Monoclonal antibody for resisting to cyprinus carpio haematopterus immune globulin (IgM) and application of monoclonal antibody

A monoclonal antibody and immunoglobulin technology, applied in the biological field, can solve problems such as single method, false negative, difficult quantitative detection and diagnosis of the disease

Active Publication Date: 2016-06-08
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these molecular biological methods all detect the presence or absence of nucleic acid of the virus. The single method often leads to false positive and false negative results, making it difficult to quantitatively detect and diagnose the disease.

Method used

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  • Monoclonal antibody for resisting to cyprinus carpio haematopterus immune globulin (IgM) and application of monoclonal antibody
  • Monoclonal antibody for resisting to cyprinus carpio haematopterus immune globulin (IgM) and application of monoclonal antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1 Preparation and purification of anti-koi immunoglobulin IGM monoclonal antibody

[0035] Materials and Methods

[0036] Experimental Materials

[0037] The test koi came from the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences, with a body length of 20 cm; other chemical reagents were of analytical grade, provided by Sinopharm Chemical Reagent Co., Ltd.

[0038] 1 Koi IgM purification

[0039] 1.1 Take the blood of koi carp by docking the tail, put it in a glass tube, solidify it at room temperature, place it at 37°C for 2 hours, then place it at 4°C overnight, after the serum is fully analyzed, centrifuge at 3000g / min for 10min, and take the supernatant .

[0040] 1.2 Purification: Centrifuge the fish serum at 12000rpm for 20min, take the supernatant; measure the volume of the supernatant; slowly add saturated ammonium sulfate solution to the supernatant while stirring, until the final saturation is 33%; put the solut...

Embodiment 2

[0070] Example 2 Establishment of Indirect ELISA Detection Method for Koi Herpes Virus

[0071] 1. Determination of antigen working concentration

[0072] Dilute the purified koi herpes virus with coating solution (pH8.6) to a concentration gradient of 50 μg / mL, 30 μg / mL, and 10 μg / mL, coat a 96-well microtiter plate, add 100 uL to each well, and pack in a wet box at 4 °C be overnight. Add 300 μL of 10% skim milk to each well to block, incubate in a 37°C incubator for 1 h, and wash with PBST 3 times, each time for 3-5 min. Dilute anti-KHV positive serum and koi negative serum 1:300 times, add 100uL to each well for ELISA square test, incubate in 37°C incubator for 1h, wash with PBST 3 times, each 3-5min. The above prepared mouse anti-koi IgM antibody 1:2000, 100uL per well, incubated in a 37°C incubator for 1h, washed 3 times with PBST, 3-5min each time. Add 50 μL chromogenic substrate (TMB), and develop color at 37°C in the dark for 10 minutes. With 50μL 2mol / LH 2 SO 4 ...

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Abstract

The invention discloses a monoclonal antibody for resisting to cyprinus carpio haematopterus IgM, a hybridoma cell strain and application of the monoclonal antibody. The monoclonal antibody for resisting to cyprinus carpio haematopterus IgM is secreted and produced by the hybridoma cell strain A5-E10 with the preservation number being CCTCC NO: C2015218. The monoclonal antibody can be applied to detection of certain pathogeny of cyprinus carpio haematopterus or common carp or a vaccine specific antibody of an aquatic product, thereby early-stage disease diagnosis and vaccine using effect evaluation are conducted, high clinical application and promotion value is achieved for epidemiological investigation and pathogeny diagnosis conducted on disease of cyprinus carpio haematopterus or common carp, and reference is provided for prevention and treatment of other cultured fish diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody against koi immunoglobulin IgM and an application thereof. Background technique [0002] As a large category of ornamental fish, koi has a beautiful body and bright colors. It has been bred in large quantities in China and is the main species of recreational fishery at present. As the breeding environment is getting worse and worse, various infectious diseases of koi, such as carp spring viremia, herpes viral papilloma, koi herpes virus disease, etc., have begun to spread widely, and the establishment of various disease detection methods is urgently needed. The successful preparation of anti-koi immunoglobulin IgM monoclonal antibody will provide important materials for the immunological diagnosis of various diseases of Koi. [0003] Antibodies are immunoglobulins produced by lymphocytes in the immune system of vertebrates that can specifically bind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42C12N5/20G01N33/577G01N33/569C12R1/91
CPCC07K16/4208G01N33/56994G01N33/577
Inventor 李莹莹王庆曾伟伟王英英刘春郑树城石存斌宋新建黄琦雯
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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