CN102914647A - Method for detecting duck flavivirus antibody by using agar diffusion method - Google Patents

Abstract

The invention provides a method for detecting a duck flavivirus antibody by using an agar diffusion method, and belongs to the field of veterinarian lemology and immunology. A detected duck flavivirus is an isolate from a duck farm in Anhui. The flavivirus is inoculated to an SPF chicken embryo and propagated for five generations to obtain a stable virus strain which is named as duck flavivirus AH-F10. Allantonic fluid is added into a white oil adjuvant through formaldehyde inactivation for emulsification, an inactivation immune is prepared, and laying ducks are immunized continuously for twice to obtain duck flavivirus positive serum. A clear sediment belt can be presented by carrying out agar diffusion reaction on the positive serum and the duck flavivirus liquid after formaldehyde inactivation. The most suitable buffer solution and the most suitable buffer solution concentration for the agar diffusion reaction are determined based on the influence of different buffer solutions and different concentrations of the buffer solutions on the agar diffusion valence of the position serum. The method can be used for diagnosing the duck flavivirus prevalent in China, and is simple and convenient to operate, obvious in effect and low in cost when being used for epidemiological investigation.

Description

A kind of agar diffusion method detects the method for duck flavivirus antibody

Technical field

The present invention relates to detect with a kind of agar diffusion method the method for duck flavivirus antibody, belong to zoonosis and field of immunology.

Background technology

Duck flavivirus occured first in the East China since 2010 infect the popular of duck egg drop reduction-death syndrome that the egg duck causes, and the main plant in the provinces such as Zhejiang, Anhui, Fujian, Jiangsu is arrived in bamboo telegraph, propagate into afterwards the provinces and cities such as Guangdong, Shandong, Henan, Hebei, the up to the present most of main aquatic bird cultivation in whole nation area all has been subject to the threat of this disease.After duck infected this virus, main clinical manifestation was: high hot, depressed, apocleisis etc. appears in sick duck; The egg duck egg production occurs and sharply descends even stopping production; Growth retardation appears in the meat duck.This sick incidence of disease is up to 100%, and mortality ratio is between 5% ~ 10%.This virus belongs to the tembusu virus of flaviviridae, Flavivirus.

This disease belongs to reported first in China, the biological characteristics of its cause of disease, the research of the aspect such as molecular epidemiology and molecular basis of the pathogenesis is all at the early-stage, and should disease lose in the tremendous economic that is widely current with and causes of China's aquatic bird culturing area in recent years, the propelling that need deepen continuously of existing progress, for plant and basic unit veterinary quarantine department technology shortcoming, the simple present situation of equipment, the method that creates a kind of simple and effective its antibody of detection is very necessary and urgent.

Summary of the invention

In order to address the above problem, the invention provides the method that a kind of fine jade expansion method detects duck flavivirus antibody.

Duck flavivirus of the present invention is duck flavivirus, is separated by Agricultural University Of Anhui zoonosis laboratory.This Strain has been deposited in Chinese Typical Representative culture collection center, preservation address: China, Wuhan, Wuhan University on April 18th, 2012; Its preservation name is called duck flavivirus AH-F10 strain, and deposit number is: CCTCC NO:V201213.

The step of the method for agar diffusion method detection duck flavivirus antibody is as follows:

(1) the sick duck oophoropathy tissue of aseptic collection infected duck flavivirus grinds after homogenate multigelation three times with it, the centrifugal 10min of 12000r/min; Get its supernatant under the aseptic condition, supernatant is inoculated 10 age in days SPF(after the degerming of 0.22um filtering with microporous membrane be no-special pathogen) the chicken embryo, 0.2mL/ piece, hatch in 38 ℃ of incubators, discard the dead chicken embryo of inoculation in 24 hours, gather in the crops dead chick embryo allantoic liquid in 48 hours to 120 hours, reached continuously for 5 generations, gather in the crops viral allantoic fluid; With the 5th generation the flavivirus allantoic fluid mix through 3 ‰ formaldehyde and shake up, deactivation is 36 hours in 37 ℃ of shaking tables, obtains the duck flavivirus agp antigen;

(2) be 7.2 with the pH value, concentration is that the PBS damping fluid of 1/15M is prepared 1% Ago-Gel, and the preparation fine jade expands dull and stereotyped, and Ago-Gel thickness is between 4.8mm～5.2mm, with the punching of Seven-hole plum card punch; The agp antigen of preparation is added interstitial hole, and its addition is every hole 25 microlitres; Fine jade to be detected is expanded the concentration of antibody after by 2 times of dilutions adds successively six holes on every side, the concentration that the fine jade that namely adds six holes expands antibody be respectively former fine jade expand antibody concentration 2 ^-1, 2 ^-2, 2 ^-3, 2 ^-4, 2 ^-5, 2 ^-6Fine jade is expanded dull and stereotyped reaction observation after 15 hours in 37 ℃ of wet boxes, read the fine jade expansion and tire.

Compared with the prior art, beneficial effect of the present invention is embodied in: the present invention has set up tiring of fine jade expanding method check antibody, the assay good reproducibility, quick and precisely.Preferably type and the concentration of Ago-Gel damping fluid have been determined.

Description of drawings

Fig. 1 is that the duck serum antibody titer is 1: 4 o'clock fine jade expansion result

Fig. 2 is that the duck serum antibody titer is 1: 16 o'clock fine jade expansion result

Embodiment

Below by specific embodiment technical solution of the present invention is done further and to be explained.

Embodiment 1

From the sick duck of Anhui zone infected duck flavivirus, gather the pathological tissues such as its ovary under the aseptic condition, it is ground after homogenate multigelation three times, the centrifugal 10min of 12000r/min.Get its supernatant under the aseptic condition, supernatant is inoculated 10 age in days SPF chicken embryos after the degerming of 0.22um filtering with microporous membrane, 0.2mL/ piece, hatch in 38 ℃ of incubators, discard the dead chicken embryo of inoculation in 24 hours, gather in the crops the allantoic fluid of dead chicken embryo in 48 hours to 120 hours, the generation virus allantoic fluid of results is inoculated 10 age in days SPF chicken embryos, 0.2mL/ piece, hatch in 38 ℃ of incubators.Gather in the crops the allantoic fluid of dead chicken embryo in 48 hours to 120 hours, obtain second generation virus allantoic fluid, reached so continuously for 5 generations, the results allantoic fluid is for subsequent use.

Add by volume 3 ‰ formaldehyde in 37 ℃ of shaking table deactivations 36 hours in the 5th generation virus allantoic fluid of results, after deactivation is finished, press 96 parts of inactivation of viruses liquid, 4 parts of Tween-80s shake up, and are antigen for vaccine as the water of emulsification of lower step; White-oil adjuvant is as emulsification of lower step oil phase.The ratio of 3 parts of oil phases, 1 part of water is got oil phase, water stirring and emulsifying by volume, is prepared into the oil-emulsion inactivated vaccinating agent of duck flavivirus, and add 1% thimerosal solution make it final concentration and be ten thousand/, shake up for subsequent use.

Fine jade expands determining of optimum condition:

Use the duck flavivirus inactivated vaccine chest muscle injecting immune of the preparation egg duck of laying eggs, immunizing dose be 1mL/ only, same approach and method are carried out immunity second time after three weeks, to the blood sampling of egg duck, extract serum after two weeks of for the second time immunity, as fine jade expansion antibody.

With the 5th generation the flavivirus allantoic fluid mix through 3 ‰ formaldehyde and shake up, deactivation is 36 hours in 37 ℃ of shaking tables, preparation duck flavivirus agp antigen.

Select PBS damping fluid (phosphate buffer that namely contains the pH7.4 of 0.05% Tween-20), (the preparation of PB damping fluid: get the NaH2PO4 of 19ml 0.2mol/L and the Na2HPO412H2O of 81ml 0.2mol/L, fully mix the PB damping fluid that is 0.2mol/L of PB damping fluid.) and three kinds of damping fluids of physiological saline of mass concentration 0.85%, under the condition of different pH values; The variable concentrations of PB and PBS damping fluid, the fine jade of part antibody expands potency ratio together, and preferably damping fluid and pH value are reacted in screening, the results are shown in Table one:

Table one

By the result as can be known: be 7.2 in the pH value,, concentration is that 1% Ago-Gel of the PBS damping fluid preparation of 1/15M has preferably that fine jade expands reaction effect.

Embodiment 2

To 60 parts of Anhui Duo Jia egg duck field censorships at least the duck serum of the flavivirus inactivated vaccine (adjacent twice immunity time interval is about 15 days) of twice of continuous immunity carry out fine jade expansion method and check its antibody.Be 1% Ago-Gel of the PBS damping fluid preparation of 1/15M with concentration, the preparation fine jade expands dull and stereotyped, and Ago-Gel thickness is between 4.8mm～5.2mm, with the punching of Seven-hole plum card punch.The agp antigen of preparation is added interstitial hole, and fine jade expands the concentration of antibody after by 2 times of dilutions and adds successively on every side six holes (concentration that fine jade expands antibody is respectively 2 ^-1, 2 ^-2, 2 ^-3, 2 ^-4, 2 ^-5, 2 ^-6).The antigen-antibody addition is about every hole 25 microlitres.Fine jade is expanded dull and stereotyped reaction observation after 15 hours in 37 ℃ of wet boxes, read the fine jade expansion and tire (as shown in Figure 1 and Figure 2).

Fine jade expands the statistics of tiring and sees Table two:

Table two

This shows that the positive rate that comprehensively detects that fine jade expansion method detects flavivirus antibody is 93.3%, the antibody titer that the duck flavivirus inactivated vaccine is crossed in immunity focuses mostly on 1: between the 4-16, account for 71.6%.This shows that fine jade expansion method detects flavivirus antibody and has higher positive rate, for the clinical analysis immune effect provides strong support.

Claims (1)

1. an agar diffusion method detects the method for duck flavivirus antibody, and it is characterized in that: the method comprises the steps:

(1) the sick duck oophoropathy tissue of aseptic collection infected duck flavivirus grinds after homogenate multigelation three times with it, the centrifugal 10min of 12000r/min; Get its supernatant under the aseptic condition, supernatant is inoculated 10 age in days SPF(after the degerming of 0.22um filtering with microporous membrane be no-special pathogen) the chicken embryo, 0.2mL/ piece, hatch in 38 ℃ of incubators, discard the dead chicken embryo of inoculation in 24 hours, gather in the crops dead chick embryo allantoic liquid in 48 hours to 120 hours, reached continuously for 5 generations, gather in the crops viral allantoic fluid; With the 5th generation the flavivirus allantoic fluid mix through 3 ‰ formaldehyde and shake up, deactivation is 36 hours in 37 ℃ of shaking tables, obtains the duck flavivirus agp antigen;

(2) be 7.2 with the pH value, concentration is that the PBS damping fluid of 1/15M is prepared 1% Ago-Gel, and the preparation fine jade expands dull and stereotyped, and Ago-Gel thickness is between 4.8mm～5.2mm, with the punching of Seven-hole plum card punch; The agp antigen of preparation is added interstitial hole, and its addition is every hole 25 microlitres; Fine jade to be detected is expanded the concentration of antibody after by 2 times of dilutions adds successively six holes on every side, the concentration that the fine jade that namely adds six holes expands antibody be respectively former fine jade expand antibody concentration 2 ^-1, 2 ^-2, 2 ^-3, 2 ^-4, 2 ^-5, 2 ^-6Fine jade is expanded dull and stereotyped reaction observation after 15 hours in 37 ℃ of wet boxes, read the fine jade expansion and tire.

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