ELISA kit for detecting iridescent virus antibody in largemouth bass and its detection method

A kit and antibody technology, applied in the direction of viruses/phages, viruses, viral peptides, etc., can solve the problems of large genome, complex pathogenicity and difficulty of frog virus, and achieve good sensitivity and specificity.

Active Publication Date: 2021-10-26
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the harm of frog virus to largemouth bass has not been taken seriously before. With the continuous improvement of the breeding level of largemouth bass, frog virus has become the biggest pain point of high-density culture of largemouth bass; the genome of frog virus is relatively large (80-120Kbp) , the pathogenicity is relatively complex, and the preparation of immunological raw materials for detecting LMBV-specific antibodies is technically difficult, and there is no accurate, simple and sensitive detection method for LMBV-specific antibodies

Method used

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  • ELISA kit for detecting iridescent virus antibody in largemouth bass and its detection method
  • ELISA kit for detecting iridescent virus antibody in largemouth bass and its detection method
  • ELISA kit for detecting iridescent virus antibody in largemouth bass and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Expression purification of recombinant proteins

[0051] The expression plasmid of the N-end domain (LVMCPMCPMCPMCP), LMBV MCP protein of LMBV is constructed, converted to E. coli expression, Rosetta (DE3) PLYSS Expressed to get recombinant proteins LVMCP, LVMCPN, and LVMCPC.

[0052] Among them, the amino acid sequences of LVMCP, LVMCPN and LVMCPC are: SEQ ID NO: 2, SEQ ID NO: 1 and SEQ ID NO: 3; LMBV MCP protein sequence structure analysis figure 1 Indicated.

[0053] The expression of three kinds of engineering bacteria is identified by Western blot, and the results are figure 2 As shown, where M is protein Ladder, lanes 1-3 are LVMCP, LVMCPN, and LVMCPC protein blot, respectively.

[0054] Three recombinant proteins were purified by affinity chromatography, and the kit was quantified with a commercial BCA protein to quantify the protein concentration.

Embodiment 2

[0055] Example 2LMBV antibody positive, negative serum and quality control

[0056] 1, LMBV antibody positive serum preparation

[0057] The LMBV virus was cultured with carp epithelial cells (EPC), and the final concentration of 0.2% formaldehyde inactivated virus was added. After the quality inspection, the vaccine was prepared with VSA201 adjuvant emulsified virus, and 4 ° C was stored at 4 ° C after quality inspection. Choose 200-300 grams of healthy large mouth 50 tail, use the resulting LMBV inactivated vaccine abdominal cavity, 200 μl / only, immunized 2 times, each immunization interval for 15 days. All fish-fin fins were blooded, combined with ELISA test assessing antibody titers. Serum samples were selected (serum dilution 40 times, and serum samples of OD450 nm at around 1.0) were used as LMBV antibody positive serum, stored, stored, spare.

[0058] 2, LMBV antibody negative serum preparation

[0059] Choose 200-300 grams of healthy large mouth 50 tail, using VSA201 adj...

Embodiment 3

[0063] Example 3 ELISA reactivity of three recombinant proteins

[0064] The immunological reaction of the large mouth serum antibody with three recombinant proteins (prepared by Example 1) was detected using an indirect ELISA method. ELISA Detects the solid phase package selection: LVMCP, LVMCPN, and LVMCPC. specific method:

[0065] (1) Three recombinant proteins were diluted with 0.05 mol / L carbonate (pH = 9.6) to 2 μg / ml, 4 μg / ml and 8 μg / ml bag by the enzyme board, 100 μl / well, 37 ° C for 1 hour. , Incubate overnight at 4 ° C;

[0066] (2) Wash the enzyme board 3 times with 0.05% Tween-20 (PBST), 5 minutes / time; each hole is added to 100 μl of closed liquid, 37 ° C is closed for 1 hour;

[0067] (3) was washed 3 times with PBST, 3 minutes / time; the serum serum (LMBV antibody positive serum or LMBV antibody) prepared in Example 2 was diluted in a hole, incubated in the hole, incubated at 37 ° C 30 minute;

[0068] (4) Wash 3 times with PBST, 3 minutes / time; add...

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Abstract

The invention relates to the technical field of immune detection, in particular to an ELISA kit for detecting largemouth bass iridescent virus antibodies and a detection method thereof. The kit includes an antibody capture agent, a solid phase support, a first antibody, an enzyme-labeled second antibody, a blocking solution, a chromogenic solution and a stop solution; wherein the antibody capture agent is recombinant largemouth bass iridescent virus LVMCPn Protein, its amino acid sequence is shown in SEQ ID NO:1. The kit of the invention has good sensitivity and specificity to the LMBV antibody, and has good repeatability and stability.

Description

Technical field [0001] The present invention relates to the field of immunoassay, and more particularly to an ELISA kit for detecting an antibody against large mouth iridescent viruses and its detection methods. Background technique [0002] Largemouth Bass, Micropterus Salmoides is the sunspot, and the black bass is a fish, commonly known as the California brew, is the important fish species of special fish breeding in South China. The black bass meat is delicious, the nutritional value is high, and the consumption increases year by year. With the change of growth habits, the large-mouth black bass is gradually forming a large-scale breeding model. When there is no disease, there is a large mouthful of black gear, and the profit is considerable. However, under high-density breeding conditions, the disease is frequent in the disease, in many diseases, the "rotten disease" caused by the frog, the rock, the body disease is the most serious, and the virus combines bacterial infectio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/01G01N33/569G01N33/543
CPCC07K14/005C12N2710/00022G01N33/543G01N33/56983G01N2333/01G01N2469/20
Inventor 李中圣刘文娜张钰薇王凤求黄锦炉尹兴强李涛罗律曹梦蕊
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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