Nucleic acid antibody kit for rapid detection of virus

A kit and antibody technology, applied in the field of virus diagnosis, can solve the problems of unfavorable patient treatment, affecting the detection rate, inability to detect hepatitis B virus early and accurately, and achieve the effect of facilitating treatment and improving accuracy

Active Publication Date: 2021-12-21
BEIJING SANROAD BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Etiological detection has high accuracy and short detection time, but the equipment is expensive and false positives are prone to occur; the detection of the five hepatitis B indicators is subject to certain restrictions under the condition of drug or virus mutation, and the status of hepatitis B virus cannot be detected early and accurately, which affects the detection rate, not conducive to early treatment of patients

Method used

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  • Nucleic acid antibody kit for rapid detection of virus
  • Nucleic acid antibody kit for rapid detection of virus
  • Nucleic acid antibody kit for rapid detection of virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Preparation of anti-HBsAg antibody

[0037] Take the HBV surface antigen protein, dilute the antigen to 1mg / ml, take 50μl of antigen solution, 50μl of 0.01M PBS and 150μl of Freund's complete adjuvant for the first immunization, mix and emulsify it, and subcutaneously immunize male BALB / c children aged 6-8 weeks at multiple points mouse. After the initial immunization, every two weeks, take 25 μl of antigen solution, 75 μl of 0.01M PBS and 100 μl of Freund's incomplete adjuvant and mix them for 5 booster immunizations. The spleen was taken four days after the last immunization for cell fusion. The splenocytes of the immunized Balb / c mice were taken and fused with the myeloma Sp2 / 0 cell line using the PEG method. The fused cells were resuspended in 20% FBS-HAT-DMEM medium, and evenly spread on 96 Inside the orifice plate, 37°C, 5% CO 2 nourish. The fused cells were cultured for about a week and replaced with 10% FBS-HT-DMEM medium in half. When the area of...

Embodiment 2

[0038] Example 2. Purification of monoclonal antibodies

[0039] BALB / c mice were taken, and 1 week before hybridoma cell inoculation, pristane was injected intraperitoneally, 0.5ml / mouse. After 1 week, each mouse was inoculated intraperitoneally with about 1X10 6 hybridoma cells; 7-10 days later, ascites was collected. Centrifuge the ascitic fluid at 10,000×g for 30 minutes, discard the precipitate, use 50% ammonium sulfate for salting out and extract roughly, dissolve it in PBS, and dialyze with running water for 5 hours; dialyze with 0.1mol / L phosphate buffer (pH8.0) overnight; load the sample , use 0.1mol / L phosphate buffer (pH8.0) to elute the impurity protein, elute with citrate eluent with different pH values, collect the elution peaks in sections, and concentrate to obtain the purified anti-HBsAg Antibody 5G7-2.

Embodiment 3

[0040] Example 3. Anti-HBsAg antibody subtype identification

[0041] Subtype determination was performed on the positive mouse monoclonal cell lines screened by indirect ELISA according to the subtype determination reagent (Sigma Company). The ELISA plate provided in the kit has been pre-coated with specific antibodies against mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, lambda light chain, and the anti-HBsAg purified in Example 2 Antibody 5G7-2 samples were added to the sample wells, 50 μl per well, without incubation. Add 1X goat anti-mouse IgA+IgM+IgG-HRP into the sample wells, 50 μl per well, mix gently, and incubate for 1 h. Remove the liquid in the well and add 1XPBST to wash the well 3 times, and absorb excess water with absorbent paper. Add chromogenic solution, 100 μl per well, and develop color for 15 minutes at room temperature in the dark. Add 100 μl stop solution to stop the color reaction. The result is as figure 1 As shown, the monoclonal a...

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Abstract

The invention discloses a nucleic acid antibody kit for rapidly detecting viruses. The present invention obtains HBsAg monoclonal antibody through screening, prepares the HBV fluorescent quantum dot rapid detection test paper by preparing the monoclonal antibody-labeled quantum dot and other antibody-labeled nitrocellulose membranes, and simultaneously analyzes the HBV-DNA sequence, selects the conservative region design RPA primers and probes are prepared into corresponding detection test strips; the combination of the two detection methods can further improve the accuracy of detection, and at the same time reflect the status of virus replication through DNA content, so as to make early diagnosis and facilitate the treatment of patients as soon as possible , suitable for large-scale promotion and use.

Description

technical field [0001] The invention relates to the field of virus diagnosis, and more particularly to a nucleic acid antibody kit for rapidly detecting viruses. Background technique [0002] Hepatitis B is a disease caused by hepatitis B virus (Hepatitis B virus, HBV) infection, mainly manifested by liver cell damage. One million people died from liver failure, liver cirrhosis and primary hepatocellular carcinoma caused by HBV infection. [0003] HBV is a hepadnavirus. The complete HBV is a double-layered capsid particle called Dane particle. The outer layer is a lipoprotein envelope, and the inside of the envelope is a viral capsid, or core particle. The envelope is composed of HBsAg. ; The nucleocapsid is composed of hepatitis B core antigen (HBcAg), and the core contains the viral genome and polymerase. [0004] At present, the main detection methods for detecting HBV include pathogenic detection and serological detection. Among them, the pathogenic detection is mainl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/08G01N33/577G01N33/569G01N33/58C12Q1/70C12Q1/6844C12N15/11
CPCC07K16/082G01N33/577G01N33/56983G01N33/582G01N33/588C12Q1/706C12Q1/6844C07K2317/56C07K2317/565C07K2317/92C12Q2600/166G01N2333/02C12Q2531/119C12Q2522/101C12Q2563/107C12Q2537/1376C12Q2521/319C12Q2565/625
Inventor 马红妙张玲
Owner BEIJING SANROAD BIOLOGICAL PROD CO LTD
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