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Monoclonal antibody of outer membrane protein of chlamydia abortus and application thereof

A monoclonal antibody, outer membrane protein technology, applied in anti-bacterial immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of high cost, narrow scope of application, unfavorable clinical popularization, etc., to achieve strong specificity, The effect of good fluorescence properties

Active Publication Date: 2011-10-19
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCR technology has a long diagnostic cycle and narrow scope of application, which is not conducive to clinical promotion. Although imported antibody detection kits are highly reliable, they are also expensive and not conducive to clinical popularization.

Method used

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  • Monoclonal antibody of outer membrane protein of chlamydia abortus and application thereof
  • Monoclonal antibody of outer membrane protein of chlamydia abortus and application thereof
  • Monoclonal antibody of outer membrane protein of chlamydia abortus and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0044] Preparation of monoclonal antibody ascites:

[0045] The screened hybridoma cells were inoculated into the peritoneal cavity of 10-week-old healthy Balb / c mice treated with inducer silica gel H (or sterilized liquid paraffin, 0.5mL / mouse) for 10 days in advance, 0.5mL / mouse (containing 5 ×10 5 ~1×10 6 hybridoma cells), after 14 days, it can be seen that the abdomen of the mouse increased significantly. At this time, the ascites was collected and the collected ascites was centrifuged at 3000r / min for 10min. Save for later.

[0046] Identification of monoclonal antibodies:

[0047] Using the Sigma mouse monoclonal antibody typing kit, the agar diffusion test was used to determine that 5 of the monoclonal antibodies secreted by the obtained 6 hybridoma cells were IgG1 type, and one was IgG3 type.

[0048] The potency of hybridoma cell culture supernatant was detected by indirect ELISA method. The results show that the C.abortus POMP18D-1 protein monoclonal antibody 1F...

Embodiment 2

[0054] Example 2C. Establishment of abortus POMP18D-1 protein monoclonal antibody direct immunofluorescence detection method

[0055] Tissue sample processing: Take suspected abortion chlamydia disease materials, take appropriate amount of fresh lung, spleen, uterine mucosa, and vaginal mucosa, make 0.1-0.3mm frozen sections, fix with cold acetone for 10 minutes, and keep away from light for inspection.

[0056] Cell sample processing: Grind the diseased tissue at 4°C, treat with 200UI / mL gentamicin at room temperature for 30min, centrifuge at 1,000r / min for 10min, discard the precipitate, and use the supernatant to infect McCoy cells that have grown to a monolayer (24 Coverslips were pre-placed in the well cell plate), infected for 48 hours, the coverslips were taken out, fixed with pre-cooled methanol or absolute ethanol for 10 minutes, taken out to dry, and stored at -20°C for later use;

[0057]Take out the above prepared glass slides, add PBS dropwise to infiltrate for 10...

Embodiment 3

[0061] Example 3C. Establishment of abortus POMP18D-1 protein monoclonal antibody indirect immunofluorescence detection method

[0062] Take suspected C.abortus disease materials, cut out appropriate amount of lung, spleen, uterine mucosa, vaginal mucosa, grind at 4°C, treat with 200UI / mL gentamicin at room temperature for 30min, centrifuge at 1.000r / min for 10min, discard the precipitate, and use The supernatant was used to infect McCoy cells that had grown to a monolayer (the coverslips were placed in the 24-well cell plate in advance), for 48 hours after infection, the coverslips were taken out, fixed with pre-cooled methanol or absolute ethanol for 10 minutes, taken out and dried in the air at -20 Store at ℃ for later use.

[0063] Take out the prepared glass slide, add 25 μL 10 μg / ml C.abortus POMP18D-1 protein monoclonal antibody 1F10D4 dropwise, put it into a wet box, incubate at 37°C for 1 hour, take it out and wash it with PBST for 3 times, each time for 5 minutes, an...

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Abstract

The invention relates to monoclonal antibody of N-terminal protein of chlamydia abortus POMP18D as well as a preparation method and application thereof. The monoclonal antibody is secreted by a hybridoma cell strain 1F10D4 with the collection number of CGMCC No.4658. The invention also provides application of the monoclonal antibody in direct fluorescence immune or indirect fluorescence immune detection of chlamydia abortus. The monoclonal antibody of N-terminal protein of chlamydia abortus POMP18D has strong specificity and fluorescent characteristic, and is suitable for being used as a fluorescence antibody to establish a direct fluorescence detection method or indirect immune fluorescence detection method.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a monoclonal antibody of chlamydia abortitropia and its preparation method and application, in particular to a monoclonal antibody of chlamydia aborophila POMP18D N-terminal protein and its preparation method and application. Background technique [0002] Chlamydia abortus (C. abortus) is a large group of obligate eukaryotic intracellular parasitic prokaryotes closely related to Gram-negative bacteria, which can cause various animal and human infections. Livestock production causes huge economic losses and affects public health. Abortion-tropic Chlamydia is a relatively common pathogen that causes abortion in sows. In recent years, Qiu Changqing et al. have conducted surveys on swine chlamydia in large-scale pig farms in various provinces in China, showing that the overall trend of swine Chlamydia infection is on the rise. For example, in 1987 The annual infection rate in Sichu...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/12G01N33/577
Inventor 何诚杨君敬凌勇潘青欧长波
Owner CHINA AGRI UNIV
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