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Hybridoma cell line Anti-CLas McAb1 and monoclonal antibody secreted by hybridoma cell line Anti-CLas McAb1 and use thereof

A technology of hybridoma cell lines and monoclonal antibodies, applied in analytical materials, anti-bacterial immunoglobulin, biochemical equipment and methods, etc., can solve the problems of poor specificity, achieve low cost, strong anti-interference, and wide application Effect

Active Publication Date: 2018-09-04
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The patent discloses a polyclonal antibody, because the polyclonal antibody itself has the unavoidable disadvantage of poor specificity, and there are many kinds of outer membrane proteins of citrus Huanglongbing bacteria, which outer membrane protein is used as the antigen to make a monoclonal antibody detection The best effect of citrus Huanglongbing has not been reported, and there is no research report on the monoclonal antibody against the specific protein of Huanglongbing

Method used

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  • Hybridoma cell line Anti-CLas McAb1 and monoclonal antibody secreted by hybridoma cell line Anti-CLas McAb1 and use thereof
  • Hybridoma cell line Anti-CLas McAb1 and monoclonal antibody secreted by hybridoma cell line Anti-CLas McAb1 and use thereof
  • Hybridoma cell line Anti-CLas McAb1 and monoclonal antibody secreted by hybridoma cell line Anti-CLas McAb1 and use thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 The establishment of hybridoma cell line Anti-CLas McAb1

[0025] 1. Preparation of experimental materials and reagents

[0026] Before operation, place the various sterilized consumables required in the table below in the ultra-clean bench for irradiation sterilization. Pipettes of various specifications and matching pipette tips, homogenizer, scissors and tweezers, glass plates, foam pads, needles, cell screens, filter paper, 96-well cell culture plates (sterile).

[0027] Table 1

[0028]

[0029] 2. Experimental method

[0030] 1. Preparation of recombinant expression plasmid p102-McAb1

[0031] Firstly design specific primers for McAb1 of Huanglongbing bacteria (the nucleotide sequence of the recombinant Huanglongbing bacteria CLas McAb1 protein is shown in SEQID NO: 1), and design the upper and lower primers for amplifying the coding region gene of Huanglongbing bacteria CLas McAb1 according to the specific structure of the vector. 5'-CACCCCAGC...

Embodiment 2

[0059] Embodiment 2: Preparation of monoclonal antibody

[0060] 1. Ascites preparation

[0061] (1) BALB / c mice over 6 weeks old were intraperitoneally injected with Freund's incomplete adjuvant, 0.5ml / only;

[0062] (2) Three days later, the hybridoma cells diluted with PBS or incomplete medium were inoculated intraperitoneally, 1-5×10 per mouse 6 / 0.5ml;

[0063] (3) After an interval of 3 days, observe the occurrence of ascites in the mice every day. If the abdomen is obviously enlarged, and the skin feels tense when touched by hand, the ascites can be collected with a 10ml needle;

[0064] (4) Centrifuge the ascitic fluid (12000 rpm for 10 min), collect the supernatant, and freeze the supernatant in a -20°C refrigerator.

[0065] 2. Ascites antibody purification

[0066] (1) Serum pretreatment: 0.22 pore size filter filters serum, mixes with equal volume PBS (pH7.4), adjusts to pH7.8, and the effect of affinity hanging column is better at this moment;

[0067] (2) Eq...

Embodiment 3

[0076] The characteristic detection of embodiment 3 monoclonal antibody

[0077] 1. Detection of monoclonal antibody titer

[0078] (1) Coating: McAb1 antigen was coated at 1ug / ml, 100ul / well, overnight at 4°C;

[0079] (2) Blocking: Pat dry the liquid in the microplate, add blocking solution (1% BSAin TBST) 150ul / well, incubate at 37°C for 60min;

[0080] (3) Primary antibody: pat dry the liquid in the microplate, 350ul / hole / time, wash 3 times with TBST, pat dry, and dilute the antibody after purification in Example 2 with 1:3000 and 3 times (1 %BSA dilution), 100ul / well, incubated at 37°C for 60min;

[0081] (4) Secondary antibody: pat dry the liquid in the microplate, 350ul / well / time, wash 3 times with TBST, pat dry, add HRP goat anti-mouse (diluted at 1:3000 blocking solution) 100ul / well, incubate at 37°C for 60min ;

[0082] (5) Color development: pat dry the liquid in the microplate, 350ul / well / time, wash 3 times with TBST, pat dry, add single substrate TMB100ul / well...

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Abstract

The invention provides a hybridoma cell line Anti-CLas McAb1 which is a mouse hybridoma cell (Mus musculus Hybridoma) Anti-CLas McAb1 and has a preservation number of CCTCC NO: C2017219. The inventionalso provides a preparation method of the hybridoma cell strain and a recombinant Candidatus Liberibacter asiaticus CLas McAb1 monoclonal antibody secreted by the hybridoma cell strain or a passage cell thereof. The invention also provides uses of the recombinant Candidatus Liberibacter asiaticus n CLas McAb1 monoclonal antibody. The uses comprise a detection kit and an immunocolloidal gold teststrip and utilizes the specific detection of the recombinant Candidatus Liberibacter asiaticus CLas McAb1 monoclonal antibody. The recombinant Candidatus Liberibacter asiaticus CLas McAb1 monoclonal antibody has the advantages of good specificity, strong interference resistance, low cost and wide application, and can quickly and effectively detect Candidatus Liberibacter asiaticus in citrus materials.

Description

technical field [0001] The invention relates to a hybridoma cell line and a monoclonal antibody, in particular to the hybridoma cell line Anti-CLasMcAb1 and the secreted monoclonal antibody and application thereof. Background technique [0002] Citrus huanglongbing is the most difficult, most harmful, and most threatening destructive disease in citrus production. It is known as the "AIDS" of citrus and is widely distributed in almost all major citrus producing areas in my country. As a devastating disease, citrus infected plants often lose fruiting ability or die within 2 to 3 years, and even cause garden destruction. The rampant harm and spread of citrus huanglongbing has posed a serious threat to the stability and development of the citrus industry, and has become a major obstacle to the development of the citrus industry. [0003] At present, there is still a lack of effective control agents and ideal resistant varieties for citrus huanglongbing, so integrated control is...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/12G01N33/569G01N33/577
CPCC07K16/1203G01N33/56911G01N33/577
Inventor 丁芳约翰·哈通邓秀新彭抒昂洪霓王国平刘永忠
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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