A kind of preparation method and application of rabbit anti-grouper serum immunoglobulin HRP-labeled antibody

A technology of serum immunization and labeling antibodies, which is applied in the direction of instruments, color/spectral characteristic measurement, and measuring devices, can solve problems such as the loss of seawater fish farming, the economic loss of grouper, and the great harm of larvae and juvenile fish. Appropriate labeling rate of the conjugate, large dilution, and high sensitivity

Active Publication Date: 2019-10-11
FISHERIES RES INST OF FUJIAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, frequent occurrence of diseases in recent years has caused huge economic losses to the grouper aquaculture industry.
[0003] Viruses, parasites and bacterial infections are extremely harmful to larvae and juvenile fish and cause huge losses to the marine fish farming industry
[0004] At present, the PCR method is usually used to detect grouper virus, parasite and bacterial infection. The PCR method is to identify the infection of diseases by detecting the expression of virus genes in the fish brain and eyes; however, in the grouper breeding , as the main body of breeding fish, it is impossible to kill the broodstock and then detect it by PCR method; therefore, there is an urgent need for a method of detecting diseases on the basis of ensuring the normal vitality of the fish

Method used

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  • A kind of preparation method and application of rabbit anti-grouper serum immunoglobulin HRP-labeled antibody
  • A kind of preparation method and application of rabbit anti-grouper serum immunoglobulin HRP-labeled antibody
  • A kind of preparation method and application of rabbit anti-grouper serum immunoglobulin HRP-labeled antibody

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Experimental program
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Effect test

Embodiment 1

[0044] 1. Preparation of rabbit anti-grouper serum immunoglobulin antibody

[0045] 1) Purification of grouper serum immunoglobulin

[0046] a. Kill the fish to take blood, let it stand still to get serum, add ammonium sulfate solution to the serum to make the final concentration 10%, centrifuge at 10000r / min for 5min, and discard the precipitate;

[0047] b. Add ammonium sulfate solution to the centrifuged solution to make the final concentration 30%, centrifuge at 10000r / min for 5min, and discard the supernatant;

[0048] c. Dissolve the above precipitate with PBS buffer, and measure the protein concentration by Bradford method;

[0049] 2) Preparation and purification of grouper immunoglobulin rabbit antibody

[0050] a. Add 2 mg of purified fish immunoglobulin to the adjuvant system and mix well, and use it to immunize rabbits, with an interval of 2 weeks between each two immunizations; If the price reaches more than 40,000, kill the rabbit and get the whole blood;

[...

Embodiment 2

[0071] A method for detecting grouper neuronecrosis virus

[0072] A kit for detecting grouper neuronecrosis virus, comprising the above-mentioned rabbit anti-grouper serum immunoglobulin HRP-labeled antibody; and an antigen, the antigen being Coat protein of grouper neuronecrosis virus.

[0073] Preparation of standard curve:

[0074] Select 100 larvae of 3-4cm grouper and divide them into two groups at random, 50 in each group, and inject 5×10 6 Copies / μL of nerve necrosis virus was 30 μL, and the control group was injected with the same amount of sterilized normal saline. On the 3rd, 6th, 9th, 12th, and 15th day of the experiment, 6 fish were randomly selected from each group to draw blood to detect the titer of the neuronecrosis virus antibody in the serum. The neuronecrosis virus is RGNNV serotype.

[0075] The detection method is as follows:

[0076] a. After diluting the antigen with PBS buffer to 5 μg / mL, add it to the ELISA plate, incubate at 37°C for 2 hours, or ...

Embodiment 3

[0085] A method for detecting cryptocystosis in grouper stimuli.

[0086] A kit for detecting grouper-stimulated Cryptocaryoniasis, comprising the above-mentioned rabbit anti-grouper serum immunoglobulin HRP-labeled antibody; and an antigen, which is the total body protein of Cryptocaryon stimuli larvae.

[0087] Stimulation of Extraction of Total Protein from Cryptocaryon larvae

[0088] According to the instructions of the whole protein extraction kit (purchased from Beijing Suolaibao Technology Co., Ltd., item number BC3640), the total protein of stimulated Cryptocaryon larvae was extracted.

[0089] Preparation of standard curve:

[0090] Select 120 larvae of 4-6cm grouper, randomly divide them into two groups, 60 in each group, and put 20 in a tank equipped with 40L aerated seawater; the test group soaks 3×10 3 The stimulated Cryptocaryon larvae of one / tail fish, the control group did not do any treatment, and other conditions were the same. On the 3rd, 6th, 9th, 12th,...

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Abstract

The invention discloses a preparation method and application of a rabbit anti-grouper serum immunoglobulin HRP-labeled antibody, and belongs to the technical field of antibody preparation. The rabbit anti-grouper serum immunoglobulin HRP-labeled antibody is an enzyme antibody conjugate comprising HRP and a rabbit anti-grouper serum immunoglobulin antibody, the rabbit anti-grouper serum immunoglobulin HRP-labeled antibody can be specifically bound with grouper serum immunoglobulin for color development of a HRP substrate. The HRP-labeled antibody prepared by the method has high purity, high enzyme activity, suitable conjugate labeling rate, great dilution degree for performing ELISA tests, and high sensitivity, and can be used for detecting viral diseases, parasitic diseases and bacterial diseases of grouper.

Description

technical field [0001] The invention belongs to the technical field of antibody preparation, and in particular relates to a preparation method and application of a rabbit anti-grouper serum immunoglobulin HRP-labeled antibody. Background technique [0002] Grouper is a high-grade and high-quality fish among seawater cultured fish. With the maturity of artificial breeding technology of grouper in my country and the industrialized large-scale production of grouper seedlings, it has become an important marine fish culture species. However, the frequent occurrence of diseases in recent years has caused huge economic losses to the grouper aquaculture industry. [0003] Viruses, parasites and bacterial infections are extremely harmful to larvae and juvenile fish, causing huge losses to the marine fish farming industry. [0004] At present, the PCR method is usually used to detect grouper virus, parasite and bacterial infection. The PCR method is to identify the infection of dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/535G01N21/31
Inventor 葛辉林克冰黄种持朱志煌
Owner FISHERIES RES INST OF FUJIAN
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