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Mycoplasma bovis monoclonal antibody, and preparation method and application thereof

A monoclonal antibody, Mycoplasma bovis technology, applied in the field of detection and identification of Mycoplasma bovis, can solve the problems of false positive, high non-specificity, low sensitivity of Mycoplasma bovis pneumonia, etc., to achieve the effect of strong variability

Active Publication Date: 2015-04-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional antibody detection methods have low sensitivity and high non-specificity in diagnosing Mycoplasma bovis pneumonia
The PCR and isothermal ring-mediated method for Mycoplasma bovis are highly sensitive, but the former requires special equipment, and both methods are prone to false positives due to nucleic acid contamination

Method used

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  • Mycoplasma bovis monoclonal antibody, and preparation method and application thereof
  • Mycoplasma bovis monoclonal antibody, and preparation method and application thereof
  • Mycoplasma bovis monoclonal antibody, and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1: the preparation of mycoplasma bovis monoclonal antibody

[0043] 1. Preparation of Mycoplasma bovis antigen

[0044] After culturing Mycoplasma bovis HB0801 in PPLO medium at 37°C for 72 hours, take an appropriate amount for counting, and inactivate the rest with pyrrole at a final concentration of 0.2% (v / v) for 24 hours, then centrifuge at 12000r / min for 30min to collect the bacteria, and then use Sterilized PBS was washed 5 times, the total protein concentration was measured with a BCA kit (purchased from Beijing Saichi Co., Ltd.), and frozen at -20°C.

[0045] Counting method: Dilute the above liquid culture of Mycoplasma bovis with PPLO medium (Bai Zhidi, 2011) to different dilutions, spread on the surface of PPLO solid medium, and add 5% (v / v) CO 2 After 2-3 days, observe the colony morphology with an optical microscope at low magnification. Mycoplasma colonies grown on solid media should have typical "fried egg-like" characteristics (see figure 1...

Embodiment 2

[0131] A kind of application of mycoplasma bovis monoclonal antibody in detection mycoplasma bovis, its step is:

[0132] 1. Preparation and purification of Mycoplasma bovis polyclonal antibody:

[0133] (1) Use the Mycoplasma bovis antigen obtained by the method for preparing the Mycoplasma bovis antigen described in Example 1 as the immunogen, and immunize 4 Japanese white rabbits with 1 mg of Mycoplasma bovis antigen per rabbit. Antigen emulsified with adjuvant, injected subcutaneously at multiple points on the back of the neck, and immunized four times with an interval of 14 days between each time. Blood was collected from the heart, placed at 37°C for 1 hour, and overnight at 4°C to allow the serum to precipitate naturally.

[0134] (2) Centrifuge the serum to be extracted at 13000r / min for 30min at 4°C or room temperature, and collect the supernatant.

[0135] (3) Take 20mL of supernatant and mix it with an equal volume of PBS solution, slowly add 40mL of saturated amm...

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Abstract

The invention discloses a mycoplasma bovis monoclonal antibody, and a preparation method and an application thereof. The preparation method of the mycoplasma bovis monoclonal antibody comprises the following steps: 1, preparing a mycoplasma bovis antigen; 2, immunizing mice by the antigen; 3, preparing a hybridomas cell secreting the mycoplasma bovis monoclonal antibody and monoclonal antibody ascites; and 4, purifying the above obtained monoclonal antibody. In the invention, mice are immunized by a mycoplasma bovis geographical strain HB0801 antigen, a hybridomas cell strain 1C11, CCTCC NO:C201218, which can efficiently secrete the mycoplasma bovis monoclonal antibody, is obtained through a hybridomas cell technology, the generated monoclonal antibody can be specifically combined with mycoplasma bovis and mycoplasma agalactiae, and the monoclonal antibody is utilized to establish sandwich ELISA for detecting the mycoplasma bovis antigen. The method has the advantages of simple operation, short required time and high sensitivity.

Description

technical field [0001] The invention relates to the technical field of detection and identification of Mycoplasma bovis, in particular to a Mycoplasma bovis monoclonal antibody, a hybridoma cell secreting a Mycoplasma bovis monoclonal antibody, and a method for preparing a Mycoplasma bovis monoclonal antibody , also relates to the application of the bovis mycoplasma monoclonal antibody in the detection of bovis mycoplasma. Background technique [0002] Worldwide, Mycoplasma bovis (M.bovis) has become one of the most common and important pathogens in beef cattle and dairy farms, causing syndromes dominated by pneumonia, including pneumonia, arthritis, And a series of clinical diseases such as conjunctivitis. Since Mycoplasma bovis was isolated from the milk of cows suffering from mastitis in the United States for the first time in the world in 1961, various countries in the world have confirmed that the pathogen and related diseases are widely circulated in cattle herds, whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/12G01N33/577C12R1/91
Inventor 郭爱珍任泽民陈颖钰胡长敏张瑞崔鹏陈焕春
Owner HUAZHONG AGRI UNIV
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