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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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 membra

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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[0024] Example 1 Establishment of Anti-CLas McAb1 Hybridoma Cell Line

[0025] 1. Prepare experimental materials and reagents

[0026] Before operation, place the sterilized consumables required in the table below in the ultra-clean table for irradiation sterilization. Various specifications of pipettes and matching pipette tips, homogenizers, 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] First, design specific primers for Huanglong pathogen McAb1 (the nucleotide sequence of recombinant Huanglong pathogen CLas McAb1 protein is shown in SEQ ID NO: 1), and design the upper and lower primers to amplify the coding region gene of Huanglong pathogen CLas McAb1 according to the specific structure of the vector. 5'-CACCCCAGCCGTCTTTACCAGTCT-3' (nucleotide sequence shown in SEQ ID NO: ...

Example Embodiment

[0059] Example 2: Preparation of monoclonal antibodies

[0060] 1. Ascites preparation

[0061] (1) Take BALB / c mice over 6 weeks of age and inject Freund's incomplete adjuvant, 0.5ml / mouse;

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

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

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

[0065] 2. Ascites antibody purification

[0066] (1) Serum pretreatment: filter serum with 0.22 pore size filter, mix with equal volume of PBS (pH 7.4), adjust to pH 7.8, at this time, the effect of affinity hanging column is better;

[0067] (2) Equilibrium affinity column: Wash the column with...

Example Embodiment

[0076] Example 3 Characterization of monoclonal antibodies

[0077] 1. Monoclonal antibody titer detection

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

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

[0080] (3) Primary antibody: Pat dry the liquid in the ELISA plate, 350ul / well / time, wash with TBST 3 times, pat dry, and dilute the purified antibody in Example 2 at a ratio of 1:3000 and 3 times (1 %BSA dilution), 100ul / well, incubate at 37°C for 60min;

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

[0082] (5) Color development: pat dry the liquid in the ELISA plate, 350ul / well / time, wash 3 times with TBST, pat dry, add 100ul / well of single substrate TMB...

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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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