly limits the detection efficiency. Epidemiological diagnosis and detection have high throughput, but nucleic acid detection is required for auxiliar...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/569G01N33/577
CPCC07K16/10G01N33/56983G01N33/577C07K2317/56C07K2317/565C07K2317/76G01N2333/165G01N2469/10
Inventor 黄建平夏大卫
Owner 厦门博昂生物技术有限公司
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