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Monoclonal antibody resisting Tilapia immunoglobulin IgM, cell strain of monoclonal antibody and application of monoclonal antibody

A monoclonal antibody and immunoglobulin technology, applied in the biological field, can solve the problems of complicated isolation operation process of tilapia lake virus, difficulty in wide application, high conditions and technical requirements, etc.

Active Publication Date: 2019-06-07
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

[0004]The isolation operation process of tilapia lake virus is relatively complicated, and the required conditions and technologies are also demanding, so it is difficult to be widely used
Existing methods to detect the presence of viral nucleic acid, the method is single, often lead to false positive and false negative results, it is difficult to achieve quantitative detection and diagnosis

Method used

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  • Monoclonal antibody resisting Tilapia immunoglobulin IgM, cell strain of monoclonal antibody and application of monoclonal antibody
  • Monoclonal antibody resisting Tilapia immunoglobulin IgM, cell strain of monoclonal antibody and application of monoclonal antibody
  • Monoclonal antibody resisting Tilapia immunoglobulin IgM, cell strain of monoclonal antibody and application of monoclonal antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Preparation and purification of embodiment 1 anti-tilapia immunoglobulin IgM monoclonal antibody

[0040] Experimental Materials:

[0041] The experimental tilapia was from the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences, with a body length of 15-20 cm; other chemical reagents were analytically pure, provided by Guangzhou Weijia Technology Co., Ltd.

[0042] 1. Purification of Tilapia IgM

[0043] 1.1 Take blood from tilapia by docking the tail, put it in a glass tube, solidify it at room temperature, put it at 37°C for 2 hours, and then put it at 4°C overnight, after the serum is fully analyzed, centrifuge at 3000g / min for 10min, take it clear.

[0044] 1.2 Purification: centrifuge the fish serum at 12000rpm for 20min, and 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%. Stir the solution on a magnet...

Embodiment 2

[0079] Embodiment 2 Tilapia lake virus indirect ELISA detection kit

[0080] The indirect ELISA kit for detecting Tilapia Lake virus consists of: the monoclonal antibody prepared in Example 1; coating solution; horseradish peroxidase-labeled goat anti-mouse antibody (purchased from Wuhan Boster Biotechnology Company); Enzyme substrate reaction solution; positive control; negative control (standard serum); washing solution; TMB chromogenic solution and reaction termination solution.

[0081] The positive control was purified tilapia lake virus, and the negative control was uninfected normal tilapia serum.

Embodiment 3

[0082] Example 3 Establishment of Tilapia Lake Virus Indirect ELISA Detection Method

[0083] 1. Determination of antigen working concentration

[0084] Dilute the purified tilapia lake 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 store in a humid chamber at 4°C Pack 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. Anti-TiLV (Tilapia Lake Virus) positive serum and tilapia negative serum were diluted 1:300 times, and 100uL was added to each well for ELISA square array test, incubated in a 37°C incubator for 1h, washed 3 times with PBST, and 3-5 minutes each time. The above prepared mouse anti-tilapia 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), ...

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Abstract

The invention discloses a monoclonal antibody resisting Tilapia immunoglobulin IgM, a cell strain of the monoclonal antibody and an application of the monoclonal antibody. The monoclonal antibody resisting Tilapia IgM is produced through secretion of a hybridoma cell strain Ti-IgM-B6 of which the preservation number is CCTCC NO:C2018244, the titer of the antibody is more than 1 to 10000, and the hybridoma cell strain is high in activity and high in specificity; and besides, through the monoclonal antibody, an indirect ELISA method for detecting Tilapia lake viruses is established, has higher value on epidemiological investigation of Tilapia diseases, pathogeny diagnosis, epidemic disease prevention and control and the like, and provides reference for disease prevention and control of otherbred fish.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody against tilapia immunoglobulin IgM, a cell line and application thereof. Background technique [0002] The tilapia industry is one of the pillar brands for fishery development and income increase of fishermen in our province. However, in recent years, tilapia diseases have occurred frequently, such as tilapia streptococcus disease, Luohu virus disease, etc., which have caused huge economic losses to the global tilapia aquaculture industry and become the main restrictive factor for the sustainable development of the tilapia industry. one. Disease diagnosis is an important tool and prerequisite for the prevention and control of these diseases. At present, there is a lack of effective on-site rapid detection methods for the main diseases of tilapia. [0003] Antibodies are immunoglobulins produced by lymphocytes in the immune system of vertebrates tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/42G01N33/569G01N33/577C12R1/91
CPCY02A40/81
Inventor 曾伟伟王英英王庆尹纪元李莹莹任燕刘春张德峰石存斌常藕勤
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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