CN103864931A - Preparation and freeze-dried storage method of pseudorabies standard positive serum - Google Patents
Preparation and freeze-dried storage method of pseudorabies standard positive serum Download PDFInfo
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- CN103864931A CN103864931A CN201410126929.9A CN201410126929A CN103864931A CN 103864931 A CN103864931 A CN 103864931A CN 201410126929 A CN201410126929 A CN 201410126929A CN 103864931 A CN103864931 A CN 103864931A
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Abstract
The invention discloses a preparation and freeze-dried storage method of pseudorabies standard positive serum. The preparation comprises the following steps: immunizing a rabbit twice with an inactivated PRV (pseudorabies virus) solution, then, immunizing the rabbit once with the inactivated PRV solution, and collecting the serum; adding a protective agent sodium chloride, trehalose, monosodium glutamate and L-arginine into the serum, fully dissolving, filtering to remove bacteria, and subpackaging; adding the subpackaged serum into a freeze dryer box, and performing freeze-drying according to a freeze-drying curve shown as fig.1 described in the specification to obtain the pseudorabies standard positive serum. For the pseudorabies standard positive serum obtained by the method disclosed by the invention, PRV neutralizing antibody titer is more than 1:512, the antibody titer remains unchanged after the pseudorabies standard positive serum is stored for 60 months at 2-8 DEG C, so that the pseudorabies standard positive serum has good stability, can fully neutralize pseudorabies virus in vaccine exogenous virus and specificity detection, and provides a good technical support for quality inspection of pseudorabies virus vaccines.
Description
Technical field
The present invention relates to animal viral lived vaccine and check field, relate in particular to a kind of preparation and freeze-drying store method thereof of pseudoabies standard positive serum.
Background technology
Porcine pseudorabies (PR) is a kind of acute infectious disease being caused by porcine pseudorabies virus (Pseudorabies Virus, PRV), and its main clinical characteristics is that Adult Pig is inapparent infection, and pregnant sow is miscarried, stillborn foetus and respiratory symptom.After low age in days Infection in Piglets, normal appearance generates heat, has loose bowels, and follows obvious nervous symptoms and expiratory dyspnea.This disease is propagated soon in swinery, piglet mortality ratio is high, is one of serious infectious diseases of facing of pig farm.China is the popular country widely of pseudoabies, and existing multiple provinces (city, district) report that this disease occurs, and has caused huge financial loss at present.
In the anti-system of porcine pseudorabies, vaccine plays an important role, because attenuated live vaccines has little, the cheap feature of immunizing dose, so use range is extensive compared with inactivated vaccine, especially the living vaccine of preparing with gE genetically deficient strain, swinery is used after this marker vaccine, by detecting the antibody horizontal of gI or gE, can distinguish vaccine antibody and wild malicious antibody, thereby provide scientific basis for pig farm purifies pseudoabies.The quality that guarantees vaccine is particularly important, and diagnostic test and exogenous virus check are one of emphasis of animal living vaccine quality test, its objective is the pure property, the specificity that guarantee vaccine (or seed culture of viruses), and pseudorabies living vaccines is no exception." People's Republic of China's biological products rules " (version in 2000) and " People's Republic of China's veterinary drug allusion quotation " (2010 editions 3) require pseudorabies living vaccines to carry out diagnostic test and exogenous virus check, but due to domestic existing pseudoabies standard positive serum technology of preparing length consuming time, success ratio is low, antibody titer is low, cause being difficult to neutralize completely corresponding virus in the diagnostic test of vaccine and exogenous virus check, make the Quality inspection of vaccine be difficult to normally carry out, to vaccine quality, supervision has left hidden danger, external pseudoabies standard positive serum buying difficulty and expensive, add that check demand is larger, the promising solution of institute is produced, check needs, the preparation method of research and development high-titer pseudoabies standard positive serum is imperative.
Summary of the invention
The object of the invention is to provide in order to overcome the defect that above-mentioned prior art exists a kind of preparation and freeze-drying store method thereof of high-titer pseudoabies standard positive serum.
Object of the present invention is achieved through the following technical solutions:
A preparation method for pseudoabies standard positive serum, comprises the following steps:
(1) antigen immune: at the clean level rabbit left side inguinal region subcutaneous injection PRV inactivation of viruses liquid (10 of isolated rearing
7.0tCID
50/ mL) 2mL, interval after 14 days at 1 PRV inactivation of viruses liquid (10 of the rabbit left side subcutaneous booster immunization of inguinal region
7.0tCID
50/ mL) 2mL, apart from the 1st immunity after 28 days at rabbit right side inguinal region subcutaneous injection PRV virus liquid (10
5.0tCID
50/ mL) 2mL.
The Japan large ear rabbit that described clean level rabbit is preferably body weight 1.5～2kg, is negative to pseudoabies, rotavirus disease, swine fever, parvovirus, rabbit pest antigen-antibody; Described PRV is preferably the sick and weak malicious C strain of pseudorabies that deposit number is CCTCC NO:V201114.
(2) serum results: apart from latter 14 days of the 3rd immunity, collect serum.
A freeze-drying store method for pseudoabies standard positive serum, comprises the following steps:
(1) protectant preparation and serum keeping: add respectively sodium-chlor, trehalose, msg powder type, L-arginine by 1%, 1%, 0.1%, 0.05% of serum volume in serum, after fully dissolving, use filter membrane degerming, then packing;
Described filter membrane is preferably the filter membrane of 0.22 μ m.
(2) freeze-drying (freeze-drying curve figure as shown in Figure 1): point serum installing is packed in body of freeze dryer, in 2～3 hours, sample is cooled to-45 ℃ by normal temperature, at this temperature, maintain after 3～4 hours, start to be evacuated to after 10～20Pa, start again to heat up, in 2 hours, rise to-10 ℃, maintain 10～12 hours with the sublimation temperature of-10 ℃ afterwards, then in 1 hour, lift temperature to 0 ℃ and maintain 2h, then in 3 hours, lift temperature to 28 ℃ and maintain tamponade outlet under 4～6 hours final vacuum states.
The preparation of pseudoabies standard positive serum and a freeze-drying store method thereof, for using respectively aforesaid method to prepare pseudoabies standard positive serum freeze-drying preservation.
The pseudoabies standard positive serum PRV NAT obtaining by method of the present invention, more than 1:512, is preserved 60 months antibody titers and is remained unchanged under 2～8 ℃ of conditions.
The present invention adopts a series of means such as serum preparation, serum freeze-drying, antibody demarcation, vaccine exogenous virus and specificity check to study preparation method, the freeze-drying store method of standard positive serum, test-results shows, this immunization route and mode can make rabbit produce very high pseudo-rabies antibody, and through freeze-drying preserve after the shelf time long, there is good stability, in can be fully in the check of vaccine exogenous virus and specificity and Pseudorabies virus, for the check of pseudoabies attenuated vaccine provides good resource.
Compared with prior art, the present invention has the following advantages:
(1) preparation method, the freeze-drying store method of standard positive serum have been studied; test-results shows; first immunization method and the immunization route of immune live virus again after immune inactivation of viruses; can make rabbit produce after antibody protection at immune inactivation of viruses; under the stimulation of live virus and produce very high pseudoabies NAT, reach 1:512.Preserve antibody titer without any loss through freeze-drying, pseudoabies NAT still maintains 1:512, and the shelf time is long, has good stability.In can be fully in the check of vaccine exogenous virus and specificity and Pseudorabies virus, for the quality test of pseudoabies attenuated vaccine provides good technical support.
(2) in serum ratio, 1% sodium-chlor, 1% trehalose, 0.1% msg powder type, 0.05%L-arginine are dissolved in positive serum, with the filter membrane degerming of 0.22 μ m, then adopt corresponding freeze-drying curve to carry out freeze-drying; Under 2～8 ℃ of conditions, preserving 60 months antibody titers remains unchanged.
Accompanying drawing explanation
Fig. 1 is the freeze-drying curve figure of pseudoabies standard positive serum;
Fig. 2 is pseudoabies standard positive serum preparation technology schema.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is done to further detailed description, but embodiments of the present invention are not limited to this.
Pseudoabies standard positive serum preparation technology schema of the present invention as shown in Figure 2.
Embodiment 1
1, the preparation of serum
5 of screening body weight 1.5～2kg Japan large ear rabbits (clean level), detect pseudoabies, rotavirus disease, swine fever, parvovirus, rabbit pest antigen-antibody through PCR and ELISA all negative.Every rabbit left side inguinal region subcutaneous injection PRV(C strain) inactivation of viruses liquid (10
7.0tCID
50/ mL) 2mL, interval is 1 PRV(C strain of every rabbit left side subcutaneous booster immunization of inguinal region after 14 days) inactivation of viruses liquid (10
7.0tCID
50/ mL) 2mL, apart from the 1st immunity every rabbit right flank butt crack subcutaneous injection PRV(C strain after 28 days) virus liquid (10
5.0tCID
50/ mL) 2mL, apart from latter 14 days of the 3rd immunity, can be by for subsequent use rabbit carotid artery bloodletting blood sampling separation of serum.Wherein, PRV(C strain) be the sick and weak malicious C strain of pseudorabies of the natural weak poison of a strain, deposit number is: CCTCC NO:V201114; Depositary institution: Chinese Typical Representative culture collection center, CHINA CENTERFOR TYPE CULTURE COLLECTION(CCTCC); Preservation address: China. Wuhan. Wuhan University.
2, serum NAT detects test
Filter membrane degerming by the rabbit anteserum collecting with 0.22 μ m, puts 56 ℃ of water-bath deactivations 30 minutes.In the each hole of Tissue Culture Plate, add 50 μ L DMEM nutrient solutions, in the 1st hole, add subsequently serum 50 μ L to be checked, after mixing, take out 50 μ L with micropipet, add in the 2nd hole, mix rear taking-up 50 μ L and add again in the 3rd hole, the like, until the 10th hole, serum dilution is 1:2,1:4,1:8 ... 1:1024, every part of serum dilution to be checked is done 3 repetitions.50 μ L are contained to 200 TCID
50/ 0.1mLPRV virus liquid is added in different dilution serum hole, in 37 ℃ and 1 hour.The PK15 cell that adds 100 μ L to disperse through trysinization in every serum hole.On each piece plate of each test, set up virus control, first by 200TCID
50/ 0.1mL virus liquid is made 10 times of serial dilutions, gets 10
0, 10
-1, 10
-2, 10
-34 extent of dilution, each extent of dilution is inoculated 4 holes, every hole 100 μ L, then every hole adds 100 μ L cell suspensions.Day by day observe, record cytopathy and non-pathology hole count, observe 5.0.2TCID in virus control
50do not cause cytopathy, positive serum, negative serum and normal cell contrast are set up, and measurement result is effective, and test conditions is set up.After observing, determine the serum greatest dilution that cell cultures 50% is protected, calculate NAT, the NAT of the rabbit anteserum collecting is 1:512.
3, the freeze-drying of serum
In serum, add respectively sodium-chlor, trehalose, msg powder type, L-arginine by 1%, 1%, 0.1%, 0.05% of serum volume, after fully dissolving, with the filter membrane degerming of 0.22 μ m, every bottle of packing 2mL.Point serum installing is packed in body of freeze dryer, in 2 hours, sample is by extremely-45 ℃ of normal temperature slow coolings, at this temperature, maintain after 3 hours, start to be evacuated to after 18Pa, start to heat up, in 2 hours, rise to-10 ℃, maintain 11 hours with the sublimation temperature of-10 ℃ afterwards, then in 1 hour, lift temperature to 0 ℃ and maintain 2h, then in 3 hours, lift temperature to 28 ℃ and maintain tamponade outlet under 5 hours final vacuum states, freeze-drying whole process is 29 hours.
4, after serum freeze-drying, NAT detects test
Aseptic freeze-dried rabbit anteserum to be checked is reduced to 2mL with the DMEM nutrient solution that does not contain serum, puts 56 ℃ of water-bath deactivations 30 minutes.In the each hole of Tissue Culture Plate, add 50 μ L DMEM nutrient solutions, in the 1st hole, add subsequently serum 50 μ L to be checked, after mixing, take out 50 μ L with micropipet, add in the 2nd hole, mix rear taking-up 50 μ L and add again in the 3rd hole, the like, until the 10th hole, serum dilution is 1:2,1:4,1:8 ... 1:1024, every part of serum dilution to be checked is done 3 repetitions.50 μ L are contained to 200 TCID
50/ 0.1mLPRV virus liquid is added in different dilution serum hole, in 37 ℃ and 1 hour.The PK15 cell that adds 100 μ L to disperse through trysinization in every serum hole.On each piece plate of each test, set up virus control, first by 200TCID
50/ 0.1mL virus liquid is made 10 times of serial dilutions, gets 10
0, 10
-1, 10
-2, 10
-34 extent of dilution, each extent of dilution is inoculated 4 holes, every hole 100 μ L, then every hole adds 100 μ L cell suspensions.Day by day observe, record cytopathy and non-pathology hole count, observe 5.0.2TCID in virus control
50do not cause cytopathy, positive serum, negative serum and normal cell contrast are set up, and measurement result is effective, and test conditions is set up.After observing, determine the serum greatest dilution that cell cultures 50% is protected, calculate NAT, the NAT of freeze-drying rabbit anteserum is 1:512.
5, diagnostic test test
By pseudorabies living vaccines that Wuhan Chopper Biology Co., Ltd. produces (Bartha-K61 strain), use containing 2% new-born calf serum DMEM cell culture fluid and be diluted to 200TCID
50/ 0.1mL.Get viral dilution liquid and positive rabbit anteserum equivalent mixes, put 37 ℃ of water-bath neutralizing effects after 1 hour, be inoculated in the 96 hole ST Tissue Culture Plates that grow up to good individual layer (discarding cell culture fluid), inoculation 6 holes, every hole 0.2mL establishes normal cell contrast and each 2 holes of virus control simultaneously.Put 37 ℃, contain 5%CO
2in incubator, cultivate and observe 5.There is CPE in virus control hole, in normal cell control wells and virus and hole does not all produce CPE, illustrates that the rabbit positive serum of preparation can be for the diagnostic test of pseudorabies living vaccines.
6, exogenous virus check test
Get pseudorabies living vaccines that Wuhan Chopper Biology Co., Ltd. produces (Bartha-K61 strain), every bottle of freeze dried vaccine adds serum-free DMEM cell culture fluid, be diluted to every 1.0mL containing 10 parts, after mixing with 37 ℃ of positive rabbit anteserums of equivalent in after 1 hour as tested inspection product, grown up to each 2 bottles of the Tissue Culture Flask of good individual layer Vero, ST, MDBK cell with the inoculation of vaccine and rabbit anteserum mixed solution, every bottle at least 1.0mL(containing at least 1 part of vaccine product to be checked).When inoculating cell individual layer, need 37 ℃ of absorption 1h, then discard inoculum, use PBS washed cell surface 2 times, then add 10mL cell maintenance medium and continue to cultivate; The sample of processing is synchronously inoculated ST cell, and inoculation bottle number, dosage of inoculation, incubation time be same Vero, ST, MDBK cell all.Separately establish 1 bottle of ST normal cell contrast.By after above-mentioned Vero, ST, the freeze thawing of MDBK cell culture 3 times, centrifugal 10 minutes of 3000g/m, be inoculated in respectively corresponding Vero, ST, MDBK cell monolayer each 2 bottles for subculture, at least 1mL of every bottle graft kind, put after 37 ℃ of absorption 1h, add cell maintenance medium, put in 37 ℃ of incubators and cultivate and observe.Centrifugal 10 minutes synchronous inoculation ST cells of 3000g/m after the freeze thawing of ST cell, inoculation bottle number, dosage of inoculation, incubation time be same Vero, ST, MDBK cell all.Each subculture is all set up normal cell contrast.The total incubation time of test sample on every kind of cell should be no less than 14.At least subculture 1 time of cell culture between incubation period.The cell monolayer quantity of last subculture and area, incubation time should meet the requirement of fluorescence antibody test procedure, cytopathogenic effect test procedure and the check of red corpuscle adsorptivity exogenous virus.In incubation period, cell culture is carried out routine examination, pathology that culture is acellular, cultivates while end, continues as follows check by above-mentioned after subculture cell culture freeze thawing 3 times.MDBK cell is selected in the inspection of BVDV fluorescence antibody, and Vero cell is selected in the inspection of PPV fluorescence antibody, and ST cell is selected in the inspection of CSFV fluorescence antibody.Should comprise three groups of cells to the check of each specific exogenous virus: the last subculture cell culture of (1) test sample; (2) the specific virus positive control (positive control that BVDV Oregon C24V strain inoculation MDBK cell checks as BVDV fluorescence antibody of setting up when the last subculture of test sample, the positive control that ST cell checks as PPV fluorescence antibody is synchronously inoculated in PPV NADL-2 strain, the positive control that CSFV Thiverval strain inoculation ST cell checks as CSFV fluorescence antibody); (3) normal cell contrast.Every group of cell monolayer (or synchronously inoculation) selected 24 porocyte culture plates, and each sample is inoculated 4 holes, every hole inoculation 0.5mL.Above cell cultures, after 5～7 days, discards nutritive medium, with PBS(pH7.2) washing 2～3 times, dry rear use 80% cold acetone and put 2～8 ℃ and fix 30 minutes, discard stationary liquid, after seasoning with suitable fluorescence antibody dye, microscopy.Optional direct immunofluorescence test procedure or indirect immunofluorescence test procedure while checking BVDV exogenous virus, select indirect immunofluorescence test procedure while checking PPV exogenous virus, select indirect immunofluorescence test procedure while checking CSFV exogenous virus.There is specific fluorescence in positive control, normal cell is without fluorescence, and test sample is without fluorescence.Result demonstration, test conditions is all set up, and illustrates that the rabbit positive serum of preparation can be for the exogenous virus check of pseudorabies living vaccines.
Embodiment 2
1, the preparation of serum
10 of screening body weight 1.5～2kg Japan large ear rabbits (clean level), detect pseudoabies, porcine reproductive and respiratory syndrome, swine fever, porcine parvovirus, rabbit pest antigen-antibody through PCR and ELISA all negative.Every rabbit left side inguinal region subcutaneous injection PRV(C strain) inactivation of viruses liquid (10
7.0tCID
50/ mL) 2mL, interval is 1 PRV(C strain of every rabbit left side subcutaneous booster immunization of inguinal region after 14 days) inactivation of viruses liquid (10
7.0tCID
50/ mL) 2mL, apart from the 1st immunity every rabbit right flank butt crack subcutaneous injection PRV(C strain after 28 days) virus liquid (10
5.0tCID
50/ mL) 2mL.Apart from latter 15 days of the 3rd immunity, can be by for subsequent use rabbit carotid artery bloodletting blood sampling separation of serum.
2, serum NAT detects test
Filter membrane degerming by the rabbit anteserum collecting with 0.22 μ m, puts 56 ℃ of water-bath deactivations 30 minutes.In the each hole of Tissue Culture Plate, add 50 μ L DMEM nutrient solutions, in the 1st hole, add subsequently serum 50 μ L to be checked, after mixing, take out 50 μ L with micropipet, add in the 2nd hole, mix rear taking-up 50 μ L and add again in the 3rd hole, the like, until the 10th hole, serum dilution is 1:2,1:4,1:8 ... 1:1024, every part of serum dilution to be checked is done 3 repetitions.50 μ L are contained to 200 TCID
50/ 0.1mLPRV virus liquid is added in different dilution serum hole, in 37 ℃ and 1 hour.The PK15 cell that adds 100 μ L to disperse through trysinization in every serum hole.On each piece plate of each test, set up virus control, first by 200TCID
50/ 0.1mL virus liquid is made 10 times of serial dilutions, gets 10
0, 10
-1, 10
-2, 10
-34 extent of dilution, each extent of dilution is inoculated 4 holes, every hole 100 μ L, then every hole adds 100 μ L cell suspensions.Day by day observe, record cytopathy and non-pathology hole count, observe 5.0.2TCID in virus control
50do not cause cytopathy, positive serum, negative serum and normal cell contrast are set up, and measurement result is effective, and test conditions is set up.After observing, determine the serum greatest dilution that cell cultures 50% is protected, calculate NAT, the NAT of the rabbit anteserum collecting is 1:512.
3, the freeze-drying of serum
Press serum volume ratio by 1% sodium-chlor, 1% trehalose, 0.1% msg powder type, 0.05%L-arginine is dissolved in positive serum, with the filter membrane degerming of 0.22 μ m, every bottle of packing 2mL, point serum installing is packed in body of freeze dryer, in 3 hours, sample is by extremely-45 ℃ of normal temperature slow coolings, at this temperature, maintain after 4 hours, start to be evacuated to after 15Pa, starting intensification, in 2 hours, rise to-10 ℃, maintain 10 hours with the sublimation temperature of-10 ℃ afterwards, then in 1 hour, lift temperature to 0 ℃ and maintain 2h, in 3 hours, lift temperature to again 28 ℃ and maintain tamponade outlet under 5 hours final vacuum states, freeze-drying whole process is 30 hours.
4, the serum NAT after freeze-drying detects test
Aseptic freeze-dried rabbit anteserum to be checked is reduced to 2mL with the DMEM nutrient solution that does not contain serum, puts 56 ℃ of water-bath deactivations 30 minutes.In the each hole of Tissue Culture Plate, add 50 μ L DMEM nutrient solutions, in the 1st hole, add subsequently serum 50 μ L to be checked, after mixing, take out 50 μ L with micropipet, add in the 2nd hole, mix rear taking-up 50 μ L and add again in the 3rd hole, the like, until the 10th hole (by mixed solution 50 μ L), serum dilution is 1:2,1:4,1:8 ... 1:1024, every part of serum dilution to be checked is done 3 repetitions.50 μ L are contained to 200 TCID
50/ 0.1mLPRV virus liquid is added in different dilution serum hole, in 37 ℃ and 1 hour.The PK15 cell that adds 100 μ L to disperse through trysinization in every serum hole.On each piece plate of each test, set up virus control, first by 200TCID
50/ 0.1mL virus liquid is made 10 times of serial dilutions, gets 10
0, 10
-1, 10
-2, 10
-34 extent of dilution, each extent of dilution is inoculated 4 holes, every hole 100 μ L, then every hole adds 100 μ L cell suspensions.Day by day observe, record cytopathy and non-pathology hole count, observe 5.0.2TCID in virus control
50do not cause cytopathy, positive serum, negative serum and normal cell contrast are set up, and measurement result is effective, and test conditions is set up.After observing, determine the serum greatest dilution that cell cultures 50% is protected, calculate NAT, the NAT of freeze-drying rabbit anteserum is 1:512.
5, diagnostic test test
By pseudorabies living vaccines that Wuhan Chopper Biology Co., Ltd. produces (Bartha-K61 strain), use containing 2% new-born calf serum DMEM cell culture fluid and be diluted to 200TCID
50/ 0.1mL.Get viral dilution liquid and positive rabbit anteserum equivalent mixes, put 37 ℃ of water-bath neutralizing effects after 1 hour, be inoculated in the 96 hole ST Tissue Culture Plates that grow up to good individual layer (discarding cell culture fluid), inoculation 6 holes, every hole 0.2mL establishes normal cell contrast and each 2 holes of virus control simultaneously.Put 37 ℃, contain 5%CO
2in incubator, cultivate and observe 5.There is CPE in virus control hole, in normal cell control wells and virus and hole does not all produce CPE, illustrates that the rabbit positive serum of preparation can be for the diagnostic test of pseudorabies living vaccines.
6, the test of freeze-dry blood serum
By the serum of the serum of case study on implementation 1,2 freeze-drying, conventional freeze-drying, (the serum quantitative separating that embodiment 1 is gathered in neutral glass bottle, sealing bottleneck, sticks label normal temperature and enters in body of freeze dryer, in 1 hour, goods is down to-42 ℃ and maintain 2 hours.Goods were risen to-5 ℃ and maintain 10 hours from-42 ℃ in 1 hour.Goods were risen to 10 ℃ and maintain 2 hours from-5 ℃ in 1 hour; Goods were risen to 28 ℃ and maintain 6 hours from 10 ℃ in 1 hour, freeze-drying finishes) and the serum of freeze-drying not, being placed under 2～8 ℃ of conditions and preserving, different time sampling, measures antibody titer and physical behavior thereof, and detected result is in table 1.
After table 1. freeze-drying, serum is in the antibody titer of 2～8 ℃ of preservation different times
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (6)
1. a preparation method for pseudoabies standard positive serum, is characterized in that comprising the following steps:
(1) antigen immune: at the clean level rabbit left side inguinal region subcutaneous injection 2mL 10 of isolated rearing
7.0tCID
50/ mL PRV inactivation of viruses liquid, interval after 14 days at 1 2mL 10 of the rabbit left side subcutaneous booster immunization of inguinal region
7.0tCID
50/ mL PRV inactivation of viruses liquid, apart from the 1st immunity after 28 days at rabbit right side inguinal region subcutaneous injection 2mL 10
5.0tCID
50/ mL PRV virus liquid;
(2) serum results: apart from latter 14 days of the 3rd immunity, collect serum.
2. the preparation method of pseudoabies standard positive serum according to claim 1, it is characterized in that: the clean level rabbit described in step (1) is the Japan large ear rabbit of body weight 1.5～2kg, and pseudoabies, rotavirus disease, swine fever, parvovirus, rabbit pest antigen-antibody are negative.
3. the preparation method of pseudoabies standard positive serum according to claim 1, is characterized in that: the PRV described in step (1) is that deposit number is the sick and weak malicious C strain of pseudorabies of CCTCC NO:V201114.
4. a freeze-drying store method for pseudoabies standard positive serum, is characterized in that comprising the following steps:
(1) protectant preparation and serum keeping: add respectively sodium-chlor, trehalose, msg powder type, L-arginine by 1%, 1%, 0.1%, 0.05% of serum volume in serum, after fully dissolving, use filter membrane degerming, then packing;
(2) freeze-drying: point serum installing is packed in body of freeze dryer, in 2～3 hours, sample is cooled to-45 ℃ by normal temperature, at this temperature, maintain after 3～4 hours, start to be evacuated to after 10～20Pa, start again to heat up, in 2 hours, rise to-10 ℃, maintain 10～12 hours with the sublimation temperature of-10 ℃ afterwards, then in 1 hour, lift temperature to 0 ℃ and maintain 2h, then in 3 hours, lift temperature to 28 ℃ and maintain tamponade outlet under 4～6 hours final vacuum states.
5. the freeze-drying store method of pseudoabies standard positive serum according to claim 4, is characterized in that: the filter membrane described in step (1) is the filter membrane of 0.22 μ m.
6. the preparation of a pseudoabies standard positive serum and freeze-drying store method thereof, it is characterized in that: right to use requires the method described in 1～3 any one to prepare pseudoabies standard positive serum, the method re-using described in claim 4 or 5 is carried out freeze-drying preservation.
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| CN105732807A (en) * | 2016-02-24 | 2016-07-06 | 福州大北农生物技术有限公司 | Porcine pseudorabies virus positive serum preparation method |
| JP2020523577A (en) * | 2017-06-13 | 2020-08-06 | スン・チュン・チンＣｈｕｎｇ Ｃｈｉｎ ＳＵＮ | Novel method for preserving serum protein activity |
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| CN105548536B (en) * | 2015-12-08 | 2018-10-16 | 天津瑞普生物技术股份有限公司 | A kind of preparation method of Latex agglutination test Positive Sera |
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| CN105732807B (en) * | 2016-02-24 | 2019-02-26 | 福州大北农生物技术有限公司 | A kind of preparation method of porcine pseudorabies virus positive serum |
| JP2020523577A (en) * | 2017-06-13 | 2020-08-06 | スン・チュン・チンＣｈｕｎｇ Ｃｈｉｎ ＳＵＮ | Novel method for preserving serum protein activity |
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