CN102901816A - 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, the formal name used at school Shelled Turtle Trionyx Sinensis always is 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, the 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 up to the lethal of 90-100%, and this disease is longer latent period, the illness that initial stage shows such as white background plate etc. are similar with the illness of some bacteriosises of finding in the past easily, cause easily mistaken diagnosis, affect treatment opportunity adversely, it is a kind of convenient therefore 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 the RT-PCR method, but the method need to be carried out the steps such as extraction of nucleic acid, and sample preparation is loaded down with trivial details, and cross pollution occurs when detecting batch samples easily.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.
Chicken yolk immune globulin G antibody is called again IgY(Yolk Immunoglobulin) be a kind of 7S immunoglobulin that is present in immune stepmother's chicken with yolk, it has the advantages such as productive rate height, good stability, high specificity, be a kind ofly be easy to produce, cheap antibody, the amount (about 200mg) of the specific IgY that contains in collected only 1 egg of 1 chicken of immunity is higher than the antibody amount in the collected whole serum of immune 1 rabbit far away, and IgY has stronger acidproof, alkaline-resisting, temperature capacity.But, at present both at home and abroad also not about the correlative study report based on the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method of Yolk antibody.
Summary of the invention
Technical matters to be solved by this invention provides a kind of stability, specificity and the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody highly sensitive, that immune interference is little, background is low.
The present invention solves the problems of the technologies described above the technical scheme that adopts: a kind of soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody may further comprise the steps:
(1) antigen is pre-coated
Be that 9.6 carbonate buffer solution is diluted to 0.1 ng/ul with soft-shelled turtle systematicness septicemia spherical viruses antigen with the pH of .01 M, be added in the polystyrene 96 hole reaction plates by the 100ul/ hole, 4 ℃ of placements are spent the night, liquid in the hole inclines next day, after hole usefulness PBS-T cleansing solution washing 3 times, every hole adds 1 % bovine serum albumin(BSA) (BSA) confining liquid, and 37 ℃ of low-speed oscillation incubation 1 h seal, sealing siphons away bovine serum albumin(BSA) confining liquid in the hole after finishing;
(2) detection reaction
After the usefulness PBS-T cleansing solution cleaning of hole, the soft-shelled turtle systematicness septicemia spherical viruses Yolk antibody 100ul that adds doubling dilution, it is 1 parallel that every gradient is established, set up simultaneously blank and negative control, after adding a cover 37 ℃ of incubation 1 h, wash 3 times with the PBS-T cleansing solution, the goat-anti chicken IgG that presses in advance horseradish peroxidase (HRP) mark of 1:10000 dilution proportion with 1% bovine serum albumin(BSA) confining liquid that in every hole, adds again 100ul, behind 37 ℃ of incubation 1 h, the PBS-T cleansing solution cleans 5 times, and after distillation was washed 3 times, every hole added the o-phenylenediamine substrate solution 100ul that newly configures, 37 ℃ of dark places, 15 min that develop the color, every hole adds the 2M H of 50ul
2SO
4Cessation reaction;
(3) result judges
Read the absorbance at each 492 nm wavelength place, hole in microplate reader, measure the hole absorbance greater than the negative control hole average more than 2.1 times the person be judged to the positive.
The preparation process of the soft-shelled turtle systematicness septicemia spherical viruses antigen described in the step (1) is as follows: choose ill soft-shelled turtle, get the TNE damping fluid that the soft-shelled turtle internal organ place ice bath in advance to cross, homogenate is until fully cracked, with homogenate at 4 ℃, under the 6000g condition behind the centrifugal 10min, get supernatant at 4 ℃, centrifugal 20min under the 8000g condition, get again centrifugal gained supernatant in 4 ℃, obtain the primary sedimentation thing behind the centrifugal 1.5h of 35000g, the loose part in the precipitation upper strata of primary sedimentation thing is carefully rinsed out with the TM damping fluid, and precipitation lower floor white portion is resuspended in the TM damping fluid of 1ml, and 4 ℃ of ice baths spend the night; The precipitation Eddy diffusion that will spend the night next day and process, under 4 ℃ of lower 3500g conditions behind the centrifugal 5min, get supernatant in 4 ℃, centrifugal 1.5h obtains secondary precipitate under the 38000g condition, after the loose part in the precipitation upper strata of secondary precipitate carefully removed, make viral suspension after lower floor's white precipitate is resuspended with a small amount of TM damping fluid, 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 the step (2) is as follows:
A. laying hen immunity and collect immunity eggs
Laying hen is carried out 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 injecting immune, described booster immunization is the incomplete Freunds adjuvant that adds equivalent in described soft-shelled turtle systematicness septicemia spherical viruses antigen, fully emulsifiedly rear laying hen is carried out injecting immune, the injection consumption is every kilogram of hen injection 500mg soft-shelled turtle systematicness septicemia spherical viruses antigen, be 15 days each immune interval time, finish rear 10 days of immunity, begin to collect egg, in 4 ℃ of Refrigerator stores;
B. the extraction of special Yolk antibody
With immunity eggs obtained above with the alcohol wipe eggshell and separate yolk, draw yolk liquid, then the pH that adds 0.05 M of 10 times of volumes of yolk liquid in yolk liquid is acetic acid-sodium acetate buffer of 5.0, process in 4 ℃ of refrigerator hold over night behind the mixing, to spend the night mixed liquor after processing in 4 ℃, get supernatant behind centrifugal 20 min under the condition of 10000g, in supernatant, add saturated (NH
4)
2SO
4Make its final concentration be 40%, 4 ℃ and leave standstill 10h, get precipitation behind centrifugal 20 min of 10000g again, with the Na of precipitation with 140g/L
2SO
4Be diluted to 100 ml, again in 4 ℃ leave standstill 10 h after, centrifugal 20 min of 10000 g, precipitation is that 7.2 PBS is resuspended with the pH of 0.01M, is that the bag filter of the 100KD desalination of dialysing obtains the systemic septicemia spherical viruses of soft-shelled turtle Yolk antibody with interception at last.
The pH of described TNE damping fluid is 7.5, and the preparation final concentration is as follows: 50 mM Tris-HCl, and 200 mM NaCl, the 5mM ethylenediamine tetraacetic acid divides (EDTA), 1mM phenylmethylsulfonyl fluoride (PMSF).
The pH of described TM damping fluid is 7.5, and the preparation final concentration is as follows: 50 mM Tris-HCl, 10 mM MgCl
2, 1mM PMSF.
Described PBS-T cleansing solution be Tween-20 and PBS damping fluid by volume the ratio of 1:2000 be mixed to get, described PBS buffer concentration is 0.1 M, 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: per 100 ml o-phenylenediamine substrate solutions contain citric acid 0.73 g, Na
2HPO
42H
2O 1.186 g, o-phenylenediamine 0.04 g, 30% H
2O
2150 ul.
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 has obtained specific anti-TSSSV-IgY take the TSSSV of purifying as the antigen immune laying hen, the anti-TSSSV-IgY of specificity of preparation can be used for the TSSSV indirect ELISA and detects, with the 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, the obvious advantage such as reduce false positive occurrence rate, utilize TSSSV-IgY to carry out ELISA detection diagnosis and have stability, specificity and highly sensitive, immune interference is little, the advantages such as background is low, simultaneously TSSSV-IgY preparation is simple, not needing to get the operations such as blood damages animal, hen can cause that after the antigen through the 20-30mg high conservative carries out immunity the IgY of high-titer secretes for a long time, and ELISA detection method is carried out the marketization and commercialization provides the foundation in order to carry out with IgY 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 the TNE damping fluid that the soft-shelled turtle internal organ place ice bath in advance to cross, homogenate is until fully cracked, with homogenate at 4 ℃, under the 6000g condition behind the centrifugal 10min, get supernatant at 4 ℃, centrifugal 20min under the 8000g condition, get again centrifugal gained supernatant in 4 ℃, obtain the primary sedimentation thing behind the centrifugal 1.5h of 35000g, the loose part in the precipitation upper strata of primary sedimentation thing is carefully rinsed out with the TM damping fluid, and precipitation lower floor white portion is resuspended in the TM damping fluid of 1ml, and 4 ℃ of ice baths spend the night; The precipitation Eddy diffusion that will spend the night next day and process, under 4 ℃ of lower 3500g conditions behind the centrifugal 5min, get supernatant in 4 ℃, centrifugal 1.5h obtains secondary precipitate under the 38000g condition, after the loose part in the precipitation upper strata of secondary precipitate carefully removed, make viral suspension after lower floor's white precipitate is resuspended with a small amount of TM damping fluid, be soft-shelled turtle systematicness septicemia spherical viruses antigen.
Wherein the pH of TNE damping fluid is 7.5, and the preparation final concentration is as follows: 50 mM Tris-HCl, 200 mM NaCl, 5mM EDTA, 1mM PMSF; The pH of TM damping fluid is 7.5, and the preparation final concentration is as follows: 50 mM Tris-HCl, 10 mM MgCl
2, 1mM PMSF.
Above-mentioned soft-shelled turtle systematicness septicemia spherical viruses (Soft-shelled turtle systemic septicemia spherical virus, STSSSV), strain is the P1015 strain, preserving number is CGMCC No.5378, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 21st, 2011, the 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 and collect immunity eggs
Laying hen is carried out 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 injecting immune, described booster immunization is the incomplete Freunds adjuvant that adds equivalent in described soft-shelled turtle systematicness septicemia spherical viruses antigen, fully emulsifiedly rear laying hen is carried out injecting immune, the injection consumption is every kilogram of hen injection 500mg soft-shelled turtle systematicness septicemia spherical viruses antigen, be 15 days each immune interval time, finish rear 10 days of immunity, begin to collect egg, in 4 ℃ of Refrigerator stores;
B. the extraction of special Yolk antibody
With immunity eggs obtained above with the alcohol wipe eggshell and separate yolk, draw yolk liquid, then the pH that adds 0.05 M of 10 times of volumes of yolk liquid in yolk liquid is acetic acid-sodium acetate buffer of 5.0, process in 4 ℃ of refrigerator hold over night behind the mixing, to spend the night mixed liquor after processing in 4 ℃, get supernatant behind centrifugal 20 min under the condition of 10000g, in supernatant, add saturated (NH
4)
2SO
4Make its final concentration be 40%, 4 ℃ and leave standstill 10h, get precipitation behind centrifugal 20 min of 10000g again, with the Na of precipitation with 140g/L
2SO
4Be diluted to 100 ml, again in 4 ℃ leave standstill 10 h after, centrifugal 20 min of 10000 g, precipitation is that 7.2 PBS is resuspended with the pH of 0.01M, is that the bag filter of the 100KD desalination of dialysing obtains the systemic septicemia spherical viruses of soft-shelled turtle Yolk antibody with interception at last.
Embodiment 3
The anti-TSSSV-IgY of specificity is used for the TSSSV indirect ELISA and detects, and concrete steps are as follows
1, antigen is pre-coated
Be that 9.6 carbonate buffer solution (CBS) is diluted to 0.1 ng/ul with soft-shelled turtle systematicness septicemia spherical viruses antigen (concrete preparation process as above-mentioned embodiment 1 as described in) with the pH of .01 M, be added in the polystyrene 96 hole reaction plates by the 100ul/ hole, 4 ℃ of placements are spent the night, liquid in the hole inclines next day, after hole usefulness PBS-T cleansing solution washing 3 times, every hole adds 1% bovine serum albumin(BSA) confining liquid, 37 ℃ of low-speed oscillation incubation 1 h seal, sealing siphons away bovine serum albumin(BSA) confining liquid in the hole after finishing; Wherein the PBS-T cleansing solution be Tween-20 and PBS damping fluid by volume the ratio of 1:2000 be mixed to get, described PBS buffer concentration is 0.1 M, pH is 7.4;
2, detection reaction
After the usefulness PBS-T cleansing solution cleaning of hole, every hole adds presses 1:640 with PBS in advance, 1:1280,1:2560,1:5120, the soft-shelled turtle of 1:10240 doubling dilution systematicness septicemia spherical viruses Yolk antibody (the concrete preparation process of TSSSV-IgY as above-mentioned embodiment 2 as described in) 100ul, it is 1 parallel that every gradient is established, other establishes the negative contrast of albumen dislysate without the egg of any cause of disease immunity, PBS is blank, after adding a cover 37 ℃ of incubation 1 h, wash 3 times with the PBS-T cleansing solution, in every hole, add again the goat-anti chicken IgG that presses in advance horseradish peroxidase (HRP) mark of 1:10000 dilution proportion with 1% bovine serum albumin(BSA) confining liquid of 100ul, behind 37 ℃ of incubation 1 h, the PBS-T cleansing solution cleans 5 times, and after distillation was washed 3 times, every hole added the o-phenylenediamine substrate solution 100ul that newly configures, 37 ℃ of dark places, 15 min that develop the color, every hole adds the 2M H of 50ul
2SO
4Cessation reaction;
Wherein the compound concentration of o-phenylenediamine substrate solution is: per 100 ml o-phenylenediamine substrate solutions contain citric acid 0.73 g, Na
2HPO
42H
2O 1.186 g, o-phenylenediamine 0.04 g, 30% H
2O
2150 ul;
3, the result judges
Read absorbance (A) value at each 492 nm wavelength place, hole in microplate reader, measure the hole absorbance greater than the negative control hole average more than 2.1 times the person be judged to the positive, the greatest dilution of positive reaction is tiring of testing sample.
Bioactivity the results are shown in Table 1, and it is very high to have illustrated that the anti-TSSSV-IgY that the present invention relates to tires, and is 1:10240, can be used for indirect ELISA and detect.
Table 1
。
Embodiment 4
Anti-TSSSV-IgY sensitivity detects
After preparing virion according to the antigen of embodiment among the present invention 1, virion is precipitated resuspended with the 500ulCBS damping fluid, 4 ℃ of 6000 centrifugal 10 min of g, get supernatant, get the 20ul supernatant and be diluted to 200ul with the CBS damping fluid, measure OD280 and the OD260 of the rear virus liquid of dilution, calculate the concentration of virus protein according to formula, measure absorbance with the 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, with the virus liquid concentration dilution to 10 after the dilution
-1Behind the ug/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
The above table explanation, the anti-TSSSV-IgY indirect ELISA method sensitivity that the present invention sets up is high, only is 10 at virus quantity
-6Just can detect accurately during ug.
Embodiment 5
Anti-TSSSV-IgY specific detection
Be 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 according to the ratio adding CBS damping fluid of 1:10, thoroughly homogenate, 4 ℃, centrifugal 10 min of 10000 g get supernatant, and repeated centrifugation once as the case may be.Above-mentioned supernatant is coated with respectively after with the dilution of CBS damping fluid according to the ratio of 1:10, the anti-TSSSV-IgY indirect ELISA method of setting up according to the present invention detects, measure hole A value〉the negative control hole average more than 2.1 times the person be judged to the positive, testing result is as shown in table 3
Table 3
The above table explanation, anti-TSSSV-special being combined with virus protein of IgY energy, not can with soft-shelled turtle histone generation reciprocation, the method that the anti-TSSSV-IgY indirect ELISA of the present invention's foundation detects TSSSV has very high sensitivity and good specificity, and what energy was successful detects virus from ill soft-shelled turtle tissue.
Embodiment 6
The Preliminary Applications of indirect ELISA method
Get that the laboratory preserves through artificial challenge's virus of RT-PCR test positive but do not show the soft-shelled turtle viscera tissue of illness, the swamp eel viscera tissue of artificial challenge's virus, the chicken embryo tissue of inoculation TSSSV success, preparation method according to sample in the embodiment 1 prepares test sample, prepares normal soft-shelled turtle tissue detection sample as negative control by same method in addition.Carrying out TSSSV by the indirect ELISA detection method that establishes detects.
Testing result sees Table 4, and above-mentioned test sample testing result except normal soft-shelled turtle tissue sample all is positive, and it is negative that normal soft-shelled turtle is organized as testing result.
Table 4
Illustrated 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 the viral soft-shelled turtle tissue.Proved that further the anti-TSSSV-IgY indirect ELISA method that the present invention relates to has very high sensitivity.
Claims (9)
1. one kind based on the soft-shelled turtle of Yolk antibody systematicness septicemia spherical viruses indirect ELISA detection method, it is characterized in that may further comprise the steps:
(1) antigen is pre-coated
Be that 9.6 carbonate buffer solution is diluted to 0.1 ng/ul with soft-shelled turtle systematicness septicemia spherical viruses antigen with the pH of .01 M, be added in the polystyrene 96 hole reaction plates by the 100ul/ hole, 4 ℃ of placements are spent the night, liquid in the hole inclines next day, after hole usefulness PBS-T cleansing solution washing 3 times, every hole adds 1 % bovine serum albumin(BSA) confining liquid, and 37 ℃ of low-speed oscillation incubation 1 h seal, sealing siphons away bovine serum albumin(BSA) confining liquid in the hole after finishing;
(2) detection reaction
After the usefulness PBS-T cleansing solution cleaning of hole, the soft-shelled turtle systematicness septicemia spherical viruses Yolk antibody 100ul that adds doubling dilution, it is 1 parallel that every gradient is established, set up simultaneously blank and negative control, after adding a cover 37 ℃ of incubation 1 h, wash 3 times with the PBS-T cleansing solution, the goat-anti chicken IgG that presses in advance the horseradish peroxidase mark of 1:10000 dilution proportion with 1% bovine serum albumin(BSA) confining liquid that in every hole, adds again 100ul, behind 37 ℃ of incubation 1 h, the PBS-T cleansing solution cleans 5 times, and after distillation was washed 3 times, every hole added the o-phenylenediamine substrate solution 100ul that newly configures, 37 ℃ of dark places, 15 min that develop the color, every hole adds the 2M H of 50ul
2SO
4Cessation reaction;
(3) result judges
Read the absorbance at each 492 nm wavelength place, hole in microplate reader, measure the hole absorbance greater than the negative control hole average more than 2.1 times the person be judged to the positive.
2. the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody according to claim 1, the preparation process that it is characterized in that the soft-shelled turtle systematicness septicemia spherical viruses antigen described in the step (1) is as follows: choose ill soft-shelled turtle, get the TNE damping fluid that the soft-shelled turtle internal organ place ice bath in advance to cross, homogenate is until fully cracked, with homogenate at 4 ℃, under the 6000g condition behind the centrifugal 10min, get supernatant at 4 ℃, centrifugal 20min under the 8000g condition, get again centrifugal gained supernatant in 4 ℃, obtain the primary sedimentation thing behind the centrifugal 1.5h of 35000g, the loose part in the precipitation upper strata of primary sedimentation thing is carefully rinsed out with the TM damping fluid, precipitation lower floor white portion is resuspended in the TM damping fluid of 1ml, and 4 ℃ of ice baths spend the night; The precipitation Eddy diffusion that will spend the night next day and process, under 4 ℃ of lower 3500g conditions behind the centrifugal 5min, get supernatant in 4 ℃, centrifugal 1.5h obtains secondary precipitate under the 38000g condition, after the loose part in the precipitation upper strata of secondary precipitate carefully removed, make viral suspension after lower floor's white precipitate is resuspended with a small amount of TM damping fluid, be soft-shelled turtle systematicness septicemia spherical viruses antigen.
3. the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody according to claim 2 is characterized in that the preparation process of the soft-shelled turtle systematicness septicemia spherical viruses Yolk antibody described in the step (2) is as follows:
A. laying hen immunity and collect immunity eggs
Laying hen is carried out 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 injecting immune, described booster immunization is the incomplete Freunds adjuvant that adds equivalent in described soft-shelled turtle systematicness septicemia spherical viruses antigen, fully emulsifiedly rear laying hen is carried out injecting immune, the injection consumption is every kilogram of hen injection 500mg soft-shelled turtle systematicness septicemia spherical viruses antigen, be 15 days each immune interval time, finish rear 10 days of immunity, begin to collect egg, in 4 ℃ of Refrigerator stores;
B. the extraction of special Yolk antibody
With immunity eggs obtained above with the alcohol wipe eggshell and separate yolk, draw yolk liquid, then the pH that adds 0.05 M of 10 times of volumes of yolk liquid in yolk liquid is acetic acid-sodium acetate buffer of 5.0, process in 4 ℃ of refrigerator hold over night behind the mixing, to spend the night mixed liquor after processing in 4 ℃, get supernatant behind centrifugal 20 min under the condition of 10000g, in supernatant, add saturated (NH
4)
2SO
4Make its final concentration be 40%, 4 ℃ and leave standstill 10h, get precipitation behind centrifugal 20 min of 10000g again, with the Na of precipitation with 140g/L
2SO
4Be diluted to 100 ml, again in 4 ℃ leave standstill 10 h after, centrifugal 20 min of 10000 g, precipitation is that 7.2 PBS is resuspended with the pH of 0.01M, is that the bag filter of the 100KD desalination of dialysing obtains the systemic septicemia spherical viruses of soft-shelled turtle Yolk antibody with interception at last.
4. the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody according to claim 3, the pH that it is characterized in that described TNE damping fluid is 7.5, the preparation final concentration is as follows: 50 mM Tris-HCl, 200 mM NaCl, the 5mM ethylenediamine tetraacetic acid divides, the 1mM phenylmethylsulfonyl fluoride.
5. the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody according to claim 3, the pH that it is characterized in that described TM damping fluid is 7.5, the preparation final concentration is as follows: 50 mM Tris-HCl, 10 mM MgCl
2, the 1mM phenylmethylsulfonyl fluoride.
6. the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody according to claim 1, it is characterized in that: described PBS-T cleansing solution be Tween-20 and PBS damping fluid by volume the ratio of 1:2000 be mixed to get, described PBS buffer concentration is 0.1 M, and pH is 7.4.
7. according to claim 1 or the indirect ELISA detection method of 3 described soft-shelled turtles systematicness septicemia spherical viruses Yolk antibodies, it is characterized in that: 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.
8. the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody according to claim 1, it is characterized in that: the compound concentration of described o-phenylenediamine substrate solution is: per 100 ml o-phenylenediamine substrate solutions contain citric acid 0.73 g, Na
2HPO
42H
2O 1.186 g, o-phenylenediamine 0.04 g, 30% H
2O
2150 ul.
9. the soft-shelled turtle systematicness septicemia spherical viruses indirect ELISA detection method based on Yolk antibody according to claim 1, it is characterized in that: described negative control is that described blank is the PBS damping fluid without the albumen dislysate of the egg of any cause of disease immunity.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104459119A (en) * | 2014-11-14 | 2015-03-25 | 宁波大学 | Soft-shelled turtle systemic septicemia virus detection test strip and preparation method thereof |
| CN103760344B (en) * | 2013-09-30 | 2015-10-14 | 辽宁医学院 | The ELISA detection method of Chinese bee sacbrood poison antibody |
| CN105891412A (en) * | 2014-11-14 | 2016-08-24 | 宁波大学 | Test strip for detecting citrobacter freundii and preparation method thereof |
-
2012
- 2012-04-13 CN CN201210107100.5A patent/CN102901816B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 张小莺等: "卵黄抗体技术中不同因素对免疫效果的影响", 《中国家禽》 * |
| 张文生等: "3种卵黄抗体分离提取方法对其生物活性影响的比较", 《中国生化药物杂志》 * |
| 胡广洲: "中华鳖暴发性败血症的研究", 《中国优秀硕士论文全文数据库 农业科技辑》 * |
Cited By (5)
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| CN104459119B (en) * | 2014-11-14 | 2016-04-13 | 宁波大学 | A kind of soft-shelled turtle systemic septicaemia Viral diagnosis test strips and preparation method thereof |
| CN105891412A (en) * | 2014-11-14 | 2016-08-24 | 宁波大学 | Test strip for detecting citrobacter freundii and preparation method thereof |
| CN105891412B (en) * | 2014-11-14 | 2018-08-21 | 宁波大学 | A kind of citrobacter freundii test strip and preparation method thereof |
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| CN102901816B (en) | 2014-12-10 |
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