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Kit for detecting new coronavirus neutralizing antibody and detection method

A coronavirus and kit technology, applied in the field of kits for the detection of new coronavirus neutralizing antibodies, can solve the problems of unsuitable quantitative evaluation, complicated operation, and long detection cycle.

Active Publication Date: 2021-04-27
BEIJING HOTGEN BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the main method for neutralizing antibody detection is the neutralizing antibody virus titer test, which is complicated to operate, has a long detection cycle, and requires high biosafety levels; it is not suitable for quantitative evaluation of large-scale population after vaccination

Method used

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  • Kit for detecting new coronavirus neutralizing antibody and detection method
  • Kit for detecting new coronavirus neutralizing antibody and detection method
  • Kit for detecting new coronavirus neutralizing antibody and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1 recombinant S-RBD-C antigen preparation

[0073] The commercialized 293T cell line was used to transfect and express the recombinant S-RBD-C-hFc antigen with the amino acid sequence shown in SEQ ID No.3, and after culturing in the cell culture box for 48h-96h, the cell culture supernatant was collected and separated. Purification, SDS-PAGE detection protein purity ≥ 90%, protein concentration ≥ 1mg / mL, antigen titer ≥ 1:10000.

[0074] Using the same method to transfect and express the recombinant S-RBD-hFc antigen whose amino acid sequence is shown in SEQ ID No.6 without adding cysteine.

[0075] Shown in SEQ ID No.6:

[0076] MDFGLSLVFLVLILKGVQCRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLE ASEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISR...

Embodiment 2

[0083] Example 2 Preparation of Neutralizing Antibody Detection Card

[0084] 1. Preparation of detection card for novel coronavirus neutralizing antibody by up-transfer luminescent competition method

[0085] 1) Preparation of coated nitrocellulose membrane:

[0086] Coating buffer: 0.01mol / L phosphate buffer, pH7.0-7.5; T line coated with commercially available human recombinant soluble ACE2, purchased from SinoBiological, with a coating concentration of 1.0 mg / mL; C line It is a commercial goat anti-mouse IgG polyclonal antibody (purchased from Invitrogen), and the coating concentration is 1.0 mg / mL. Coat the quality control line and detection line on the nitrocellulose membrane with a fully automatic three-dimensional scribing device, dry at 18-25°C for 30-60min, soak and seal the nitrocellulose with 0.01mol / L PBST buffer solution containing 2% BSA concentration Plain film for 30-60min, dry at 37°C for 30-60min, dry at room temperature and store for later use.

[0087] ...

Embodiment 3

[0099] Example 3 Preparation of Sample Diluent and New Coronavirus Neutralizing Antibody Parameter Card

[0100] 1. The sample diluent is PBS-T buffer solution with pH 7.4, the formula is as follows:

[0101] Potassium dihydrogen phosphate (KH 2 PO 4 ): 0.24g / L;

[0102] Disodium hydrogen phosphate (Na 2 HPO 4 ): 1.44g / L;

[0103] Sodium chloride (NaCl): 8g / L;

[0104] Potassium chloride (KCl): 0.2g / L;

[0105] Tween-20: 0.1%.

[0106] 2. New coronavirus neutralizing antibody parameter card

[0107] The new coronavirus neutralizing antibody parameter card contains kit information such as reagent batch number, production date, expiration date, and standard curve parameters; the standard curve parameters are determined by using the conventional neutralizing antibody titer test method to determine the antibody titer and dilution of the standard sample And it is obtained by comparing and fitting with the detection signal data of the detection signal of the new coronavirus...

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Abstract

The invention relates to the field of antibody detection, in particular to a kit for detecting a new coronavirus neutralizing antibody and a detection method. The kit comprises a competition method kit and a double-sandwich method kit. The competition method kit comprises a detection card containing a recombinant S-RBD-C antigen marked by up-conversion luminescent particles, the double-sandwich method kit comprises a detection card containing a recombinant S-RBD-C antigen marked by up-conversion luminescent particles or an S1 antigen marked by up-conversion luminescent particles, and the recombinant S-RBD-C antigen contains at least one segment of additional amino acid sequence, and is used for forming disulfide bonds with free cysteine in the S-RBD antigen. The neutralizing antibody in a subject inoculated with the novel coronavirus vaccine or a human body infected with the novel coronavirus can be detected, quantitative evaluation can be carried out, the operation is simple, and the accuracy is high.

Description

technical field [0001] The invention relates to the field of antibody detection, in particular to a kit and a detection method for detecting neutralizing antibodies against a new coronavirus. Background technique [0002] Up-conversion luminescent material (UCP particles) is composed of rare earth metal elements doped in the crystal lattice, and has three main components: host matrix, absorber and emitter. The crystal materials used as the main matrix are: oxysulfide (such as Y 2 o 2 S, GdO 2 S, La 2 o 2 S, etc.), fluoride (such as YF 3 , GdF 3 、LaF 3 etc.), gallates (such as YGaO 3 , Y 3 Ga 5 o 12 etc.) and silicates (such as YSi 2 o 5 、YSi 3 o 7 etc.), etc.; rare earth metal ions commonly used as absorbers include: ytterbium ion (Yb 3+ ), Erbium ion (Er 3+ ), samarium ions (Sm 3+ ) etc.; the rare earth metal ions commonly used as emitters are: erbium ion (Er 3+ ), holmium ion (Ho 3+ ), Thulium ion (Tm 3+ ), terbium ions (Tb 3+ )wait. The appropriate ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/58G01N33/569G01N33/558G01N33/543
CPCG01N33/6854G01N33/558G01N33/58G01N33/543G01N33/56983G01N2333/165G01N2469/20Y02A50/30
Inventor 闵微李艳召高琦徐磊郄霜张军柳晓利余韶华孙志伟林长青
Owner BEIJING HOTGEN BIOTECH CO LTD
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