CN105548536B - A kind of preparation method of Latex agglutination test Positive Sera - Google Patents
A kind of preparation method of Latex agglutination test Positive Sera Download PDFInfo
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Abstract
The present invention provides a kind of preparation method of Latex agglutination test Positive Sera, this method includes the preparation of vaccine, screening, immunity inoculation, the harvest of positive serum and the identification of negative pig.The present invention carries out carrying out fundamental immunity to negative pig first by pig japanese b encephalitis live vaccine (14 2 plants of SA14), and the pig japanese b encephalitis inactivated vaccine (14 2 plants of SA14) of the high-titer then prepared using concentrated purification technique is carried out reinforced immunological and prepares Latex agglutination test Positive Sera.With Latex agglutination test Positive Sera prepared by the present invention, relative to using the strong poison of encephalitis to prepare positive serum, the risk using strong poison is avoided, is had the advantages that safe；It is big compared with the serum amount that mouse harvest positive serum to be prepared using pig, is of relatively low cost, with scale application foreground outstanding simultaneously.
Description
Technical field
The present invention relates to biological product technical fields, and in particular to a kind of system of Latex agglutination test Positive Sera
Preparation Method.
Background technology
Japanese Type-B encephalitis is by the B-mode brain of flaviviridae (Flaviviridae) Flavivirus (Flavivirus)
A kind of Zoonosis entomophila sexually transmitted disease caused by scorching virus (Japanese Encephalitis Virus, JEV).Pig infects
Encephalitis B forms viremia virusemia, it is considered to be the major source of infection of mankind's encephalitis B prevalence, therefore, anti-pig japanese b encephalitis processed
The loss of pig breeding industry can be not only reduced, and is also the important measures for preventing people and suffering from encephalitis B.Its mainly cause sow and
The breeding difficulty of boar is mainly shown as farrowing sow miscarriage, stillborn foetus, the weak son of production, Testis of Boar Pig inflammation etc., is usually made to pig breeding industry
At serious economic loss.
Vaccine is one of the main method for preventing the disease.Currently, the pig japanese b encephalitis vaccine of Chinese commodity is mainly mouse
Brain inactivated vaccine and attenuated vaccine, for the specificity for ensureing vaccine and pure property, diagnostic test and exogenous virus verify as the epidemic disease
The essential items for inspection of seedling quality inspection.Currently, Latex agglutination test Positive Sera is mainly used in pig japanese b encephalitis work epidemic disease
Diagnostic test, the exogenous virus of seedling are examined and the purposes such as diagnosis detection.
Have not yet to see the report of Latex agglutination test Positive Sera preparation method.The system of general hyper-immune serum
Preparation Method is：It uses vaccine or low virulent strain to carry out fundamental immunity first, then carries out reinforced immunological using strong poison to stimulate body
Strong immune response is generated, hyper-immune serum is obtained, such as the preparation method of swine fever hyper-immune serum.Pig japanese b encephalitis is passed as Zoonosis
Catch an illness, using strong virus attack, prepare the protection in link for people and to attack malicious facility requirements higher, and to human health exist compared with
Big potential threat.
Invention content
The present invention is directed to the technological deficiency for the prior art, a kind of Latex agglutination test Positive Sera is provided
Preparation method, with solve in the prior art lack correlation technique the technical issues of.
Another technical problem to be solved by the present invention is that Latex agglutination test Positive Sera prepares link safety
It is relatively low.
To realize that the above technical purpose, the present invention use following technical scheme：
A kind of preparation method of Latex agglutination test Positive Sera, includes the following steps：
1) pig japanese b encephalitis vaccine is prepared；
2) take the step 1) vaccine to swine fever virus and its antibody, porcine reproductive and respiratory syndrome virus and its antibody,
The pig that porcine pseudorabies virus and its antibody, II type of pig annulus virus and its antibody are negative carries out immunity inoculation；
3) serum is harvested out of pig body after step 2) immunity inoculation；
On this basis, the step 1) vaccine is prepared for antigen for SA14-14-2 plants with Latex agglutination test.
Preferably, the pig described in step 2) is 2~3 monthly age health pigs, pig japanese b encephalitis HI potency is not higher than 1:4.
Preferably, the methods of vaccination described in step 2) is：
A) fundamental immunity：The pig japanese b encephalitis live vaccine for taking step 1) to prepare, every time with the dosage injection 2 of 1~5 part
It is secondary, per 13~17d of minor tick；
B) reinforced immunological：After 13~17d of fundamental immunity, the pig japanese b encephalitis inactivated vaccine for taking step 1) to prepare, with muscle
The mode of injection is inoculated with 3~5 times, and the adjacent time interval being inoculated with twice is 7~10d, and the dosage being once inoculated with afterwards is preceding primary
1.5~2 times of dosage of inoculation.
On this basis it is further preferred that step B) in the dosage injected for the first time be 3~5mL.
Preferably, the neutralize antibody titers of serum described in step 3) are higher than 1:400.
Preferably, the vaccine described in step 1) includes pig japanese b encephalitis live vaccine, Latex agglutination test titre is high
In 106.0TCID50/ head part.
Preferably, the vaccine described in step 1) includes pig japanese b encephalitis live vaccine, which is that JEV venom is added
Made of freeze drying protectant is freeze-dried.
Preferably, the vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, which is by pig japanese b encephalitis
Virus liquid is concentrated by ultrafiltration after purification, is inactivated using beta-propiolactone, reuse ISA760VG adjuvants carry out emulsification be prepared
's；The aperture being wherein concentrated by ultrafiltration is 300KD, and cycles of concentration is 5~10 times.
Preferably, the vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, the potency of the vaccine is higher than
109.0TCID50/ml。
Preferably, the vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, which is by pig japanese b encephalitis
Virus liquid is concentrated by ultrafiltration after purification, is inactivated using beta-propiolactone, reuse ISA760VG adjuvants carry out emulsification be prepared
's；Wherein inactivation includes the following steps：With 1:2000~1:The ratio of 8000 (w/w) is sick to the pig japanese b encephalitis after ultrafiltration concentration
Beta-propiolactone is added in venom, 24~96h is inactivated in 2~8 DEG C, and is placed on 35~39 DEG C of water-baths and hydrolyzes 1.5~2.5h.
Preferably, the vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, which is by pig japanese b encephalitis
Virus liquid is concentrated by ultrafiltration after purification, is inactivated using beta-propiolactone, reuse ISA760VG adjuvants carry out emulsification be prepared
's；The mixed proportion of wherein Latex agglutination test liquid and ISA760VG adjuvants is 1:2~1:3,5~10min of emulsification times.
Preferably, in step 3) before harvesting serum, first use Latex agglutination test ELISA antibody kits with
And plaque reduction neutralization test measures serum neutralization titer；On this basis it is further preferred that the minute of above-mentioned potency
It is 5~10d after step 2) last time immunity inoculation.
Preferably, being to harvest serum by way of vena cava anterior bloodletting in step 3).
In above technical scheme, described Latex agglutination test SA14-14-2 plants are conventional strains, and poison is planted by our company
Room identification preserves；ISA760VG adjuvants are trade names, are refered in particular to by match BIC Corp of France product, model ISA760VG
Vaccine adjuvant product can also be bought from market.
Latex agglutination test Positive Sera prepared by the method for the present invention can be used for pig japanese b encephalitis live vaccine
Diagnostic test and exogenous virus are examined and the purposes such as diagnosis detection.In the technical scheme, japanese encephalitis virus SA14-14-2
Strain has reliable safety and a good immunogenicity, encephalitis B only there are one serotype, different strains in virulence and
It is had differences in blood clotting characteristic, but the no significant difference in antigenicity.Therefore, pig second is prepared using SA14-14-2 plants of immune swines
Type encephalitis viruses Positive Sera avoids, using strong poison, having the advantages that safe.In addition, encephalitis B is dynamic in experiment
Object is most sensitive with mouse, and cavy, rat and rabbit are insensitive, however prepares positive serum, the serum amount of harvest using mouse
It is less, be not suitable for large-scale application.It is big compared with the serum amount that mouse harvests that positive serum is prepared using pig, is of relatively low cost, and is had
There is scale application foreground outstanding.
Specific implementation mode
The specific implementation mode of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.
Approximating language used in following embodiment can be used for quantitative expression, show in the feelings for not changing basic function
Quantity is allowed to have certain variation under condition.Therefore, it is not limited to this accurately with the modified numerical value of the language such as " about ", " left and right " institute
Numerical value itself.In some embodiments, the range for allowing its modified numerical value in positive and negative 10 (10%) " about " is indicated
Interior variation, for example, what " about 100 " indicated can be any numerical value between 90 to 110.In addition, " the about first numerical value arrives
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
It may be related with the precision of measuring instrument.
In addition to being defined, technical and scientific term used in following embodiment has and fields technology people of the present invention
The identical meanings that member is commonly understood by.
Test reagent consumptive material used in following embodiment is unless otherwise specified conventional biochemical reagent；The experiment
Method is unless otherwise specified conventional method；Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result
It is averaged；% in following embodiment is unless otherwise instructed mass percentage.
Embodiment 1 (preparation of Latex agglutination test Positive Sera)
The preparation of 1 vaccine
The preparation and inspection of 1.1 pig japanese b encephalitis live vaccines (SA14-14-2 plants)
1.1.1 live vaccine is prepared the JEV venom through steriling test qualification, and suitable freeze drying protectant is added, and is added simultaneously
Enter suitable antibiotic, full and uniform, after regulation head part quantitative separating, the B-mode brain of pig is made in the rapid vacuum freezedrying that carries out
Scorching live vaccine (SA14-14-2 plants).
1.1.2 live vaccine is examined carries out physical behavior, vacuum degree, residue by pig japanese b encephalitis live vaccine made of 1.1.1
Moisture, steriling test, mycoplasma inspection, exogenous virus inspection, diagnostic test, safety examination and efficacy test (viral level
Measure), it is as a result all qualified.Wherein 10 are determined to the viral level of JEV venom with seedling8.0TCID50/ ml, it is B-mode to be made pig
Viral level after encephalitis live vaccine (SA14-14-2 plants) is determined to 106.6TCID50/ head part.
The preparation of 1.2 pig japanese b encephalitis inactivated vaccines (SA14-14-2 plants)
1.2.1 prepared by inactivated vaccine
1.2.1.1 venom concentration, inactivation carry out the JEV venom through steriling test qualification using the ultrafiltration membrane packet of 300KD
Beta-propiolactone is added in 1: 4000 ratio after 7 times of concentrations, after setting 2~8 DEG C of stirring inactivation 44h, sets 37 DEG C of hydrolysis 2h.
1.2.1.2 emulsify by above-mentioned 1.2.1.1 hydrolysis complete venom with match Bick novel adjuvant ISA760VG with
2: 3 ratio is emulsified using cutter, and the time of shearing is altogether 6min, and pig is made in the vaccine quantitative separating emulsified
Encephalitis B inactivated vaccine (SA14-14-2 plants).
1.2.1 inactivated vaccine examine pig japanese b encephalitis inactivated vaccine progress dosage form, stability made of 1.2.1 and
Steriling test etc., it is as a result every to examine all qualifications.With seedling 10 are determined to the viral level of JEV venom8.6TCID50/ ml,
The viral level of venom is determined to 10 after concentrated9.25TCID50/ml.Venom after inactivation is subjected to inactivation inspection, in cell
Upper blind passage three generations, the results showed that cytopathogenic effect, i.e. inactivation be not complete for the venom.
(the pig japanese b encephalitis HI potency of weanling pig 1 of screening 3 monthly ages of the selection susceptible eutrophy of health of 2 negative pigs
Not higher than 1: 4), and swine fever virus and its antibody, porcine reproductive and respiratory syndrome virus and its antibody, porcine pseudorabies virus after testing
And its antibody, II type of pig annulus virus and its antibody are feminine gender, are transferred to experiment swinery isolated rearing and observe 7, as a result try
It tests pig to be in a good state of nutrition, not finding stress be with the generation of other diseases.
3 immunity inoculations
3.1 live vaccine fundamental immunities：Totally 2 times.Every pig musculi colli injects the pig japanese b encephalitis work of 5 parts for the first time
Vaccine (SA14-14-2 plants) is spaced 14, and booster immunization is carried out 1 time with Isodose.
3.2 reinforced immunological：Totally 3 times.Carry out first time pig japanese b encephalitis inactivated vaccine within 15 days after the completion of fundamental immunity
Immune, every pig musculi colli injection 4ml of (SA14-14-2 plants)；Interval carries out second of inactivated vaccine booster immunization in 10 days,
Every pig muscle injection 6ml；Interval carries out third time inactivated vaccine booster immunization, every pig muscle injection 8ml on 7th.
4 positive serums harvest and identification
A small amount of Swine serum is acquired within 10 days after last 1 immunity inoculation of detection of 4.1 positive serum potency, it is B-mode using pig
Encephalitis viruses ELISA antibody kits and plaque reduction neutralization test measure neutralization titer, confirm that Latex agglutination test is anti-
Body is that positive and neutralize antibody titers are 1: 636.
4.1.1 the detection of Latex agglutination test ELISA antibody is tried using Latex agglutination test ELISA antibody tests
The serum that agent box (being purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) takes different immunization periods is detected, and is grasped
Make to carry out with reference to product description.It the results are shown in Table 1.
Front and back pig blood final proof ELISA antibody is immunized in the detection of table 1
Note：The ＜ 0.2 of negative control OD value=0.08, P values are equal to 1.539 and are more than 1.0.Value >=0.21 sample S/P, is judged to pig
B encephalitis virus antibody is positive, and sample S/P values ＜ 0.21 is judged to Latex agglutination test negative antibody.
4.1.2 plaque reduction neutralization test measures neutralizing antibody by 10 days after third time booster immunization serum, in 0.22
This experiment is carried out after 30 minutes through 56 DEG C of inactivations after μm membrane filtration, and steps are as follows.
4.1.2.1 choose the 500 μ l of virus liquid (about 1000PFU/ml) of appropriate dilution and equivalent doubling dilution (1: 10,
1: 20,1: 40,1: 80,1: 160,1: 320,1: 640,1: 1280) serum mixing to be checked is separately added with the virus liquid of same dilution
Upper equivalent is as above stated diluted dilution normal serum and is compared, and sets 37 DEG C of effect 90min, during which shook up one every 15~20 minutes
It is secondary.
4.1.2.2 6 porocyte culture plates of BHK-21 are covered in inoculation, and each titre connects 2 holes, per hole 0.2ml, absorption 90
Minute.
4.1.2.3 addition methylcellulose covering, 37 DEG C, 5%CO2After being cultivated 5 in incubator, covering is sucked out,
With violet staining, plaque counting is carried out.
4.1.2.4 it is to neutralize to imitate that result, which calculates the serum dilution for making plaque reduce 50% compared with normal serum compares,
Valence.In serum neutralization titer Lg=L+d (S-0.5) L=1, d=0.3, S=to be checked and the sum of ratio.It is computed surveyed serum
Neutralization titer is 1: 636.
4.2 positive serums be harvested from third time booster immunization after harvest sun by the way of vena cava anterior bloodletting within 15th
Property serum 500ml.The positive serum of harvest is subjected to steriling test, mycoplasma is examined and exogenous virus is examined, as a result every inspection
Test whole qualifications.
The positive serum of harvest is carried out following experiments by the application effect of 4.3 positive serums after 56 DEG C inactivate 30 minutes.
4.3.1 specific test (diagnostic test) is by the positive serum inactivated PBS (0.015mol/L, pH value 7.4
~7.6) it is diluted to the positive serum and 2 × 10 of different dilutions2.0TCID50The venom mixed in equal amounts of/0.1ml, at 37 DEG C
After neutralizing 90 minutes, it is inoculated with suslik kidney primary cell, each dilution is inoculated with 6 holes, sets 37 DEG C of cultures, be observed continuously 7, judges
As a result.It the results are shown in Table 2.The result shows that 1: the 40 times of dilution of the positive serum of harvest can meet pig japanese b encephalitis live vaccine and differentiate inspection
The needs tested.
2 various concentration positive serum specific test result of table
4.3.2 exogenous virus examines neutralization test that 3 bottles of pig japanese b encephalitis live vaccines is taken to be diluted to 10 part/ml with DMEM
After mix, 10000rpm centrifuges 10min, the positive serum mixing for taking supernatant and equivalent to inactivate, and sets 37 DEG C and neutralizes 90min.So
After take suitable corrective inoculation suslik kidney primary cell, discard adsorption liquid after adsorbing 60min, the training of DMEM cell maintenance mediums be added
It supports, 3 generation of such blind passage, each generation does not occur cytopathy, shows to neutralize completely, i.e., positive serum can meet exogenous virus
The needs of inspection.
4.3.2 the positive that we harvest in Latex agglutination test ELISA antibody kits positive serum 4.1.1
The OD values of serum are compareed higher than its standard positive serum, i.e., should can by calibration appropriate and the positive serum for examining us to prepare
It is used as diagnosis detection.
Embodiment 2 (preparation of Latex agglutination test Positive Sera)
The preparation of 1 vaccine
The preparation and inspection of 1.1 pig japanese b encephalitis live vaccines (SA14-14-2 plants)
1.1.1 live vaccine is prepared the JEV venom through steriling test qualification, and suitable freeze drying protectant is added, and is added simultaneously
Enter suitable antibiotic, full and uniform, after regulation head part quantitative separating, the B-mode brain of pig is made in the rapid vacuum freezedrying that carries out
Scorching live vaccine (SA14-14-2 plants).
1.1.2 live vaccine is examined carries out physical behavior, vacuum degree, residue by pig japanese b encephalitis live vaccine made of 1.1.1
Moisture, steriling test, mycoplasma inspection, exogenous virus inspection, diagnostic test, safety examination and efficacy test (viral level
Measure), it is as a result all qualified.Wherein 10 are determined to the viral level of JEV venom with seedling8.6TCID50/ ml, it is B-mode to be made pig
Viral level after encephalitis live vaccine (SA14-14-2 plants) is determined to 107.0TCID50/ head part.
The preparation of 1.2 pig japanese b encephalitis inactivated vaccines (SA14-14-2 plants)
1.2.1 prepared by inactivated vaccine
1.2.1.1 venom concentration, inactivation carry out the JEV venom through steriling test qualification using the ultrafiltration membrane packet of 300KD
Beta-propiolactone is added in 1: 2000 ratio after 7 times of concentrations, after setting 2~8 DEG C of stirring inactivation 36h, sets 37 DEG C of hydrolysis 2h.
1.2.1.2 emulsify by above-mentioned 1.2.1.1 hydrolysis complete venom with match Bick novel adjuvant ISA760VG with
1: 2 ratio is emulsified using cutter, and the time of shearing is altogether 7min, and pig is made in the vaccine quantitative separating emulsified
Encephalitis B inactivated vaccine (SA14-14-2 plants).
1.2.1 inactivated vaccine examine pig japanese b encephalitis inactivated vaccine progress dosage form, stability made of 1.2.1 and
Steriling test etc., it is as a result every to examine all qualifications.With seedling 10 are determined to the viral level of JEV venom8.6TCID50/ ml,
The viral level of venom is determined to 10 after concentrated9.25TCID50/ml.Venom after inactivation is subjected to inactivation inspection, in cell
Upper blind passage three generations, the results showed that cytopathogenic effect, i.e. inactivation be not complete for the venom.
(the pig japanese b encephalitis HI potency of weanling pig 1 of screening 3 monthly ages of the selection susceptible eutrophy of health of 2 negative pigs
Not higher than 1: 4), and swine fever virus and its antibody, porcine reproductive and respiratory syndrome virus and its antibody, porcine pseudorabies virus after testing
And its antibody, II type of pig annulus virus and its antibody are feminine gender, are transferred to experiment swinery isolated rearing and observe 10, as a result try
It tests pig to be in a good state of nutrition, not finding stress be with the generation of other diseases.
3 immunity inoculations
3.1 live vaccine fundamental immunities：Totally 2 times.Every pig musculi colli injects the pig japanese b encephalitis work of 2 parts for the first time
Vaccine (SA14-14-2 plants) is spaced 14, and booster immunization is carried out 1 time with Isodose.
3.2 reinforced immunological：Totally 4 times.Carry out first time pig japanese b encephalitis inactivated vaccine within 15 days after the completion of fundamental immunity
Immune, every pig musculi colli injection 2ml of (SA14-14-2 plants)；Interval carries out second of inactivated vaccine booster immunization in 10 days,
Every pig muscle injection 4ml；Interval carries out third time inactivated vaccine booster immunization, every pig muscle injection 9ml, interval 7 on the 10th
Day carries out the 4th inactivated vaccine booster immunization, every pig muscle injection 8ml.
4 positive serums harvest and identification
A small amount of Swine serum is acquired after last 1 immunity inoculation of detection of 4.1 positive serum potency within 7 days, uses the B-mode brain of pig
Scorching virus ELISA antibody kit and plaque reduction neutralization test measure neutralization titer, confirm Latex agglutination test antibody
It is 1: 568 for positive and neutralize antibody titers.
4.1.1 the detection of Latex agglutination test ELISA antibody is tried using Latex agglutination test ELISA antibody tests
The serum that agent box (being purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) takes different immunization periods is detected, and is grasped
Make to carry out with reference to product description.It the results are shown in Table 3.
Front and back pig blood final proof ELISA antibody is immunized in the detection of table 3
Note：The ＜ 0.2 of negative control OD value=0.08, P values are equal to 1.539.Value >=0.21 sample S/P is judged to the B-mode brain of pig
Scorching virus antibody positive, sample S/P values ＜ 0.21, is judged to Latex agglutination test negative antibody.
4.1.2 plaque reduction neutralization test measures neutralizing antibody by 10 days after third time booster immunization serum, in 0.22
This experiment is carried out after 30 minutes through 56 DEG C of inactivations after μm membrane filtration, and steps are as follows.
4.1.2.1 choose the 500 μ l of virus liquid (about 1000PFU/ml) of appropriate dilution and equivalent doubling dilution (1: 10,
1: 20,1: 40,1: 80,1: 160,1: 320,1: 640,1: 1280) serum mixing to be checked is separately added with the virus liquid of same dilution
Upper equivalent is as above stated diluted dilution normal serum and is compared, and sets 37 DEG C of effect 90min, during which shook up one every 15~20 minutes
It is secondary.
4.1.2.2 6 porocyte culture plates of BHK-21 are covered in inoculation, and each titre connects 2 holes, per hole 0.2ml, absorption 90
Minute.
4.1.2.3 addition methylcellulose covering, 37 DEG C, 5%CO2After being cultivated 5 in incubator, covering is sucked out,
With violet staining, plaque counting is carried out.
4.1.2.4 it is to neutralize to imitate that result, which calculates the serum dilution for making plaque reduce 50% compared with normal serum compares,
Valence.In serum neutralization titer Lg=L+d (S-0.5) L=1, d=0.3, S=to be checked and the sum of ratio.It is computed surveyed serum
Neutralization titer is 1: 568.
4.2 positive serums be harvested from the 4th booster immunization after harvest sun by the way of vena cava anterior bloodletting within 12nd
Property serum about 520ml.The positive serum of harvest is carried out steriling test, mycoplasma inspection and exogenous virus to examine, it is as a result every
It is all qualified to examine.
The positive serum of harvest is carried out following experiments by the application effect of 4.3 positive serums after 56 DEG C inactivate 30 minutes.
4.3.1 specific test (diagnostic test) is by the positive serum inactivated PBS (0.015mol/L, pH value 7.4
~7.6) it is diluted to the positive serum and 2 × 10 of different dilutions2.0TCID50The venom mixed in equal amounts of/0.1ml, at 37 DEG C
After neutralizing 90 minutes, it is inoculated with suslik kidney primary cell, each dilution is inoculated with 6 holes, sets 37 DEG C of cultures, be observed continuously 7, judges
As a result.It the results are shown in Table 4.The result shows that 1: the 32 times of dilution of the positive serum of harvest can meet pig japanese b encephalitis live vaccine and differentiate inspection
The needs tested.
4 various concentration positive serum specific test result of table
4.3.2 exogenous virus examines neutralization test that 3 bottles of pig japanese b encephalitis live vaccines is taken to be diluted to 10 part/ml with DMEM
After mix, 10000rpm centrifuges 10min, the positive serum mixing for taking supernatant and equivalent to inactivate, and sets 37 DEG C and neutralizes 90min.So
After take suitable corrective inoculation suslik kidney primary cell, discard adsorption liquid after adsorbing 60min, the training of DMEM cell maintenance mediums be added
It supports, 3 generation of such blind passage, each generation does not occur cytopathy, shows to neutralize completely, i.e., positive serum can meet exogenous virus
The needs of inspection.
4.3.2 the positive that we harvest in Latex agglutination test ELISA antibody kits positive serum 4.1.1
The OD values of serum are compareed higher than its standard positive serum, i.e., should can by calibration appropriate and the positive serum for examining us to prepare
It is used as diagnosis detection.
Embodiment 3 (preparation of Latex agglutination test Positive Sera)
The preparation of 1 vaccine
The preparation and inspection of 1.1 pig japanese b encephalitis live vaccines (SA14-14-2 plants)
1.1.1 live vaccine is prepared the JEV venom through steriling test qualification, and suitable freeze drying protectant is added, and is added simultaneously
Enter suitable antibiotic, full and uniform, after regulation head part quantitative separating, the B-mode brain of pig is made in the rapid vacuum freezedrying that carries out
Scorching live vaccine (SA14-14-2 plants).
1.1.2 live vaccine is examined carries out physical behavior, vacuum degree, residue by pig japanese b encephalitis live vaccine made of 1.1.1
Moisture, steriling test, mycoplasma inspection, exogenous virus inspection, diagnostic test, safety examination and efficacy test (viral level
Measure), it is as a result all qualified.Wherein 10 are determined to the viral level of JEV venom with seedling8.4TCID50/ ml, it is B-mode to be made pig
Viral level after encephalitis live vaccine (SA14-14-2 plants) is determined to 106.75TCID50/ head part.
The preparation of 1.2 pig japanese b encephalitis inactivated vaccines (SA14-14-2 plants)
1.2.1 prepared by inactivated vaccine
1.2.1.1 venom concentration, inactivation carry out the JEV venom through steriling test qualification using the ultrafiltration membrane packet of 300KD
Beta-propiolactone is added in 1: 6000 ratio after 10 times of concentrations, after setting 2~8 DEG C of stirring inactivation 72h, sets 37 DEG C of hydrolysis 2h.
1.2.1.2 emulsify by above-mentioned 1.2.1.1 hydrolysis complete venom with match Bick novel adjuvant ISA760VG with
2: 3 ratio is emulsified using cutter, and the time of shearing is altogether 7min, and pig is made in the vaccine quantitative separating emulsified
Encephalitis B inactivated vaccine (SA14-14-2 plants).
1.2.1 inactivated vaccine examine pig japanese b encephalitis inactivated vaccine progress dosage form, stability made of 1.2.1 and
Steriling test etc., it is as a result every to examine all qualifications.With seedling 10 are determined to the viral level of JEV venom8.4TCID50/ ml,
The viral level of venom is determined to 10 after concentrated9.25TCID50/ml.Venom after inactivation is subjected to inactivation inspection, in cell
Upper blind passage three generations, the results showed that cytopathogenic effect, i.e. inactivation be not complete for the venom.
(the pig japanese b encephalitis HI potency of weanling pig 1 of screening 2 monthly ages of the selection susceptible eutrophy of health of 2 negative pigs
Not higher than 1: 4), and swine fever virus and its antibody, porcine reproductive and respiratory syndrome virus and its antibody, porcine pseudorabies virus after testing
And its antibody, II type of pig annulus virus and its antibody are feminine gender, are transferred to experiment swinery isolated rearing and observe 15, as a result try
It tests pig to be in a good state of nutrition, not finding stress be with the generation of other diseases.
3 immunity inoculations
3.1 live vaccine fundamental immunities：Totally 2 times.Every pig musculi colli injects the pig japanese b encephalitis work of 5 parts for the first time
Vaccine (SA14-14-2 plants) is spaced 15, and booster immunization is carried out 1 time with Isodose.
3.2 reinforced immunological：Totally 3 times.Carry out first time pig japanese b encephalitis inactivated vaccine within 15 days after the completion of fundamental immunity
Immune, every pig musculi colli injection 1ml of (SA14-14-2 plants)；Interval carries out second of inactivated vaccine booster immunization in 10 days,
Every pig muscle injection 2ml；Interval carries out third time inactivated vaccine booster immunization, every pig muscle injection 4ml on 10th；Interval 10
Day carries out the 4th inactivated vaccine booster immunization, every pig muscle injection 6ml；Interval carries out the 5th inactivated vaccine on the 7th and reinforces
It is immune, every pig muscle injection 10ml.
4 positive serums harvest and identification
A small amount of Swine serum is acquired after last 1 immunity inoculation of detection of 4.1 positive serum potency within 5 days, uses the B-mode brain of pig
Scorching virus ELISA antibody kit and plaque reduction neutralization test measure neutralization titer, confirm Latex agglutination test antibody
It is 1: 600 for positive and neutralize antibody titers.
4.1.1 the detection of Latex agglutination test ELISA antibody is tried using Latex agglutination test ELISA antibody tests
The serum that agent box (being purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) takes different immunization periods is detected, and is grasped
Make to carry out with reference to product description.It the results are shown in Table 5.
Front and back pig blood final proof ELISA antibody is immunized in the detection of table 5
Note：The ＜ 0.2 of negative control OD value=0.08, P values are equal to 1.539.Value >=0.21 sample S/P is judged to the B-mode brain of pig
Scorching virus antibody positive, sample S/P values ＜ 0.21, is judged to Latex agglutination test negative antibody.
4.1.2 plaque reduction neutralization test measures neutralizing antibody by 5 days after the 5th booster immunization serum, in 0.22 μ
This experiment is carried out after 56 DEG C inactivate 30 minutes after m membrane filtrations, steps are as follows.
4.1.2.1 choose the 500 μ l of virus liquid (about 1000PFU/ml) of appropriate dilution and equivalent doubling dilution (1: 10,
1: 20,1: 40,1: 80,1: 160,1: 320,1: 640,1: 1280) serum mixing to be checked is separately added with the virus liquid of same dilution
Upper equivalent is as above stated diluted dilution normal serum and is compared, and sets 37 DEG C of effect 90min, during which shook up one every 15~20 minutes
It is secondary.
4.1.2.2 6 porocyte culture plates of BHK-21 are covered in inoculation, and each titre connects 2 holes, per hole 0.2ml, absorption 90
Minute.
4.1.2.3 addition methylcellulose covering, 37 DEG C, 5%CO2After being cultivated 5 in incubator, covering is sucked out,
With violet staining, plaque counting is carried out.
4.1.2.4 it is to neutralize to imitate that result, which calculates the serum dilution for making plaque reduce 50% compared with normal serum compares,
Valence.In serum neutralization titer Lg=L+d (S-0.5) L=1, d=0.3, S=to be checked and the sum of ratio.It is computed surveyed serum
Neutralization titer is 1: 600.
4.2 positive serums be harvested from the 5th booster immunization after harvest sun by the way of vena cava anterior bloodletting within 10th
Property serum 550ml.The positive serum of harvest is subjected to steriling test, mycoplasma is examined and exogenous virus is examined, as a result every inspection
Test whole qualifications.
The positive serum of harvest is carried out following experiments by the application effect of 4.3 positive serums after 56 DEG C inactivate 30 minutes.
4.3.1 specific test (diagnostic test) is by the positive serum inactivated PBS (0.015mol/L, pH value 7.4
~7.6) it is diluted to the positive serum and 2 × 10 of different dilutions2.0TCID50The venom mixed in equal amounts of/0.1ml, at 37 DEG C
After neutralizing 90 minutes, it is inoculated with suslik kidney primary cell, each dilution is inoculated with 6 holes, sets 37 DEG C of cultures, be observed continuously 7, judges
As a result.It the results are shown in Table 6.The result shows that 1: the 32 times of dilution of the positive serum of harvest can meet pig japanese b encephalitis live vaccine and differentiate inspection
The needs tested.
6 various concentration positive serum specific test result of table
4.3.2 exogenous virus examines neutralization test that 3 bottles of pig japanese b encephalitis live vaccines is taken to be diluted to 10 part/ml with DMEM
After mix, 10000rpm centrifuges 10min, the positive serum mixing for taking supernatant and equivalent to inactivate, and sets 37 DEG C and neutralizes 90min.So
After take suitable corrective inoculation suslik kidney primary cell, discard adsorption liquid after adsorbing 60min, the training of DMEM cell maintenance mediums be added
It supports, 3 generation of such blind passage, each generation does not occur cytopathy, shows to neutralize completely, i.e., positive serum can meet exogenous virus
The needs of inspection.
4.3.2 the positive that we harvest in Latex agglutination test ELISA antibody kits positive serum 4.1.1
The OD values of serum are compareed higher than its standard positive serum, i.e., should can by calibration appropriate and the positive serum for examining us to prepare
It is used as diagnosis detection.
Embodiment 4
A kind of preparation method of Latex agglutination test Positive Sera, includes the following steps：
1) pig japanese b encephalitis vaccine is prepared；
2) take the step 1) vaccine to swine fever virus and its antibody, porcine reproductive and respiratory syndrome virus and its antibody,
The pig that porcine pseudorabies virus and its antibody, II type of pig annulus virus and its antibody are negative carries out immunity inoculation；
3) serum is harvested out of pig body after step 2) immunity inoculation；
Wherein：Step 1) the vaccine is prepared for antigen for SA14-14-2 plants with Latex agglutination test.
On the basis of above technical scheme, meet the following conditions：
Pig described in step 2) is 2 monthly age health pigs, and pig japanese b encephalitis HI potency is not higher than 1:4.
Methods of vaccination described in step 2) is：
A) fundamental immunity：The pig japanese b encephalitis live vaccine for taking step 1) to prepare, every time with the dosage injection of 1 part 2 times, often
Minor tick 13d；
B) reinforced immunological：After fundamental immunity 13d, the pig japanese b encephalitis inactivated vaccine for taking step 1) to prepare, with intramuscular injection
Mode be inoculated with 3 times, the adjacent time interval being inoculated with twice be 7d, the dosage being once inoculated with afterwards is a preceding dosage of inoculation
1.5 again.
Step B) in the dosage injected for the first time be 3mL.
The neutralize antibody titers of serum described in step 3) are higher than 1:400.
Vaccine described in step 1) includes pig japanese b encephalitis live vaccine, and Latex agglutination test titre is higher than
106.0TCID50/ head part.
Vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, which is to carry out Latex agglutination test liquid
It is concentrated by ultrafiltration after purification, is inactivated using beta-propiolactone, reuse ISA760VG adjuvants and carry out what emulsification was prepared；Wherein ultrafiltration
The aperture of concentration is 300KD, and cycles of concentration is 5 times.
Vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, and the potency of the vaccine is higher than 109.0TCID50/ml。
Vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, which is to carry out Latex agglutination test liquid
It is concentrated by ultrafiltration after purification, is inactivated using beta-propiolactone, reuse ISA760VG adjuvants and carry out what emulsification was prepared；Wherein inactivate
Include the following steps：With 1:Beta-propiolactone is added into the Latex agglutination test liquid after ultrafiltration concentration in the ratio of 2000 (w/w),
For 24 hours in 2 DEG C of inactivations, 35 DEG C of water-bath hydrolysis 1.5h are placed on.
Vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, which is to carry out Latex agglutination test liquid
It is concentrated by ultrafiltration after purification, is inactivated using beta-propiolactone, reuse ISA760VG adjuvants and carry out what emulsification was prepared；Wherein pig second
The mixed proportion of type encephalitis viruses liquid and ISA760VG adjuvants is 1:2, emulsification times 5min.
Embodiment 5
A kind of preparation method of Latex agglutination test Positive Sera, includes the following steps：
1) pig japanese b encephalitis vaccine is prepared；
2) take the step 1) vaccine to swine fever virus and its antibody, porcine reproductive and respiratory syndrome virus and its antibody,
The pig that porcine pseudorabies virus and its antibody, II type of pig annulus virus and its antibody are negative carries out immunity inoculation；
3) serum is harvested out of pig body after step 2) immunity inoculation；
Wherein：Step 1) the vaccine is prepared for antigen for SA14-14-2 plants with Latex agglutination test.
On the basis of above technical scheme, meet the following conditions：
Pig described in step 2) is 3 monthly age health pigs, and pig japanese b encephalitis HI potency is not higher than 1:4.
Methods of vaccination described in step 2) is：
A) fundamental immunity：The pig japanese b encephalitis live vaccine for taking step 1) to prepare, every time with the dosage injection of 5 parts 2 times, often
Minor tick 17d；
B) reinforced immunological：After fundamental immunity 17d, the pig japanese b encephalitis inactivated vaccine for taking step 1) to prepare, with intramuscular injection
Mode be inoculated with 5 times, the adjacent time interval being inoculated with twice be 10d, the dosage being once inoculated with afterwards is the 2 of a preceding dosage of inoculation
Times.
Step B) in the dosage injected for the first time be 5mL.
Vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, which is to carry out Latex agglutination test liquid
It is concentrated by ultrafiltration after purification, is inactivated using beta-propiolactone, reuse ISA760VG adjuvants and carry out what emulsification was prepared；Wherein ultrafiltration
The aperture of concentration is 300KD, and cycles of concentration is 10 times.
Vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, which is to carry out Latex agglutination test liquid
It is concentrated by ultrafiltration after purification, is inactivated using beta-propiolactone, reuse ISA760VG adjuvants and carry out what emulsification was prepared；Wherein inactivate
Include the following steps：With 1:Beta-propiolactone is added into the Latex agglutination test liquid after ultrafiltration concentration in the ratio of 8000 (w/w),
96h is inactivated in 8 DEG C, and is placed on 39 DEG C of water-bath hydrolysis 2.5h.
Vaccine described in step 1) includes pig japanese b encephalitis inactivated vaccine, which is to carry out Latex agglutination test liquid
It is concentrated by ultrafiltration after purification, is inactivated using beta-propiolactone, reuse ISA760VG adjuvants and carry out what emulsification was prepared；Wherein pig second
The mixed proportion of type encephalitis viruses liquid and ISA760VG adjuvants is 1:3, emulsification times 10min.
Embodiment 6
A kind of preparation method of Latex agglutination test Positive Sera, includes the following steps：
1) pig japanese b encephalitis vaccine is prepared；
2) take the step 1) vaccine to swine fever virus and its antibody, porcine reproductive and respiratory syndrome virus and its antibody,
The pig that porcine pseudorabies virus and its antibody, II type of pig annulus virus and its antibody are negative carries out immunity inoculation；
3) serum is harvested out of pig body after step 2) immunity inoculation；
Wherein：Step 1) the vaccine is prepared for antigen for SA14-14-2 plants with Latex agglutination test.
The embodiments of the present invention have been described in detail above, but content is only the preferred embodiment of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement etc. done in the application range of the present invention, should all
It is included within protection scope of the present invention.
Claims (7)
1. a kind of preparation method of Latex agglutination test Positive Sera, includes the following steps：
1) pig japanese b encephalitis vaccine is prepared；
2) take the step 1) vaccine pseudo- to swine fever virus and its antibody, porcine reproductive and respiratory syndrome virus and its antibody, pig
The pig that rabies viruses and its antibody, II type of pig annulus virus and its antibody are negative carries out immunity inoculation；
3) serum is harvested out of pig body after step 2) immunity inoculation；
It is characterized in that：Step 1) the vaccine is prepared for antigen for SA14-14-2 plants with Latex agglutination test；
Simultaneously：
Pig described in step 2) is 2~3 monthly age health pigs, and pig japanese b encephalitis HI potency is not higher than 1:4；
Methods of vaccination described in step 2) is：
A) fundamental immunity：The pig japanese b encephalitis live vaccine for taking step 1) to prepare, every time with the dosage injection of 1~5 part 2 times, often
13~17d of minor tick；
B) reinforced immunological：After 13~17d of fundamental immunity, the pig japanese b encephalitis inactivated vaccine for taking step 1) to prepare, with intramuscular injection
Mode be inoculated with 3~5 times, the adjacent time interval being inoculated with twice be 7~10d, the dosage being once inoculated with afterwards is preceding primary inoculation
1.5~2 times of dosage；
Step B) in the dosage injected for the first time be 3~5mL.
2. preparation method according to claim 1, it is characterised in that the neutralize antibody titers of serum described in step 3) are high
In 1:400.
3. preparation method according to claim 1, it is characterised in that the vaccine described in step 1) includes that pig japanese b encephalitis is lived
Vaccine, Latex agglutination test titre are higher than 106.0TCID50/ head part.
4. preparation method according to claim 1, it is characterised in that the vaccine described in step 1) includes that pig japanese b encephalitis goes out
Live vaccine, the vaccine are that Latex agglutination test liquid is concentrated by ultrafiltration after purification, are inactivated, are reused using beta-propiolactone
ISA760VG adjuvants carry out what emulsification was prepared；The aperture being wherein concentrated by ultrafiltration is 300KD, and cycles of concentration is 5~10 times.
5. preparation method according to claim 1, it is characterised in that the vaccine described in step 1) includes that pig japanese b encephalitis goes out
The potency of live vaccine, the vaccine is higher than 109.0TCID50/ml。
6. preparation method according to claim 1, it is characterised in that the vaccine described in step 1) includes that pig japanese b encephalitis goes out
Live vaccine, the vaccine are that Latex agglutination test liquid is concentrated by ultrafiltration after purification, are inactivated, are reused using beta-propiolactone
ISA760VG adjuvants carry out what emulsification was prepared；Wherein inactivation includes the following steps：With 1:2000~1:8000 mass ratio
Beta-propiolactone is added into the Latex agglutination test liquid after ultrafiltration concentration, 24~96h are inactivated in 2~8 DEG C, and it is placed on 35~
39 DEG C of water-baths hydrolyze 1.5~2.5h.
7. preparation method according to claim 1, it is characterised in that the vaccine described in step 1) includes that pig japanese b encephalitis goes out
Live vaccine, the vaccine are that Latex agglutination test liquid is concentrated by ultrafiltration after purification, are inactivated, are reused using beta-propiolactone
ISA760VG adjuvants carry out what emulsification was prepared；Wherein Latex agglutination test liquid and the mixed proportion of ISA760VG adjuvants is
1:2~1:3,5~10min of emulsification times.
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| TWI690592B (en) * | 2017-04-28 | 2020-04-11 | 高端疫苗生物製劑股份有限公司 | Japanese encephalitis vaccine and method for production thereof |
| CN111018970B (en) * | 2019-12-27 | 2021-09-14 | 中牧实业股份有限公司 | Specific positive serum for porcine encephalitis B virus and preparation method thereof |
| CN112062836B (en) * | 2020-08-31 | 2022-07-29 | 中牧实业股份有限公司 | High-titer positive serum of porcine Seneca valley virus and preparation method thereof |
| CN112094339A (en) * | 2020-09-27 | 2020-12-18 | 中国兽医药品监察所 | Porcine circovirus type 2 positive serum and preparation method thereof |
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