SARS-CoV-2 neutralization antibody detection kit

A detection kit, sars-cov-2 technology, applied in the direction of biological testing, measuring devices, immunoassays, etc., can solve the problems of low sensitivity, inability to accurately detect antibodies, and inability to realize large-scale screening, so as to improve sensitivity and specific effect

Pending Publication Date: 2021-07-09
苏州携创生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is not equipped with fully automated instruments and equipment, and requires manual sample loading and other operations. It takes 2 hours to complete a test, and it still cannot achieve the purpose of mass screening.
Moreover, ELISA has a narrow linear range and low sensitivity, making it impossible to accurately detect SARS-CoV-2 neutralizing antibodies

Method used

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  • SARS-CoV-2 neutralization antibody detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0039] 1. Preparation of RBD recombinant protein

[0040] The RBD domain gene of SARS-CoV-2 (306-527 amino acid sequence of S protein (GeneID: 43740568)) was transformed into competent Escherichia coli DH5α, ice-bathed for 30 minutes, and cultured in LB medium at 37°C for 60 minutes Recover, and finally take the bacterial solution and cultivate it on the LB agar plate containing Amp antibiotics to select positive colonies for identification;

[0041] Select the correctly recombined plasmid to transform Escherichia coli DH10 Bac competent cells, recover for 2-4 hours, and culture in LB medium containing Kan+Gent+Tet to select positive colonies for identification;

[0042] Transform the correctly recombined bacmid into insect cell sf9, centrifuge to collect the supernatant after 3-5 days, and use it as the first-generation virus; re-infect the insect cell sf9 with the first-generation virus, collect the supernatant after 2-4 days, and use it as the second-generation virus The s...

Embodiment 2

[0051] This embodiment provides a SARS-CoV-2 neutralization detection kit 1, including M reagent, R2 reagent, R1 reagent and calibrator.

[0052] M reagent: the recombinant RBD protein prepared in Example 1 was coated on the surface of the tosyl magnetic beads, and the tosyl magnetic beads coated with the recombinant RBD protein were diluted to a working concentration of 0.5ug / mL with PBS buffer (in the form of recombinant RBD protein concentration meter);

[0053] R2 reagent: label alkaline phosphatase (AP) on the surface of ACE2 antigen, use Sulfo-SMCC and 2-IT as the joint cross-linking agent, dilute the ACE2 antigen labeled AP enzyme to 1ug / mL working concentration with MES buffer;

[0054] R1 reagent: add a stabilizer to the PBS buffer solution, the stabilizer is 1% of the total mass of the R1 reagent glycine and 0.5% of the total mass of the R1 reagent Tween 20.

[0055] Calibrator: Use the recombinant RBD protein prepared in Example 1 as an immunogen to screen out high...

Embodiment 3

[0057] This embodiment provides a SARS-CoV-2 neutralization detection kit 2, including M reagent, R2 reagent, R1 reagent and calibrator.

[0058] M reagent: ACE2 antigen is coated on the surface of tosyl magnetic beads, and the tosyl magnetic beads coated with ACE2 antigen are diluted to a working concentration of 0.5ug / mL (based on the concentration of ACE2 antigen) with PBS buffer;

[0059] R2 reagent: the recombinant RBD protein surface labeled alkaline phosphatase (AP) prepared in Example 1, using Sulfo-SMCC and 2-IT as a joint cross-linking agent, using MES buffer to dilute the recombinant RBD protein labeled AP enzyme into 1ug / mL working concentration;

[0060] R1 reagent: add a stabilizer to the PBS buffer solution, the stabilizer is glycine 1% of the total mass of the R1 reagent and Tween 20 accounting for 0.5% of the total mass of the R1 reagent;

[0061] Calibrator: Use the recombinant RBD protein prepared in Example 1 as an immunogen to screen out high-affinity IgG...

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Abstract

The invention relates to an SARS-CoV-2 neutralization detection kit, which comprises a solid phase carrier, a screening protein, a competitive binding protein, alkaline phosphatase and an alkaline phosphatase luminescent substrate, wherein the screening protein comprises an RBD recombinant protein having an amino acid sequence represented by SEQ IDNO.1 or having at least 85% homology, and / or the fusion protein of the S protein and the N protein with the amino acid sequence as shown in SEQ IDNO.2 or having at least 85% of homology with the fusion protein. The kit developed by the invention can be used for efficiently, safely, quantitatively and accurately detecting the SARS-CoV-2 neutralization antibody, and large-batch rapid detection can be realized. And the RBD protein recombined by the eukaryotic expression system or the fusion protein of the S protein and the N protein is selected as the screening protein of the competitive reaction system, so that the sensitivity and the specificity of the detection kit can be further improved.

Description

technical field [0001] The invention belongs to the technical field of antibody engineering, in particular to a SARS-CoV-2 neutralizing antibody detection kit. Background technique [0002] The development of a preventive vaccine against SARS-CoV-2 is the most fundamental means to prevent the infection of the new coronavirus SARS-CoV-2. The key to judging the success of a preventive vaccine is whether it can induce an effective humoral immune response. Therefore, establishing a stable and effective neutralizing antibody (NAbs) detection method is an important means to evaluate the immunogenicity of SARS-CoV-2 vaccine. [0003] SARS-CoV-2 is a type of single-stranded positive-sense RNA coronavirus, and its main structural proteins include spike protein (S protein), nucleocapsid protein (Nucleocapsid, N protein), membrane protein (Membrane protein, M protein). ), envelope protein (envelope protein, E protein). For a virus to enter a cell, the cell must have its corresponding...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569G01N33/58G01N33/543
CPCG01N33/54326G01N33/56983G01N33/581G01N33/6854G01N2333/165G01N2469/20
Inventor 赵婷王健李芳向雪花秦枫黄敬双万文琴张伟任伟超刘萍
Owner 苏州携创生物技术有限公司
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