Anti-Listeria monocytogenes monoclonal antibody and its application

A technology of mononucleosis and monoclonal antibody, which is applied in the biological field, can solve the problems of increased detection cost, long detection cycle, and extended detection time, and achieve strict prevention of missed detection and false detection, good sensitivity, improved detection sensitivity and The effect of the detection rate

Inactive Publication Date: 2016-04-06
中华人民共和国吉林出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection of LM in my country mainly adopts traditional separation culture detection and identification, PCR, ELISA and automatic identification system and other detection technologies. Traditional separation culture and biochemical identification generally take 3-7 days, the detection cycle is long, and the operation is cumbersome; PCR technology needs to extract nucleic acid and then amplify. The DNA extraction process is easily affected by exogenous nucleic acid. At the same time, impurities and bacteria in food will inhibit PCR amplification, resulting in false negatives. There is no LM in the domestic market. ELISA kit, so LM’s ELISA test needs to buy imported kits, which undoubtedly increases the cost of testing and limits the wide application of ELISA; the price of automatic identification system is high, and it is difficult to popularize in grassroots laboratories
Moreover, there is a common problem in the above tests - in the actual measurement of the actual samples, the food needs to be pre-cultured in order to increase the number of LM through the process of enrichment, but this will undoubtedly prolong the detection time and is not suitable for food-borne diseases. Rapid detection of pathogenic bacteria

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  • Anti-Listeria monocytogenes monoclonal antibody and its application
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  • Anti-Listeria monocytogenes monoclonal antibody and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Preparation of Example 1 Listeria monocytogenes Antigen Bacteria Liquid and BALB / c Mouse Immunization

[0020] Take the Listeria monocytogenes ( ATCC19111 ), inoculated on Trypticase Soy Yeast Extract Agar (TSA) medium (pH7.3±0.1) by streaking method, cultured in conventional method, centrifuged at 6000r / min for 10min, collected bacterial precipitate, and sterilized Colony counting (2.4×10 9 CFU / mL), adding formaldehyde with a final concentration of 0.3% overnight at 4°C to inactivate the bacteria. The next day, the eluted bacterial liquid was subjected to ultrasonic waves under the conditions of 20KHZ and 150W ice bath to break the bacterial cells, each time for 10 seconds, with an interval of 10 seconds, and a total time of 20 minutes. BCA protein assay kit was used to measure the bacterial protein content, and the result was 2.548 mg / mL.

[0021] Eight-week-old female BALB / c mice were used for immunization. The above-mentioned inactivated bacteria solution (2×...

Embodiment 2

[0022] The mensuration of embodiment 2 immune mouse serum antibody titer

[0023] After 3 immunizations, blood was collected from the tail of the mice, and the serum titer was detected by conventional indirect ELISA method. Wherein, the coated antigen is the Listeria monocytogenes bacterium liquid prepared in Example 1, the dilution is 1:40, the positive serum dilution is 1:1600, and the enzyme-labeled secondary antibody is HRP (horseradish peroxidase )-labeled rabbit anti-mouse IgG (1:15000 dilution). The assay results showed that the antibody titer was 1:12800.

Embodiment 3

[0024] Example 3 Preparation of Listeria monocytogenes monoclonal antibody

[0025] (1) Establishment of hybridoma cell lines

[0026] ①Preparation of feeder cells: Normal BABL / C mice were killed by neck dislocation, 8 mL of HAT culture solution was injected into the peritoneal cavity, and the culture solution was drawn out after gently shaking the mouse for a few times, and the cell concentration was adjusted to 10 5 Add 100 μL per well to a 96-well cell culture plate, place at 37°C, 5% CO 2 Cultivate overnight in a cell culture incubator for use as feeder cells.

[0027] ②Cultivation of myeloma cells and preparation of cell suspension: Myeloma cells (SP2 / 0) were resuscitated one week before fusion, passaged every other day, and fusion was carried out one day after passage. Take about 4 bottles (25cm 2 ) SP2 / 0 cells, blow down the cells in each bottle with RPMI-1640 culture medium, centrifuge at 1200r / min for 10min, repeat 2-3 times, and count with a cell counting board....

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Abstract

The invention discloses an anti-Listeria monocytogenes monoclonal antibody hybridoma cell strain, which solves the problem that reliable LM monoclonal antibodies are not easy to obtain. The cloned antibody is applied to the detection of Listeria monocytogenes in food by liquid chip and colloidal gold method, and has the advantages of good sensitivity, specificity, stability and repeatability. Pathogens can also be detected at very low concentrations, further improving the detection sensitivity and detection rate, and strictly preventing missed and false detections.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically anti-Listeria monocytogenes monoclonal antibody and its application. Background technique [0002] Listeria monocytogenes (Listeriamonocytogenes, LM) is one of the most common food-borne pathogens. WHO listed it as one of the four major food-borne bacteria in the 1990s along with Escherichia coli O157:H7, Salmonella and Staphylococcus aureus. Origin pathogenic bacteria. LM is the most pathogenic bacterium in the genus Listeria, and it is also the only typical intracellular parasite that is pathogenic to humans. It can pass through the three barriers of the host and cause serious zoonotic infectious diseases, such as Inflammation, meningitis, sepsis, miscarriage, etc. Pregnant women, newborns, the elderly, and those with immunocompromise have a fatality rate of 20-30% after infection with the bacteria, and neonates and other immunocompromised persons have a fatality rate as high as 70%....

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 孟日增刘韬刘金华赵庆松王宁聂丹丹
Owner 中华人民共和国吉林出入境检验检疫局
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