New coronavirus single-chain antibody, quality control substance and preparation method

A single-chain antibody, quality control technology, applied in the field of immunodetection, can solve the problems of inability to perform accurate and effective measurement, lack of stability and sensitivity, achieve easy reconstitution and mixing, and improve molecular stability and specificity Binding capacity, low viscosity effect

Active Publication Date: 2022-04-29
深圳市昭蓝生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2019-nCoV antibody detection has been widely used in ELISA, colloidal gold, lateral flow chromatography, chemiluminescence and other platforms, but the general quality control antibody cannot be accurately and effectively measured due to insufficient stability and sensitivity

Method used

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  • New coronavirus single-chain antibody, quality control substance and preparation method
  • New coronavirus single-chain antibody, quality control substance and preparation method
  • New coronavirus single-chain antibody, quality control substance and preparation method

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preparation example Construction

[0032] The preparation method of the new coronavirus antibody quality control product of an embodiment of the present invention comprises the following steps:

[0033] The new coronavirus antibody is mixed with the freeze-drying protection solution to obtain the antibody solution.

[0034] The antibody solution was subjected to pre-freezing treatment, primary drying treatment and secondary drying treatment in sequence to obtain the new coronavirus antibody quality control product.

[0035]The condition parameters of the above pre-freezing treatment are as follows: partition temperature -5℃~-15℃ for 35~45min, partition temperature -45℃~-55℃ for 80~100min, partition temperature -20℃~-30℃ Treat for 80 to 100 minutes, and the partition temperature is -50°C to -60°C for 80 to 100 minutes. The condition parameters of the above-mentioned one-time drying treatment are as follows: partition temperature -50℃~-60℃, vacuum degree 0.2mbar~0.4mbar for 6~10s, partition temperature -50℃~-60℃...

Embodiment 1

[0042] 1. Preparation of whole mouse IgM monoclonal antibody

[0043] Animal immunization: Protein synthesis was carried out according to the N gene sequence published by China CDC, and N protein was synthesized by Anhui General Biotechnology. The N gene sequence is shown below. Mice were immunized with the prepared 2019-nCoV N protein as an immunogen to prepare whole mouse IgM monoclonal antibodies. Equal amounts of N protein and Freund's adjuvant were mixed and fully emulsified, and three Balb / c mice were injected with 25 μg each, and immunized three times with an interval of 15 days.

[0044] N gene sequence:

[0045] GGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGG

[0046] Cell fusion: 3 days before fusion, mice were intraperitoneally injected with 25 μg of coupling protein, and the spleen cells of immunized mice were taken out, and 2.0×10 7 2.0×10 SP2 / 0 myeloma cells 8 The splenocytes of 1 immunized Balb / c mouse wer...

Embodiment 2

[0080] Example 2 Preparation of new coronavirus IgM antibody quality control product

[0081]Raw material processing and concentration confirmation: take the above-mentioned IgM chimeric antibody raw materials out of the refrigerator under storage conditions and thaw them, and process the raw materials in the biological safety cabinet of the 100,000-level production workshop. Virus Antibody Assay Kit, Human Immunodeficiency Virus Antigen Antibody Assay Kit, and Treponemal pallidum Antibody Assay Kit are tested, and raw materials with negative or non-reactive test results are selected, and then the concentration of 2019-nCoV IgM chimeric antibody raw materials is determined. Then centrifuge and filter to obtain clarified antibody raw materials, and confirm the antibody concentration in a 100,000-level production workshop.

[0082] Solution preparation: The freeze-dried solution is prepared in a 100,000-level production workshop. The preparation process is as follows: taking 1L...

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Abstract

The present invention relates to a new coronavirus single-chain antibody, a quality control product and a preparation method, which include a light chain variable region and a heavy chain variable region in sequence from the N segment to the C terminal, and the amino acid sequence of the light chain variable region is as shown in SEQ As shown in ID NO:1, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2. The novel coronavirus single-chain antibody of the present invention has high sensitivity and good stability, and is very suitable for the preparation of quality control products. Moreover, on the basis of this variable region sequence, further use gene recombination technology to construct a human-mouse chimeric IgM antibody without a CH1 fragment, thereby improving molecular stability and specific binding ability. The novel coronavirus single-chain antibody prepared by the invention can be applied to a chemiluminescence platform for quality control detection, can effectively measure activity, and can overcome the defects of poor detection sensitivity and low specificity of general 2019‑nCoV IgM antibodies.

Description

technical field [0001] The invention relates to the technical field of immunoassay, in particular to a novel coronavirus single-chain antibody, a quality control product and a preparation method. Background technique [0002] 2019 Novel Coronavirus (2019-nCoV), named "2019-nCoV" by the World Health Organization on January 12, 2020, and "Severe Acute Respiratory Syndrome Coronavirus 2" by the International Committee on Taxonomy of Viruses on February 11, 2020 (SARS-CoV-2)”. The novel coronavirus is a new strain of coronavirus that has never been found in humans before113. Coronaviruses are a large family of viruses known to cause colds as well as more serious illnesses such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The similarity between 2019-nCoV and SARS virus in the whole genome is about 80%. From the current cognition, in terms of toxicity, 2019-nCoV is weaker than SARS, and in terms of infectivity, because 2019-nCoV has a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13G01N33/531G01N33/569
CPCC07K16/10G01N33/531G01N33/56983C07K2317/622C07K2317/52C07K2317/56G01N2496/80G01N2333/765G01N2333/165
Inventor 江章涛何雨禧魏道舜林晓涛邢智浩钱纯亘王刚林军李一荣潘运宝胡鹍辉
Owner 深圳市昭蓝生物科技有限公司
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