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Preparation method of novel coronavirus 2019-nCoV IgM/IgG antibody combined detection kit

A 2019-ncovigm, 2019-ncov technology, applied in measuring devices, immunoassays, instruments, etc., can solve the problems of expensive equipment, false positives, secondary infections, etc., and achieve the effects of short detection time, simple operation, and high sensitivity

Pending Publication Date: 2020-08-14
浙江峻山生物科技有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method also has certain defects and limitations, such as the need for a sophisticated fluorescent quantitative PCR instrument, the easy degradation of RNA, and the amplification efficiency of reverse transcriptase and polymerase, which easily lead to false positives and false negatives. risk of infection
Another gene sequencing method based on a high-throughput sequencer has also been applied to the detection of 2019-nCoV. Although this method has the characteristics of high efficiency and accuracy, its use is also limited due to expensive equipment

Method used

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  • Preparation method of novel coronavirus 2019-nCoV IgM/IgG antibody combined detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A novel coronavirus 2019-nCoV IgM / IgG antibody combined detection kit of the present invention, the specific preparation steps are as follows:

[0029](1) Select the optimal recombinant new coronavirus antigen: The recombinant new coronavirus antigens selected from three suppliers are NP-301 (Hangzhou Boyue Biotechnology Co., Ltd.), Ag8# (Fepeng Biological Co., Ltd.), 2019- nCOV-01 (Shenzhen Heavy Chain Biotechnology Co., Ltd.), and used to prepare latex test strips with an initial concentration of 2 mg / mL, by analyzing the specificity of these four test strips to IgG and IgM antibodies in test samples Based on the combination degree and the minimum detection limit, the recombinant SARS-CoV-2 antigen (Hangzhou Boyue Biotechnology Co., Ltd.) was determined to be the most suitable recombinant antigen for kit preparation.

[0030] (2) Select the optimal concentration of recombinant SARS-CoV-2 antigen for the preparation of antigen-latex complex: select five concentrations ...

Embodiment 2

[0038] A method for using a novel coronavirus 2019-nCoV IgM / IgG antibody combined detection kit comprises the following steps:

[0039] (1) Preliminary preparation. Before testing, return the latex test strips, samples to be tested and 0.5% BSA 20mM phosphate buffer solution to room temperature 20-30°C in the kit, and the kit can be used immediately after disassembly.

[0040] (2) For detection, put the latex test strip on the laboratory bench, use a pipette to draw 20 μL of the sample to be tested, drop it into the sample pad of the latex test strip, and then add 2 drops of 0.5% of PH7.4 in a dropper bottle BSA 20mM phosphate buffer saline was added to the latex test strip sample pad, and timed for 15min.

[0041] Optimization of the diluent formula: By adding 0.1%, 0.2%, 0.4%, and 0.5% BSA to the phosphate buffer solution, the samples were detected respectively. As a result, the addition of 0.5% BSA phosphate buffer solution greatly improved the detection sensitivity .

[...

Embodiment 3

[0044] Embodiment 3: Optimization of antigen-emulsion preparation process

[0045] The invention reduces the uncrosslinked recombinant new coronavirus antigen by adding two times of washing the antigen-latex complex, and improves the detection sensitivity of the latex chromatography strip. Compare the comparison experiment before optimization (that is, the antigen-latex complex has not been washed twice, and other conditions are the same as in Example 1) with the results after optimization (that is, in Example 1), as shown in image 3 As shown, after optimization, both IgM and IgG sensitivity samples can be detected, which improves the detection sensitivity of the product compared with before optimization.

[0046] The specific steps of the unoptimized comparison experiment are as follows: Take 20 μL of 4% 300 nm carboxy red microspheres into a 1.5 mL centrifuge tube, mix quickly and add pH 6.0, 20 mM phosphate buffer, then add 10 μL of 10 mg / mL EDC solution and 10 μL 10 mg / m...

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Abstract

The invention discloses a preparation method of a novel coronavirus 2019-nCoV IgM / IgG antibody combined detection kit, and belongs to the technical field of biological detection. According to the kit,latex chromatography is adopted, a PVC plate is used as a carrier, a sample pad, a compound pad, a nitrocellulose membrane and absorbent paper are sequentially overlapped and attached from left to right to form a latex test strip, and the latex test strip is loaded in a plastic clamping shell. The kit disclosed by the invention has the beneficial effects that the kit adopts latex chromatography,and IgM antibodies and IgG antibodies of the novel coronavirus 2019-nCoV in human whole blood, serum or plasma are detected by an immunocapture method. The situation that the body immune system responds to the new coronavirus can be rapidly mastered, and great reference significance is achieved for early diagnosis and post-cure reexamination of patients. In addition, the detection kit has the characteristics of high sensitivity, strong specificity, simplicity in operation, no need of complex equipment, short detection time and the like, and can play an important assistant role in eliminating the new coronavirus for human beings.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a preparation method of a novel coronavirus 2019-nCoV IgM / IgG antibody combined detection kit. Background technique [0002] The novel coronavirus (2019-nCoV) is a linear single-stranded positive-strand RNA virus that can cause acute respiratory disease. Structurally, the 30 kb single-stranded RNA genome is its core component, which is wrapped by an envelope protein shell with spines. In terms of infection mechanism, the virus uses the S protein on its spine to bind to the ACE2 protein on human lung type II epithelial cells and small intestinal endothelial cells, and then invades the host cells. According to reports from relevant authoritative organizations, it will take quite a long time for humans to completely eliminate 2019-nCoV. Therefore, before specific drugs and vaccines are successfully developed and marketed, it is still a good idea to increase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/558G01N33/543
CPCG01N33/577G01N33/56983G01N33/558G01N33/54313G01N2469/20G01N2333/165
Inventor 王迪张锋
Owner 浙江峻山生物科技有限公司
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