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Nucleic acid antibody combined detection kit for pathogenic bacteria

A detection kit and the technology of the kit are applied in the field of kits for the combined detection of pathogenic bacteria with nucleic acid antibodies, which can solve problems such as inconsistency, false negatives, and variations, and achieve the effects of improving accuracy, facilitating treatment, and early diagnosis.

Active Publication Date: 2021-11-30
湖南艾科瑞生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the DNA mutation of Helicobacter pylori is very high, there are a large number of variations, and it is highly inconsistent, which can easily cause false negative results
Therefore, the clinical sensitivity of existing methods is not very ideal

Method used

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  • Nucleic acid antibody combined detection kit for pathogenic bacteria
  • Nucleic acid antibody combined detection kit for pathogenic bacteria
  • Nucleic acid antibody combined detection kit for pathogenic bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1. Preparation of antibody against Helicobacter pylori

[0036] Take stored -70 ℃ H.Pylori To 5.1% O 2 , 10% CO 2 Cultured under microaerobic conditions 37 ℃ 48h, washed with physiological saline, sonicated cells, ultrasonic and 14000 rpm, centrifuged for 15 min, the supernatant was transferred to a new tube, precipitated in PBS buffer containing 8M urea resuspended, on SD precipitate bacterial proteins stored in -20 ℃. After the supernatant and the precipitate bacterial proteins were used Freund's complete adjuvant (CFA) emulsified 1: 1 ratio of Balb / c female mice 6-8 weeks old were immunized abdominal injection at multiple sites, the injected dose It is 60μg / only. Boosted once every 14 days, non-antigen using Freund's complete adjuvant (IFC) emulsion at a dose of 30μg / only. 3rd 7 days after the boost immunization the mice sera taken titers, titers of the highest impact intravenous injection to mice immunized with saline and mix antigen, a dose of 50μg / only...

Embodiment 2

[0037] Example 2. Purification of the monoclonal antibodies of embodiments

[0038] Take BALB / c mice, hybridoma one week before inoculation, were injected intraperitoneally with pristane, 0.5ml / only. After 1 week, each mouse was inoculated intraperitoneally with about 1X10 6 Hybridoma cells; after 7 ~ 10d, ascites was collected. After ascites was centrifuged at 10000 × g 30min, the precipitate was discarded, washed with 50% ammonium sulfate precipitation The crude extract, dissolving PBS, 5H dialysis water; dialyzed overnight equilibration with 0.1mol / L phosphate buffer (pH 8.0); the sample after, after elution with 0.1mol / L phosphate buffer (pH8.0), and the protein eluted with different pH citrate eluent, segmented eluted peaks were collected, concentrated, purified against Helicobacter pylori antibody 3F8.

Embodiment 3

[0039] Example 3. Antibodies against Helicobacter pylori subtypes identified

[0040] Be determined according to subclass subclass assay reagent (sigma Co.) screened by indirect ELISA positive monoclonal murine cell line. ELISA plates provided in the kit has been precoated specific antibody against mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, lambda light chains, purified in Example 2 of the embodiment against Helicobacter pylori antibody 3F8 sample was added to sample wells, 50 l per well, incubated without. After 1X goat anti-mouse IgA + IgM + IgG-HRP added to sample wells, 50 l per well, gently mix and incubated 1h. Deduction of the liquid added 1XPBST bore wells were washed three times with excess moisture absorbent paper. Color reagent was added, 100 l per well, at room temperature in the dark color 15min. 100μl stop solution was added to terminate the color reaction. Such as figure 1 , The present invention is a monoclonal antibody IgG2a subtype.

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Abstract

The invention discloses a nucleic acid antibody combined detection kit for pathogenic bacteria. The present invention screens and obtains monoclonal antibodies that specifically recognize Helicobacter pylori, and prepares antibody fluorescent quantum dot rapid detection test paper by using the monoclonal antibody-labeled quantum dots and other antibody-labeled nitrocellulose membranes. analysis, select the conserved region to design RPA primers and probes, and prepare corresponding test strips; the combination of the two detection methods can further improve the accuracy of detection, so as to make early diagnosis and facilitate the treatment of patients as soon as possible. Large-scale promotion and use.

Description

Technical field [0001] The present invention relates to the field of biological detection, and more particularly to nucleic acid antibodies in combination with a kit of pathogenic bacteria. Background technique [0002] Helicobacter pylori Helicobacter pylori, h.pylori ) In 1983, the Australian scholar Warren and Marshall were successfully separated from the gastric mucosa of patients with chronic gastritis. H. Pylori It is a Gram-negative spiral bacterium that retreats in gastric epithelial cells. A large number of research confirmed, H. Pylori It is the main pathogenic factor of chronic gastritis, peptic ulcer and gastrointestinal tract lymphoma, and closely related to gastric cancer. 1994 International Cancer Research Center (IARC) will H.Pylori It is classified as a Class I to cause carcinogenic factors. Currently, H. Pylori The infection rate reached 30% to 50% in developed countries, as as high as 80% in developing countries, and the adult infection rate over 20 years old a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/12G01N33/558G01N33/569G01N33/58G01N33/577C12Q1/689C12Q1/6844C12Q1/06C12N15/11C12R1/01
CPCC07K16/121C07K2317/565C12Q1/6844C12Q1/689G01N33/558G01N33/56922G01N33/577G01N33/588G01N2333/205G01N2469/10C12Q2531/119C12Q2563/107C12Q2565/625
Inventor 马红妙张玲
Owner 湖南艾科瑞生物工程有限公司
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