Monoclonal antibody of anti-rhabdovirus glucoprotein of mandarin fish, preparation and use thereof

A technology of monoclonal antibody and rhabdovirus, applied in antiviral immunoglobulin, instruments, analytical materials, etc., can solve the problem of no mandarin fish rhabdovirus, etc., and achieve enhanced immune effect, good effect and specificity strong effect

Inactive Publication Date: 2009-08-12
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although domestic and foreign reports (Dixon and Longshaw, Dis Aquat Org, 2005, 67: 25-29; Zhou et al., Viral Immunol, 2006, 19: 637-645; Chen et al.

Method used

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  • Monoclonal antibody of anti-rhabdovirus glucoprotein of mandarin fish, preparation and use thereof
  • Monoclonal antibody of anti-rhabdovirus glucoprotein of mandarin fish, preparation and use thereof
  • Monoclonal antibody of anti-rhabdovirus glucoprotein of mandarin fish, preparation and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0036] A kind of anti-mandarin fish rhabdovirus glycoprotein monoclonal antibody, its preparation steps are:

[0037] 1. Immunization of mice

[0038] The mice used for immunization were 8-week-old female BALB / c mice. After mixing 150 μl of purified mandarin fish rhabdovirus (100 μg of protein) with the same volume of Freund’s complete adjuvant, the mice were injected intraperitoneally for initial immunization After 2 weeks, mix the purified mandarin rhabdovirus of the same dose (150 μl) with Freund’s incomplete adjuvant of the same volume and perform the second immunization; after 2 weeks, use the same dose (150 μl) of The purified virus was mixed with the same volume of Freund's incomplete adjuvant for the third immunization; 1 week after the third immunization, three days before the splenocytes of the immunized mice were collected, the purified mandarin fish bombs without adjuvant Virus (75 μg protein) was directly injected into the spleen of mice for booster immunization....

Embodiment 2

[0053] Characterization of monoclonal antibodies against mandarin fish rhabdovirus glycoprotein:

[0054] 1. Ascites titer identification of monoclonal antibody

[0055] Method: Ascites titer of monoclonal antibody was identified by indirect ELISA

[0056] Result: the ascites titer of the monoclonal antibody of the present invention is 1:10 5 , indicating that the SCRV-G-1B2 cell line has the ability to secrete high-titer antibodies.

[0057] 2. Subtype identification of monoclonal antibodies

[0058] Method: The Ig subtype of the monoclonal antibody of the present invention was identified by using the Mouse mAb Isotyping Kit (Mouse mAb Isotyping Kit) of Sigma Company according to its instructions.

[0059] Results: The subtype of the monoclonal antibody of the present invention is IgG2b.

[0060] 3. Specific identification of monoclonal antibodies

[0061] Western blot:

[0062] Purified mandarin fish rhabdovirus was used as the detection group, normal EPC cell lysate w...

Embodiment 3

[0065] Monoclonal antibody against mandarin fish rhabdovirus glycoprotein for immunodetection of mandarin fish rhabdovirus infection:

[0066] 1. Indirect immunofluorescence assay (IFA) detection

[0067] Methods: EPC cells were cultured in a 6-well plate covered with glass flyers, and the cells were grown on the glass flyers. After 24 hours, the mandarin fish rhabdovirus was inoculated, and normal cells were set as negative controls. After obvious cell lesions appeared, the cells were fixed with 4% paraformaldehyde at 4°C for 1 hour, and then permeabilized with 100% alcohol at -20°C for 7 minutes. After washing with PBS, the culture supernatant of SCRV-G-1B2 cells in Example 1 was added, and incubated at room temperature for 2 hours. After washing with PBS, goat anti-mouse IgG labeled with fluorescein (FITC) was used as the secondary antibody and incubated at room temperature for 2 h. The slide was mounted with a PBS solution containing 50% glycerol by volume, and placed un...

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Abstract

The invention discloses a monoclonal antibody of anti-mandarin fish rhabdovirus glycosidoprotein and a preparation method and the application thereof, and hybridoma cell strain SCRV-G-1B2,CCTCC No:C200918 for producing the monoclonal antibody. The preparation method comprises the following steps: taking purified mandarin fish rhabdovirus as antigen, immunizing a BALB/c mouse, producing fused hybridoma cells through cell fusion and obtaining hybridoma cell strain which stably secretes anti-mandarin fish rhabdovirus glycosidoprotein monoclonal antibody through indirection ELIS screening and limited dilution monocloning. The invention can obtain murine spleen lymphocytes with higher activity, stronger specification and more quantities so as to ensure that the obtained fused hybridoma cells have better effect. By using the murine spleen strengthening immunization technique, the fusion rate and the positive rate respective reach is 96.3 percent and 97 percent. The monoclonal antibody can be used for immunodetection of mandarin fish rhabdovirus infection and can also be used for quantitative and positioning analysis of mandarin fish rhabdovirus glycosidoprotein in host tissues or cells.

Description

technical field [0001] The present invention relates to the technical field of monoclonal antibodies against aquatic animal viruses, more specifically to a monoclonal antibody against mandarin fish rhabdovirus glycoprotein, and also to a method for preparing the monoclonal antibody against mandarin fish rhabdovirus glycoprotein, secreting Fusion, isolation, identification and expansion culture of hybridoma cells with monoclonal antibody against rhabdovirus glycoprotein of mandarin fish, involving immunodetection of rhabdovirus infection of mandarin fish with monoclonal antibody against rhabdovirus glycoprotein of mandarin fish , and also relates to the use of monoclonal antibodies against mandarin fish rhabdovirus glycoprotein in the quantitative and localization analysis of mandarin fish rhabdovirus glycoprotein in host tissues or cells. Background technique [0002] Fish rhabdovirus is an important viral pathogen that causes fatal and epidemic diseases of fish and other aq...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12P21/08G01N33/577
Inventor 张奇亚陈中元
Owner INST OF AQUATIC LIFE ACAD SINICA
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