Method for yolk antibody-based enzyme-linked immunoadsordent assay (ELISA) detection of turtle systemic sepsis spherical viruses

A spherovirus, egg yolk antibody technology, applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of systemic hemorrhagic septicemia, delayed treatment timing, long incubation period, etc., to reduce the false positive rate, low cost, Minimal immune interference effect

Inactive Publication Date: 2014-12-10
NINGBO UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Since 2007, a kind of systemic sepsis has broken out in the Zhejiang area, especially the soft-shelled turtle ecological farms in Hangzhou and Ningbo. Through researches such as artificial infection experiments and other studies, it was determined that a new type of spherical virus was the primary pathogenic factor, and the new type of spherical virus was Turtle systemic sepsis spherical virus (TSSSV), which has strong infectivity. And the fatality rate is as high as 90-100%, and the incubation period of this disease is long, and the initial symptoms such as white background are easy to be similar to the symptoms of some bacterial diseases found in the past, which is easy to cause misdiagnosis and delay the timing of treatment. Therefore, it is established A convenient and sensitive virus detection method is quite important
However, there is no relevant research report on the indirect ELISA detection method of soft-shelled turtle systemic septicemia spheroid virus based on egg yolk antibody at home and abroad

Method used

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  • Method for yolk antibody-based enzyme-linked immunoadsordent assay (ELISA) detection of turtle systemic sepsis spherical viruses
  • Method for yolk antibody-based enzyme-linked immunoadsordent assay (ELISA) detection of turtle systemic sepsis spherical viruses

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Preparation of Spheroidal Virus Antigen of Turtle Systemic Septicemia

[0028] Select the diseased soft-shelled turtle, take the viscera of the soft-shelled turtle and place it in the pre-ice-bathed TNE buffer, homogenate until it is completely broken, centrifuge the homogenate at 4°C, 6000g for 10min, and take the supernatant at 4°C, 8000g Centrifuge for 20 minutes under the same conditions, take the supernatant obtained by centrifuging again at 4 ° C, 35000g for 1.5 hours to obtain a precipitate, and carefully wash off the loose part of the upper layer of the precipitate with TM buffer, and resuspend the white part of the lower layer of the precipitate in In 1ml of TM buffer, keep overnight at 4°C in an ice bath; the next day, resuspend the overnight treated pellet, centrifuge at 3500g at 4°C for 5min, then take the supernatant and centrifuge at 38000g at 4°C for 1.5h to obtain two For the secondary precipitate, carefully remove the loose part of the upper layer of th...

Embodiment 2

[0032] Preparation of egg yolk antibody against soft-shelled turtle systemic septicemia virus

[0033] a. Immune laying hens and collect immunized eggs

[0034] Carry out 1 basic immunization and 2 to 3 booster immunizations for laying hens. The basic immunization is to add an equal amount of Freund's complete adjuvant to the soft-shelled turtle systemic septicemia spheroid virus antigen, and fully emulsify the laying hens. Carry out immunization by injection, and the booster immunization is to add an equal amount of Freund's incomplete adjuvant to the antigen of soft-shelled turtle systemic septicemia spheroid virus, and carry out immunization by injection to laying hens after fully emulsifying, and the injection dosage is per kilogram of hen Inject 500mg soft-shelled turtle systemic septicemia globular virus antigen, and the interval between each immunization is 15 days. 10 days after the completion of immunization, start to collect eggs and store them in a refrigerator at 4...

Embodiment 3

[0038] Specific anti-TSSSV-IgY is used for TSSSV indirect ELISA detection, the specific steps are as follows

[0039] 1. Antigen pre-coating

[0040] The soft-shelled turtle systemic septicemia spheroid virus antigen (the specific preparation process is as described in the above-mentioned Example 1) is diluted to 0.1ng / μl with .01M pH of 9.6 carbonate buffer solution (CBS), and added to the polymer by 100 μl / well. Place in a styrene 96-well reaction plate overnight at 4°C, pour out the liquid in the wells the next day, wash the wells with PBS-T washing solution for 3 times, add 1% bovine serum albumin blocking solution to each well, shake at a low speed at 37°C Incubate for 1 hour for blocking, after the sealing is completed, suck away the bovine serum albumin blocking solution in the well;

[0041] Wherein the PBS-T washing solution is obtained by mixing Tween-20 and PBS buffer solution in a volume ratio of 1:2000, the concentration of the PBS buffer solution is 0.1M, and th...

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Abstract

The invention discloses a method for yolk antibody-based enzyme-linked immunoadsordent assay (ELISA) detection of turtle systemic sepsis spherical viruses. The method is characterized by comprising the following steps of 1, adding 0.1 nanograms per microliter of turtle systemic sepsis spherical virus antigens into a polystyrene 96-hole reaction plate according to a ratio of 100 microliters per hole, carrying out pre-coating, and carrying out blocking by a bovine serum albumin blocking liquid, 2, adding turtle systemic sepsis spherical virus yolk antibodies diluted according to a certain ratio into each one of holes, and adding horseradish peroxidase-marked goat anti-chicken IgG and an o-phenylenediamine substrate solution into each one of the holes for a color reaction, and 3, reading absorbance values at the wavelength 492 nanometers of all the holes by an ELISA device, and when the absorbance value of the detected hole is more than 2.1 times of an average absorbance value of a negative control hole, determining that the detected result is positive. The method has the advantages of high stability, high specificity, high sensitivity, small immunity interference, and low background signal-noise ratio.

Description

technical field [0001] The invention relates to the field of preparation technology and application of biological products, in particular to an indirect ELISA detection method for soft-shelled turtle systemic septicemia spheroid virus based on yolk antibody. Background technique [0002] Soft-shelled turtle, scientific name Chinese soft-shelled turtle, has always been a medicinal food and nourishing good product. In the 1980s, the rise of greenhouse farming greatly increased the production of soft-shelled turtles, which were originally considered rare, followed by the wanton prevalence of diseases. Since 2007, a kind of systemic sepsis has broken out in the Zhejiang area, especially the soft-shelled turtle ecological farms in Hangzhou and Ningbo. Through researches such as artificial infection experiments and other studies, it was determined that a new type of spherical virus was the primary pathogenic factor, and the new type of spherical virus was Turtle systemic sepsis s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
Inventor 李登峰彭娇周永强刘联国陈炯顾晓英
Owner NINGBO UNIV
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