CN102901816B - Method for yolk antibody-based enzyme-linked immunoadsordent assay (ELISA) detection of turtle systemic sepsis spherical viruses - Google Patents
Method for yolk antibody-based enzyme-linked immunoadsordent assay (ELISA) detection of turtle systemic sepsis spherical viruses Download PDFInfo
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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
The present invention relates to biological products technology of preparing and application, especially relate to a kind of soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody.
Background technology
Soft-shelled turtle, formal name used at school Shelled Turtle Trionyx Sinensis, is always herbal cuisine and excellent tonic product.The eighties in 20th century, the rise of greenhouse cultivation, significantly improves the output of regarding as rare and precious soft-shelled turtle originally, and what rise is the wantonly popular of disease thereupon.Since 2007, in Zhejiang area, especially with Hangzhou, Ningbo area soft-shelled turtle ecological cultivation farm breaks out a kind of systemic septicemia, cardinal symptom is systemic hemorrhagic sepsis, researchist is through epidemiology survey, the researchs such as histotomy and ultra-thin section observation and Experimental infection, determine that a kind of novel globular virus is its primary virulence factor, this novel globular virus is soft-shelled turtle systematicness septicemia spherical viruses (Turtle systemic sepsis spherical virus TSSSV), this virus has stronger infectiousness and the lethal up to 90-100%, and this disease is longer latent period, the illness that initial stage shows is as similar in the illness of easy some bacteriosises with found in the past such as white background plate, easily cause mistaken diagnosis, affect treatment opportunity adversely, therefore it is a kind of convenient to set up, sensitive method for detecting virus is rather important.
At present, the laboratory diagnostic method of soft-shelled turtle systematicness septicemia spherical viruses is mainly RT-PCR method, but the method need to be carried out the steps such as the extraction of nucleic acid, and sample preparation is loaded down with trivial details, and cross pollution easily occurs in the time detecting batch samples.That enzyme linked immunosorbent assay (ELISA) has is simple to operate, susceptibility, high fast, the easy advantage such as standardization, be suitable for large-scale sample detection.
It is a kind of 7S immunoglobulin being present in immune stepmother's chicken with yolk that chicken yolk immune globulin G antibody is called again IgY (Yolk Immunoglobulin), it has the advantages such as productive rate is high, good stability, high specificity, a kind of antibody that is easy to production, cheapness, the amount (about 200mg) of the specific IgY containing in collected only 1 egg of 1 chicken of immunity is far away higher than the antibody amount in the collected whole serum of 1 rabbit of immunity, and IgY has stronger acidproof, alkaline-resisting, temperature capacity.But, also do not report about the correlative study of the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody both at home and abroad at present.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of stability, specificity and the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody highly sensitive, immune interference is little, background is low.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody, comprises the following steps:
(1) antigen is pre-coated
Soft-shelled turtle systematicness septicemia spherical viruses antigen is diluted to 0.1ng/ μ l with the carbonate buffer solution that the pH of .01M is 9.6, be added in polystyrene 96 hole reaction plates by 100 μ l/ holes, 4 DEG C of placements are spent the night, liquid in hole inclines next day, by hole, with after PBS T cleansing solution washing 3 times, every hole adds 1% bovine serum albumin(BSA) (BSA) confining liquid, and 37 DEG C of low-speed oscillation incubation 1h seal, after sealing finishes, siphon away bovine serum albumin(BSA) confining liquid in hole;
(2) detection reaction
After hole is cleaned with PBS-T cleansing solution, add the soft-shelled turtle systematicness septicemia spherical viruses Yolk antibody 100 μ l of doubling dilution, it is 1 parallel that every gradient is established, set up blank and negative control simultaneously, add a cover after 37 DEG C of incubation 1h, wash 3 times with PBS-T cleansing solution, in every hole, add again that 100 μ l's use 1% bovine serum albumin(BSA) confining liquid by the goat-anti chicken IgG of the horseradish peroxidase of 1:10000 dilution proportion (HRP) mark in advance, after 37 DEG C of incubation 1h, PBS-T cleansing solution cleans 5 times, after distillation washing 3 times, every hole adds the o-phenylenediamine substrate solution 100 μ l that newly configure, 37 DEG C of dark place colour developing 15min, every hole adds the 2M H of 50 μ l
2sO
4cessation reaction,
(3) result is judged
In microplate reader, read the absorbance at 492nm wavelength place, each hole, mensuration hole absorbance is greater than the more than 2.1 times person of negative control hole average and is judged to the positive.
The preparation process of the soft-shelled turtle systematicness septicemia spherical viruses antigen described in step (1) is as follows: choose ill soft-shelled turtle, get soft-shelled turtle internal organ and be placed in the TNE damping fluid that ice bath is crossed in advance, homogenate is until completely cracked, by homogenate at 4 DEG C, under 6000g condition after centrifugal 10min, get supernatant at 4 DEG C, centrifugal 20min under 8000g condition, get again centrifugal gained supernatant in 4 DEG C, after the centrifugal 1.5h of 35000g, obtain primary sedimentation thing, loose the precipitation upper strata of primary sedimentation thing part is carefully rinsed out with TM damping fluid, precipitation lower floor white portion is resuspended in the TM damping fluid of 1ml, 4 DEG C of ice baths spend the night, next day is by the precipitation Eddy diffusion that spends the night and process, at 4 DEG C under 3500g condition after centrifugal 5min, get supernatant in 4 DEG C, under 38000g condition, centrifugal 1.5h obtains secondary precipitate, after loose the precipitation upper strata of secondary precipitate part is carefully removed, after lower floor's white precipitate is resuspended with a small amount of TM damping fluid, make viral suspension, be soft-shelled turtle systematicness septicemia spherical viruses antigen.
The preparation process of the soft-shelled turtle systematicness septicemia spherical viruses Yolk antibody described in step (2) is as follows:
A. laying hen immunity collect immunity eggs
Laying hen is carried out to 1 fundamental immunity and 2～3 booster immunizations, described fundamental immunity is the Freund's complete adjuvant that adds equivalent in described soft-shelled turtle systematicness septicemia spherical viruses antigen, fully emulsifiedly rear laying hen is carried out to injecting immune, described booster immunization is the incomplete Freund's adjuvant that adds equivalent in described soft-shelled turtle systematicness septicemia spherical viruses antigen, fully emulsifiedly rear laying hen is carried out to injecting immune, injection consumption is every kilogram of hen injection 500mg soft-shelled turtle systematicness septicemia spherical viruses antigen, be 15 days each immune interval time, complete latter 10 days of immunity, start to collect egg, in 4 DEG C of Refrigerator stores,
B. the extraction of special Yolk antibody
By immunity eggs obtained above with alcohol wipe eggshell and separate yolk, draw yolk liquid, then acetic acid-sodium acetate buffer that the pH that adds the 0.05M of 10 times of volumes of yolk liquid in yolk liquid is 5.0, after mixing in the processing of 4 DEG C of refrigerator hold over night, to spend the night mixed liquor after treatment in 4 DEG C, under the condition of 10000g, after centrifugal 20min, get supernatant, in supernatant, add saturated (NH
4)
2sO
4making its final concentration is 40%, 4 DEG C of standing 10h, then gets precipitation after the centrifugal 20min of 10000g, the Na by precipitation with 140g/L
2sO
4be diluted to 100ml, after 4 DEG C of standing 10h, the centrifugal 20min of 10000g, precipitates the PBS that is 7.2 with the pH of 0.01M resuspended again, and the bag filter that is finally 100KD with interception dialysis desalination obtains soft-shelled turtle systematicness septicemia spherical viruses Yolk antibody.
The pH of described TNE damping fluid is 7.5, and preparation final concentration is as follows: 50mM Tris-HCl, and 200mMNaCl, 5mM ethylenediamine tetraacetic acid divides (EDTA), 1mM phenylmethylsulfonyl fluoride (PMSF).
The pH of described TM damping fluid is 7.5, and preparation final concentration is as follows: 50mM Tris-HCl, 10mMMgCl
2, 1mM PMSF.
Described PBS-T cleansing solution is that Tween-20 and the PBS damping fluid ratio of 1: 2000 are by volume mixed to get, and described PBS buffer concentration is 0.1M, and pH is 7.4.
The dilution multiple proportions of described soft-shelled turtle systematicness septicemia spherical viruses Yolk antibody is 1:640,1:1280,1:2560,1:5120,1:10240.
The compound concentration of described o-phenylenediamine substrate solution is: every 100ml o-phenylenediamine substrate solution is containing citric acid 0.73g, Na
2hPO
42H
2o 1.186g, o-phenylenediamine 0.04g, 30%H
2o
2150 μ l.
Compared with prior art, the invention has the advantages that: the present invention discloses the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody first, the present invention taking the TSSSV of purifying as antigen immune laying hen has obtained specific anti-TSSSV-IgY, the anti-TSSSV-IgY of specificity of preparation can be used for TSSSV indirect ELISA and detects, with rabbit source, the antibody in mouse source is compared, it is stable that IgY has physical property, preparation is simple, output is high, cost is low, can obviously reduce the advantages such as false positive occurrence rate, utilize TSSSV-IgY to carry out ELISA detection diagnosis and there is stability, specificity and highly sensitive, immune interference is little, the advantages such as background is low, TSSSV-IgY preparation is simultaneously simple, not needing to get the operations such as blood damages animal, hen can cause that after the antigen of 20-30mg high conservative carries out immunity the IgY of high-titer secretes for a long time, in order to carry out with IgY, ELISA detection method is carried out the marketization and commercialization provides the foundation for this.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Embodiment 1
The preparation of soft-shelled turtle systematicness septicemia spherical viruses antigen
Choose ill soft-shelled turtle, get soft-shelled turtle internal organ and be placed in the TNE damping fluid that ice bath is crossed in advance, homogenate is until completely cracked, by homogenate at 4 DEG C, under 6000g condition after centrifugal 10min, get supernatant at 4 DEG C, centrifugal 20min under 8000g condition, gets again centrifugal gained supernatant in 4 DEG C, after the centrifugal 1.5h of 35000g, obtain primary sedimentation thing, loose the precipitation upper strata of primary sedimentation thing part is carefully rinsed out with TM damping fluid, and precipitation lower floor white portion is resuspended in the TM damping fluid of 1ml, and 4 DEG C of ice baths spend the night; Next day is by the precipitation Eddy diffusion that spends the night and process, at 4 DEG C under 3500g condition after centrifugal 5min, get supernatant in 4 DEG C, under 38000g condition, centrifugal 1.5h obtains secondary precipitate, after loose the precipitation upper strata of secondary precipitate part is carefully removed, after lower floor's white precipitate is resuspended with a small amount of TM damping fluid, make viral suspension, be soft-shelled turtle systematicness septicemia spherical viruses antigen.
Wherein the pH of TNE damping fluid is 7.5, and preparation final concentration is as follows: 50mM Tris-HCl, 200mM NaCl, 5mM EDTA, 1mM PMSF; The pH of TM damping fluid is 7.5, and preparation final concentration is as follows: 50mM Tris-HCl, 10mM MgCl
2, 1mM PMSF.
Above-mentioned soft-shelled turtle systematicness septicemia spherical viruses (Soft-shelled turtle systemic septicemia spherical virus, STSSSV), strain is P1015 strain, preserving number is CGMCC No.5378, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 21st, 2011, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Embodiment 2
The preparation of the Yolk antibody of anti-soft-shelled turtle systematicness septicemia spherical viruses
A. laying hen immunity collect immunity eggs
Laying hen is carried out to 1 fundamental immunity and 2～3 booster immunizations, described fundamental immunity is the Freund's complete adjuvant that adds equivalent in described soft-shelled turtle systematicness septicemia spherical viruses antigen, fully emulsifiedly rear laying hen is carried out to injecting immune, described booster immunization is the incomplete Freund's adjuvant that adds equivalent in described soft-shelled turtle systematicness septicemia spherical viruses antigen, fully emulsifiedly rear laying hen is carried out to injecting immune, injection consumption is every kilogram of hen injection 500mg soft-shelled turtle systematicness septicemia spherical viruses antigen, be 15 days each immune interval time, complete latter 10 days of immunity, start to collect egg, in 4 DEG C of Refrigerator stores,
B. the extraction of special Yolk antibody
By immunity eggs obtained above with alcohol wipe eggshell and separate yolk, draw yolk liquid, then acetic acid-sodium acetate buffer that the pH that adds the 0.05M of 10 times of volumes of yolk liquid in yolk liquid is 5.0, after mixing in the processing of 4 DEG C of refrigerator hold over night, to spend the night mixed liquor after treatment in 4 DEG C, under the condition of 10000g, after centrifugal 20min, get supernatant, in supernatant, add saturated (NH
4)
2sO
4making its final concentration is 40%, 4 DEG C of standing 10h, then gets precipitation after the centrifugal 20min of 10000g, the Na by precipitation with 140g/L
2sO
4be diluted to 100ml, after 4 DEG C of standing 10h, the centrifugal 20min of 10000g, precipitates the PBS that is 7.2 with the pH of 0.01M resuspended again, and the bag filter that is finally 100KD with interception dialysis desalination obtains soft-shelled turtle systematicness septicemia spherical viruses Yolk antibody.
Embodiment 3
The anti-TSSSV-IgY of specificity detects for TSSSV indirect ELISA, and concrete steps are as follows
1, antigen is pre-coated
The carbonate buffer solution (CBS) that is 9.6 with the pH of .01M by soft-shelled turtle systematicness septicemia spherical viruses antigen (concrete preparation process is as described in above-described embodiment 1) is diluted to 0.1ng/ μ l, be added in polystyrene 96 hole reaction plates by 100 μ l/ holes, 4 DEG C of placements are spent the night, liquid in hole inclines next day, hole is washed after 3 times with PBS-T cleansing solution, every hole adds 1% bovine serum albumin(BSA) confining liquid, 37 DEG C of low-speed oscillation incubation 1h seal, after sealing finishes, siphon away bovine serum albumin(BSA) confining liquid in hole;
Wherein PBS-T cleansing solution is that Tween-20 and the PBS damping fluid ratio of 1: 2000 are by volume mixed to get, and described PBS buffer concentration is 0.1M, and pH is 7.4;
2, detection reaction
After hole is cleaned with PBS-T cleansing solution, every hole adds in advance with PBS by 1:640, 1:1280, 1:2560, 1:5120, soft-shelled turtle systematicness septicemia spherical viruses Yolk antibody (the concrete preparation process of TSSSV-IgY is as described in above-described embodiment 2) the 100 μ l of 1:10240 doubling dilution, it is 1 parallel that every gradient is established, separately establish the negative contrast of albumen dislysate without the egg of any cause of disease immunity, PBS is blank, , add a cover after 37 DEG C of incubation 1h, wash 3 times with PBS-T cleansing solution, in every hole, add again that 100 μ l's use 1% bovine serum albumin(BSA) confining liquid by the goat-anti chicken IgG of the horseradish peroxidase of 1:10000 dilution proportion (HRP) mark in advance, after 37 DEG C of incubation 1h, PBS-T cleansing solution cleans 5 times, after distillation washing 3 times, every hole adds the o-phenylenediamine substrate solution 100 μ l that newly configure, 37 DEG C of dark place colour developing 15min, every hole adds the 2M H of 50 μ l
2sO
4cessation reaction,
Wherein the compound concentration of o-phenylenediamine substrate solution is: every 100ml o-phenylenediamine substrate solution is containing citric acid 0.73g, Na
2hPO
42H
2o 1.186g, o-phenylenediamine 0.04g, 30%H
2o
2150 μ l;
3, result is judged
In microplate reader, read absorbance (A) value at 492nm wavelength place, each hole, mensuration hole absorbance is greater than the more than 2.1 times person of negative control hole average and is judged to the positive, and the greatest dilution of positive reaction is tiring of testing sample.
Bioactivity the results are shown in Table 1, has illustrated that the anti-TSSSV-IgY the present invention relates to tires very high, is 1:10240, can detect for indirect ELISA.
Table 1
Embodiment 4
Anti-TSSSV-IgY sensitivity detects
Prepare after virion according to the antigen of embodiment in the present invention 1, virion is precipitated resuspended with 500ulCBS damping fluid, 4 DEG C of centrifugal 10min of 6000g, get supernatant, get 20 μ l supernatant CBS damping fluids and be diluted to 200 μ l, measure OD280 and the OD260 of the rear virus liquid of dilution, calculate the concentration of virus protein according to formula, measure absorbance with nucleic acid-protein quantitative determination instrument, calculate virus protein concentration according to following formula:
Protein content (mg/ml)=(1.45 × OD
280-0.74 × OD
260) × Sample Dilution degree
According to calculating gained virus protein concentration, by the virus liquid concentration dilution to 10 after dilution
-1after μ g/ml, dilute with CBS by 10 times of gradients, do altogether 5 concentration gradients.Above-mentioned viral dilution liquid is coated with respectively, and each concentration gradient is done a repetition.The anti-TSSSV-IgY indirect ELISA method of setting up according to the present invention detects, and testing result is as shown in table 2,
Table 2
Above table explanation, the anti-TSSSV-IgY indirect ELISA method sensitivity that the present invention sets up is high, is only 10 at virus quantity
-6when μ g, just can detect accurately.
Embodiment 5
Anti-TSSSV-IgY specific detection
For the specificity of verifying that IgY is combined with virus protein in ELISA detects, the present invention detects normal soft-shelled turtle viscera tissue, ill soft-shelled turtle viscera tissue, get respectively above-mentioned tissue and add CBS damping fluid according to the ratio of 1:10, thoroughly homogenate, 4 DEG C, the centrifugal 10min of 10000g, gets supernatant, and repeated centrifugation once as the case may be.After being diluted with CBS damping fluid according to the ratio of 1:10, above-mentioned supernatant is coated with respectively, the anti-TSSSV-IgY indirect ELISA method of setting up according to the present invention detects, measure the more than 2.1 times person of hole A value > negative control hole average and be judged to the positive, testing result is as shown in table 3
Table 3
| Detect sample | Blank | Normal soft-shelled turtle | Ill soft-shelled turtle | Purified virus |
| Average OD value | 0.077 | 0.053 | 2.100 | 2.459 |
| +/- | - | - | + | + |
Above table explanation, special being combined with virus protein of anti-TSSSV-IgY energy, not can with soft-shelled turtle histone generation reciprocation, the method that the anti-TSSSV-IgY indirect ELISA that the present invention sets up detects TSSSV has very high sensitivity and good specificity, can successfully from ill soft-shelled turtle tissue, detect virus.
Embodiment 6
The Preliminary Applications of indirect ELISA method
Get the artificial challenge through the RT-PCR test positive virus of preserving in laboratory but do not show soft-shelled turtle viscera tissue, the swamp eel viscera tissue of artificial challenge's virus, the successful chicken embryo of the inoculation TSSSV tissue of illness, preparation method according to sample in embodiment 1 prepares detection sample, separately prepares normal soft-shelled turtle tissue detection sample as negative control by same method.Carry out TSSSV detection by the indirect ELISA detection method establishing.
Testing result is in table 4, and above-mentioned detection sample testing result except normal soft-shelled turtle tissue sample is all positive, and it is negative that normal soft-shelled turtle is organized as testing result.
Table 4
| Detect sample | Blank | Normal soft-shelled turtle | Purified virus | Swamp eel tissue | Chicken embryo tissue | Soft-shelled turtle does not fall ill |
| Average OD value | 0.076 | 0.094 | 1.767 | 2.281 | 2.462 | 0.776 |
| +/- | - | - | + | + | + | + |
Illustrate that the anti-TSSSV-IgY indirect ELISA detection method that the present invention sets up can detect virus from infected tissue's sample, and can detect the trace virus of carrying in viral soft-shelled turtle tissue.Further prove that the anti-TSSSV-IgY indirect ELISA method the present invention relates to has very high sensitivity.
Claims (4)
1. the preparation method of a soft-shelled turtle systematicness septicemia spherical viruses antigen, it is characterized in that detailed process is as follows: choose ill soft-shelled turtle, get soft-shelled turtle internal organ and be placed in the TNE damping fluid that ice bath is crossed in advance, homogenate is until completely cracked, by homogenate at 4 DEG C, under 6000g condition after centrifugal 10min, get supernatant at 4 DEG C, centrifugal 20min under 8000g condition, get again centrifugal gained supernatant in 4 DEG C, after the centrifugal 1.5h of 35000g, obtain primary sedimentation thing, loose the precipitation upper strata of primary sedimentation thing part is carefully rinsed out with TM damping fluid, precipitation lower floor white portion is resuspended in the TM damping fluid of 1ml, 4 DEG C of ice baths spend the night, next day is by the precipitation Eddy diffusion that spends the night and process, at 4 DEG C under 3500g condition after centrifugal 5min, get supernatant in 4 DEG C, under 38000g condition, centrifugal 1.5h obtains secondary precipitate, after loose the precipitation upper strata of secondary precipitate part is carefully removed, after lower floor's white precipitate is resuspended with a small amount of TM damping fluid, make viral suspension, be soft-shelled turtle systematicness septicemia spherical viruses antigen, wherein the pH of TNE damping fluid is 7.5, preparation final concentration is as follows: 50mM Tris-HCl, 200mM NaCl, 5mM EDTA, 1mM PMSF, the pH of TM damping fluid is 7.5, and preparation final concentration is as follows: 50mM Tris-HCl, 10mM MgCl
2, 1mM PMSF.
2. the preparation method of soft-shelled turtle systematicness septicemia spherical viruses antigen according to claim 1, the pH that it is characterized in that described TNE damping fluid is 7.5, and preparation final concentration is as follows: 50mM Tris-HCl, 200mM NaCl, 5mM ethylenediamine tetraacetic acid, 1mM phenylmethylsulfonyl fluoride.
3. the preparation method of soft-shelled turtle systematicness septicemia spherical viruses antigen according to claim 1, is characterized in that the pH of described TM damping fluid is 7.5, and preparation final concentration is as follows: 50mM Tris-HCl, 10mM MgCl
2, 1mM phenylmethylsulfonyl fluoride.
4. utilize soft-shelled turtle systematicness septicemia spherical viruses antigen described in claim 1 to prepare a method for the Yolk antibody of anti-soft-shelled turtle systematicness septicemia spherical viruses, it is characterized in that concrete steps are as follows:
A. laying hen immunity collect immunity eggs
Laying hen is carried out to 1 fundamental immunity and 2～3 booster immunizations, described fundamental immunity is the Freund's complete adjuvant that adds equivalent in soft-shelled turtle systematicness septicemia spherical viruses antigen claimed in claim 1, fully emulsifiedly rear laying hen is carried out to injecting immune, described booster immunization is the incomplete Freund's adjuvant that adds equivalent in described soft-shelled turtle systematicness septicemia spherical viruses antigen, fully emulsifiedly rear laying hen is carried out to injecting immune, injection consumption is every kilogram of hen injection 500mg soft-shelled turtle systematicness septicemia spherical viruses antigen, be 15 days each immune interval time, complete latter 10 days of immunity, start to collect egg, in 4 DEG C of Refrigerator stores,
B. the extraction of special Yolk antibody
By immunity eggs obtained above with alcohol wipe eggshell and separate yolk, draw yolk liquid, then acetic acid-sodium acetate buffer that the pH that adds the 0.05M of 10 times of volumes of yolk liquid in yolk liquid is 5.0, after mixing in the processing of 4 DEG C of refrigerator hold over night, to spend the night mixed liquor after treatment in 4 DEG C, under the condition of 10000g, after centrifugal 20min, get supernatant, in supernatant, add saturated (NH
4)
2sO
4making its final concentration is 40%, 4 DEG C of standing 10h, then gets precipitation after the centrifugal 20min of 10000g, the Na by precipitation with 140g/L
2sO
4be diluted to 100ml, after 4 DEG C of standing 10h, the centrifugal 20min of 10000g, precipitates the PBS that is 7.2 with the pH of 0.01M resuspended again, and the bag filter that is finally 100KD with interception dialysis desalination obtains soft-shelled turtle systematicness septicemia spherical viruses Yolk antibody.
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