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Method for detecting staphylococcus aureus and monoclonal antibodies of staphylococcus aureus

A staphylococcus, golden yellow technology, applied in the field of microbial detection, can solve the problems of cumbersome operation, long detection cycle, and dependence on imports of detection products

Active Publication Date: 2015-07-08
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection of Staphylococcus aureus mainly relies on the biochemical identification stipulated by the national standard. The disadvantage is that the operation is cumbersome, the detection cycle is long, and it cannot adapt to the screening of a large number of samples.
Immunological detection is the fastest, accurate and stable rapid detection method that has emerged in recent years, but all detection products rely on imports. At present, there is no rapid detection product with completely independent intellectual property rights in my country that can be used in detection practice. The patent is based on Staphylococcus aureus is the detection target, and the claimed technology involves rapid detection products of Staphylococcus aureus

Method used

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  • Method for detecting staphylococcus aureus and monoclonal antibodies of staphylococcus aureus
  • Method for detecting staphylococcus aureus and monoclonal antibodies of staphylococcus aureus
  • Method for detecting staphylococcus aureus and monoclonal antibodies of staphylococcus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1. Preparation of anti-Staphylococcus aureus monoclonal antibodies 5D2G2D9C1 and 5D6D9G3B4

[0077] 1. Preparation of immunogen and positive standard

[0078] Staphylococcus aureus (ATCC No.27660) was inoculated in Brain Heart Infusion Broth (BHI), cultured with shaking at 37°C and 150r / min for 17h, counted, and inactivated by adding 0.3% formaldehyde solution at room temperature for 1 day. Adjust the concentration of Staphylococcus aureus (ATCC No.27660) to 5×10 with normal saline 9 CFU / ml was used as immunogen; the concentration was adjusted to 10 with normal saline 8 cfu / ml Staphylococcus aureus liquid was used as a positive control standard, and Brain Heart Infusion Broth (BHI) was used as a negative control standard.

[0079] 2. Preparation of monoclonal antibodies

[0080] 1) Experimental animals: Three 8-week-old, weighing about 20g female Balb / c mice were selected as experimental animals.

[0081] 2) Immunization method: each mouse was intraperitonea...

Embodiment 2

[0106] Example 2. Characterization of monoclonal antibodies 5D2G2D9C1 and 5D6D9G3B4

[0107] 1. Monoclonal Antibody Subclass Identification

[0108] 1. Antigen coating: Coat goat anti-mouse secondary antibody IgG+A+M with 0.01M PBS, 50 μl per well, coat overnight at 4°C, discard the liquid in the well the next day, and wash the plate 3 times.

[0109] 2. Blocking: add 200 μl of 1% BSA to each well, and block overnight at 4°C. Pat the board dry the next day without washing it.

[0110] 3. Add monoclonal antibody hybridoma cell supernatant, 8 microwells for each sample, 50 μl per well. Incubate for 1 hour at 37°C.

[0111] 4. After washing the plate 4 times, add specific binding rabbit anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, κ, λ, and incubate at 37°C for 1 hour.

[0112] 5. After washing the plate 4 times, add diluted horseradish peroxidase-labeled anti-rabbit secondary antibody IgG (H+L) to each well, and incubate at 37°C for 30 minutes.

[0113] After washing the ...

Embodiment 3

[0126] Example 3. The composition, preparation and application of the ELISA kit for detecting Staphylococcus aureus

[0127] 1. The enzyme-linked immunosorbent assay kit consists of the following materials:

[0128](1) ELISA plate with pre-coated antibody: Dilute with 0.02M acetic acid buffer (pH2.0) solution, coat 96-well ELISA plate with anti-Staphylococcus aureus monoclonal antibody 5D6D9G3B4, 100 μl per well. Incubate overnight at 4°C, block and wash according to conventional ELISA methods.

[0129] (2) Staphylococcus aureus positive control standard and negative control standard.

[0130] (3) Anti-Staphylococcus aureus monoclonal antibody 5D2G2D9C1 labeled with horseradish peroxidase.

[0131] (4) Enzyme-labeled antibody diluent: 0.01M PBS, pH7.6.

[0132] (5) 10× concentrated lotion: 0.1M phosphate buffer containing 0.5% Tween-20 and 0.2% sodium azide, pH 7.4, just dilute the concentrated lotion 10 times before use.

[0133] (6) Chromogenic solution A, and chromogeni...

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Abstract

The invention belongs to the field of microbiological detection, and in particular relates to a method for detecting staphylococcus aureus and two monoclonal antibodies of the staphylococcus aureus which are produced by the same monoclonal antibody preparation process. The monoclonal antibodies can be specifically combined with the staphylococcus aureus. The monoclonal antibodies are generated by mouse hybridoma cell lines 5D2G2D9C1, CGMCCNo.8763 or 5D6D9G3B4, CGMCCNo.8764. The invention also provides application of the monoclonal antibodies for resisting the staphylococcus aureus.

Description

technical field [0001] The invention belongs to the field of microorganism detection. Specifically, the present invention relates to a method for detecting Staphylococcus aureus, two strains of monoclonal antibodies against Staphylococcus aureus used in the method, and hybridoma cells producing the monoclonal antibodies. Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is the most common food-borne pathogen, widely present in the natural environment. Staphylococcus aureus mainly contaminates animal products such as milk, meat, eggs, fish and their products, including beverages made of milk, cold drinks, cakes, cooked meat, canned meat products, leftovers, fried eggs, glutinous rice cakes, jelly, Rice and rice wine are left, commonly known as "flesh-loving bacteria". After Staphylococcus aureus infects the human body, it will mainly cause the following human diseases. The first is invasive diseases such as causing suppurative infections, such as w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577C07K16/12C12N5/20C12R1/91
CPCC07K16/1271G01N33/56911G01N33/577
Inventor 刘箐曾海娟
Owner UNIV OF SHANGHAI FOR SCI & TECH
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