CN103830748B - The detection method of CA16 antigen immunogenicity in a kind of inactivated vaccine - Google Patents
The detection method of CA16 antigen immunogenicity in a kind of inactivated vaccine Download PDFInfo
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Abstract
The invention provides the detection method of CA16 antigen immunogenicity in a kind of inactivated vaccine, belong to vaccine immunity technical field, that application rat is as laboratory animal, by the inactivated vaccine immune rat containing CA16 antigen, evaluate CA16 viral inactivation vaccine immunogenicity by the NAT measuring rat blood serum.The inventive method have sensitivity strong, can repeatedly immunity, sustainable advantage of observing antibody variation trend after immunity, the immunogenic evaluation of CA16 viral inactivation vaccine has a good application prospect.
Description
Technical field
The present invention relates to vaccine immunity technical field, specifically, relate to the detection method of CA16 antigen immunogenicity in a kind of inactivated vaccine.
Background technology
Hand-foot-mouth disease is the infectious disease caused by enterovirus, and world's most area all has this sick popular report.Enterovirus EV 71 and Coxsackie virus CoxA16(are called for short CA16) infect alternately appearance, become the main pathogens of hand-foot-mouth disease.
In recent years, China's hand-foot-mouth disease epidemic situation is day by day serious, and morbidity, serious symptom and death are in ascendant trend year by year, and within 2008, hand-foot-mouth disease case 489,073 example is reported in the whole nation altogether, dead 126 examples; 2009 annual report morbidity 1,155,525 examples, dead 353 examples; 2010 annual report morbidity 1,774,669 examples, dead 905 examples; 2011 annual report morbidity 1,638,743 examples, dead 506 examples.Wherein serious symptom and death cause primarily of EV71 viral infection, and the dead is generally 0.5-3 year the age, the course of disease only 2 days.This disease seriously jeopardizes our people, particularly the health and lives of infant crowd, brings huge Disease Spectrum to society, and Class C infectious disease is listed on May 2nd, 2008 by China.
Although serious symptom and death cause primarily of EV71 viral infection, it is worth noting that CA16 is relevant to comprehensive diseases such as myocarditis, pericarditis, Refractory Shocks, and become hand-foot-mouth disease advantage cause of disease in some areas.In addition, Ministry of Public Health related data shows, upper one year popular CA16 area, reported cases number, serious symptom and death number during the popular EV71 of Second Year all obviously increase, and both promptings may exist conspiracy relation.Coxsackie virus (Coxasckievirus) A16 type (CoxA16, CA16) is the single strand plus RNA virus of Picornaviridae, and spherical in 20 cubic symmetry, diameter is about 23-30nm.Mainly propagate through excrement-mouth, respiratory tract and close contact, pass to fetus by Placenta Hominis and cause intrauterine infection.The pathogen of the hand-foot-mouth disease of early discovery is mainly CoxA16 type, after 20 century 70s, infects and alternately occurs, become the main pathogens of hand-foot-mouth disease with EV71.All there is report in multiple provinces such as China Beijing, Fujian, Tianjin, Jilin, Shandong, Guangdong, Shenzhen, Zhejiang, Sichuan, Anhui.There is no the report of CA16 viral vaccine at present, therefore the preclinical study of CAl6 viral inactivation vaccine is carried out and its immunogenicity of effective evaluation is very necessary, but how effectively to evaluate the immunogenicity of CAl6 viral inactivation vaccine, do not have the report of its method so far.
Summary of the invention
The object of the present invention is to provide the detection method of CA16 antigen immunogenicity in a kind of inactivated vaccine.
The detection method of CA16 antigen immunogenicity in inactivated vaccine provided by the invention, is by the inactivated vaccine immune rat containing CA16 antigen, detects CA16 viral inactivation vaccine immunogenicity by the NAT measuring rat blood serum.
Described rat is Wistar rat, SD rat or BrownNorway rat.
In the inventive method, immunization route is lumbar injection, intramuscular injection or subcutaneous injection.Wherein, immunizing dose is that every rat injects the inactivated vaccine containing 25 ~ 1600UCA16 antigen at every turn.
Wherein, immune programme for children is 0 day, 14 days immune 2 times respectively, blood sampling in the 28th day, and separation of serum, adopts conventional microneutralization to measure the NAT of serum.
Concrete steps are as follows: by serum doubling dilution, and CA16 virus is (purchased from ATCC, 100CCID
50/ 0.05ml) after 37 DEG C of neutralization reactions, inoculate sensitive cells (Vero/RD cell, derives from ATCC) from different dilution serum, the infection conditions according to cell judges tiring of serum neutralizing antibody, calculates the GMT(geometrical mean of each group of serum).
The inventive method is also optional uses following immune programme for children: immune programme for children is 0 day, 14 days, 28 days immune 3 times respectively, and blood sampling in the 42nd day, measures the NAT of serum.
Inactivated vaccine of the present invention, its adjuvant is aluminium hydroxide.
Further, the invention provides the immunogenic detection method of CA16 viral inactivation vaccine, by CA16 viral inactivation vaccine immunity Wistar rat, SD rat or BrownNorway rat, immunization route is lumbar injection, intramuscular injection or subcutaneous injection, and immunizing dose is that every rat injects the inactivated vaccine containing 25 ~ 1600UCA16 antigen at every turn; Immune programme for children is 0 day, 14 days immune 2 times respectively, blood sampling in the 28th day, or 0 day, 14 days, 28 days immune 3 times respectively, blood sampling in the 42nd day, the NAT of survey serum.
Present invention also offers a kind of detection method of CA16 viral immunogenic, by CA16 virus inoculation rat, detected the immunogenicity of CA16 virus by the NAT measuring rat blood serum.
Further, the method detecting CA16 viral immunogenic is that route of inoculation is lumbar injection, intramuscular injection or subcutaneous injection by CA16 virus inoculation Wistar rat, SD rat or BrownNorway rat; Immune programme for children is 0 day, 14 days immune 2 times respectively, blood sampling in the 28th day, or, difference immunity in the 0th day, 14 days, 28 days 3 times, blood sampling in the 42nd day, the NAT of survey serum.
Above-mentioned immunizing dose, those skilled in the art can experimentally the sex of rat, weight, age in days carry out routine in conjunction with prior art and select.
For serum NAT, those skilled in the art think that antibody horizontal is not less than 1:40 and then thinks that generation has the antibody of protectiveness under normal circumstances, and also namely immunogen has good immunogenicity.
The inactivated vaccine immunogenicity that the present invention utilizes experimental rat to carry out containing CA16 antigen carries out evaluation study, the immunogenic effect that early-stage Study result shows to apply mice, rabbit, sheep evaluate CA16 vaccine is all undesirable, and it is relatively responsive as the immunogenicity of model evaluation CA16 vaccine to apply rat, and have can repeatedly immunity, sustainable advantage of observing antibody variation trend after immunity.The inventive method has a good application prospect in the immunogenic detection of CA16 viral inactivation vaccine.
Detailed description of the invention
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art.If do not specialize, biochemical reagents used in embodiment are commercially available.The present invention use CA16 Strain (strain preserving number is: CGMCCNo.5373) open in Chinese patent CN102559606A.The CA16 inactivated vaccine related in embodiment and EV71-CA16 inactivated vaccine are produced by Beijing Kexing Biotech Products Co., Ltd.
The foundation of embodiment 1 immunogenicity methods and checking
The present invention is the laboratory animals such as rat, mice, sheep, rabbit by the immunity simultaneously of the three batches of CA16 inactivated vaccines, and carry out evaluation antibody horizontal in conjunction with the serum neutralizing antibody detection method of cellular level, shown in being specifically implemented as follows:
1, applying mice is that laboratory animal detects CA16 inactivated vaccine immunogenicity
Laboratory animal: ICR mice, B/c mice, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
Adjuvant types: aluminum hydroxide adjuvant
Immunization ways: lumbar injection
Immune programme for children: two pin immunity in 0 day, 14 days, blood sampling in 28 days, measures serum titer.
Immunizing dose: 0.5ml//time, containing CA16 antigen 400U in 0.5ml vaccine.Result of the test is in table 1:
Serum NAT after table 1CA16 inactivated vaccine immune mouse
Found that, CA16 vaccine immunogenicity is evaluated in application mice (ICR, B/c), two pin immunity, and 28 days blood sampling separation of serum, measure the neutralizing antibody of blood, by serum doubling dilution, and CA16 virus (100CCID
50/ 0.05ml) after 37 DEG C of neutralization reactions, inoculate sensitive cells (Vero/RD cell, derives from ATCC) from different dilution serum, the infection conditions according to cell judges tiring of serum neutralizing antibody, calculates the GMT(geometrical mean of each group of serum).In this area; it has been generally acknowledged that neutralizing antibody level is more than or equal to 1:8 is antibody positive; be more than or equal to 1:40 to protect for there being antibody; the neutralizing antibody total water that the immunogenicity that this application two Strains of Mouses evaluate CA16 vaccine measures on average is less than 1:40, shows that mice is not suitable for the animal model evaluated as CA16 vaccine immunogenicity in this test.
2, applying new zealand rabbit is that laboratory animal detects CA16 vaccine immunogenicity
Laboratory animal: new zealand rabbit, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
Adjuvant types: aluminum hydroxide adjuvant
Immunization ways: neck dorsal sc multi-point injection
Immune programme for children: two pin immunity in 0 day, 14 days, blood sampling in 28 days, measures serum titer.
Immunizing dose: 2ml//time, containing CA16 antigen 1 600U in 2mlCA16 inactivated vaccine.
Result of the test is in table 2:
Serum titer after table 2CA16 vaccine immunity new zealand rabbit
Found that: application new zealand rabbit evaluates 3 batches of CA16 vaccine immunogenicities, two pin immunity, blood sampling in 28 days measures the neutralizing antibody that NAT method measures with immunogenicity that above-mentioned 1, this application new zealand rabbit of application rat evaluation CA16 vaccine immunogenicity evaluate CA16 vaccine and is all less than or equal to 1:40, overall GMT level is not high, this test shows that the sensitivity of new zealand rabbit to CA16 vaccine is poor, rabbit individuality is larger simultaneously, cost is higher, is not suitable for the laboratory animal evaluated as CA16 vaccine immunogenicity.
3, applying sheep is that laboratory animal detects CA16 vaccine immunogenicity
Laboratory animal: sheep, female, about 20kg, the market of farm produce is bought.
Adjuvant types: aluminum hydroxide adjuvant
Immunization ways: intramuscular injection
Immune programme for children: two pin immunity in 0 day, 14 days, blood sampling in 28 days, measures serum titer.
Immunizing dose: 2ml//time, containing CA16 antigen 1 600U in 2mlCA16 inactivated vaccine.
Result of the test is in table 3:
Serum NAT after table 3CA16 vaccine immunity sheep
Result of study finds: application sheep evaluates 3 batches of CA16 vaccine immunogenicities, two pin immunity, within 28 days, measure neutralizing antibody, overall neutralizing antibody level is all less than or equal to 1:40, show the sensitivity of CA16 vaccine inadequate, sheep belongs to large individual animals simultaneously, and cost is higher, is not suitable for the laboratory animal evaluated as CA16 vaccine immunogenicity.
4, applying rat is that laboratory animal detects CA16 inactivated vaccine immunogenicity
Laboratory animal: Wistar rat and SD rat, purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
Adjuvant types: aluminum hydroxide adjuvant
Immunization ways: lumbar injection
Immune programme for children: two pin immunity in 0 day, 14 days, blood sampling in 28 days, measures serum titer.
Immunizing dose: 0.5ml//time, 0.5mlCA16 inactivated vaccine is containing CA16 antigen 400U.
Result of the test is in table 4:
Serum titer after table 4CA16 vaccine immunity rat
Found that: application rat evaluates many batches of CA16 vaccine immunogenicities, measure NAT respectively, result shows that overall neutralizing antibody level is all more than or equal to 1:40, show the sensitivity of CA16 vaccine better, rat individuality is moderate simultaneously, and cost is lower, and is suitable for repeatedly immunity, the dynamic antibody variation trend of observable is that CA16 vaccine immunogenicity evaluates desirable laboratory animal.
Embodiment 2 is applied rat and is detected the immunogenic method of CA16 viral inactivation vaccine
Experimental animal: Wistar
Vaccines classes: CA16 viral inactivation vaccine, its adjuvant is aluminium hydroxide, is produced by Beijing Kexing Biotech Products Co., Ltd.
Immunization ways: lumbar injection
Immune programme for children: a pin immunity in 0 day, blood sampling in 14 days, measures serum titer; Two pin immunity in 0 day, 14 days, blood sampling in 28 days, measures serum titer; Three pin immunity in 0 day, 14 days, 28 days, blood sampling in 42 days, measures the NAT of serum.
Immunizing dose: 0.5ml/ only/time, in 0.5mlCA16 inactivated vaccine containing CA16 antigen amount and result of the test in table 5:
The GMT of the serum titer after table 5CA16 viral inactivation vaccine immune rat
Result illustrates: application rat evaluates the CA16 inactivated vaccine immunogenicity under Different immunizing schedule, adopt three kinds of immune programme for children, measure neutralizing antibody respectively, under the identical immune programme for children of Isodose, comparatively mice, sheep, rabbit are high for neutralizing antibody level, and present good dose-effect relationship with immunizing dose.Show the sensitivity of CA16 vaccine better, rat individuality is moderate simultaneously, and cost is lower, and is suitable for repeatedly immunity, and the dynamic antibody variation trend of observable, illustrates that rat is that CA16 vaccine immunogenicity evaluates desirable laboratory animal.
The immunogenicity that embodiment 3 applies mice, rabbit detects CA16 antigen in EV71-CA16 inactivated vaccine
Experimental animal: ICR mice, new zealand rabbit, all purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
Adjuvant types: aluminum hydroxide adjuvant
Immunization ways: mice adopts lumbar injection, new zealand rabbit adopts neck dorsal sc multi-point injection
Immune programme for children: two pin immunity in 0 day, 14 days, blood sampling in 28 days, measures the NAT of serum.
Immunizing dose: mice 0.5ml/ only/time, new zealand rabbit 2ml/ only/time, in every 0.5mlCA16 inactivated vaccine containing CA16 antigen and serum titer geometrical mean in table 6:
The GMT of the serum titer for CA16 antigen after table 6EV71-CA16 inactivated vaccine immunity ICR mice, new zealand rabbit
Result shows: application ICR mice and new zealand rabbit evaluate the immunogenicity of CA16 in EV71-CA16 bivalent inactivated vaccine, overall neutralizing antibody level is all less than or equal to 1:40, show the sensitivity that CA16 antigen immunogenicity is detected equally inadequate, reconfirm mice, new zealand rabbit is not suitable as CA16 inactivated vaccine or the experimental animal of CA16 Evaluation of Immunogenicity in EV71-CA16 bivalent inactivated vaccine.
Embodiment 4 applies the immunogenicity that rat detects CA16 antigen in EV71-CA16 inactivated vaccine
Experimental animal: SD rat
Adjuvant types: aluminum hydroxide adjuvant
Immunization ways: lumbar injection
Immune programme for children: two pin immunity in 0 day, 14 days, blood sampling in 28 days, measures serum NAT.
Immunizing dose: 0.5ml//time, 0.5ml is containing CA16 antigen 400U.Immunizing dose and serum titer geometrical mean are in table 7.
The GMT of the serum titer for CA16 antigen after table 7EV71-CA16 inactivated vaccine immune rat
Result shows: application rat evaluates the immunogenicity of CA16 in EV71-CA16 bivalent inactivated vaccine, compared to mice and new zealand rabbit, has higher sensitivity.This method can be special the neutralizing antibody detected for CA16, antibody horizontal and immunizing dose present relevant dose-effect relationship, and suitable with the antibody horizontal produced under the identical immune programme for children of CA16 vaccine Isodose.May be used for the immunogenicity evaluating CA16 in EV71-CA16 bivalent inactivated vaccine.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (4)
1. the detection method of CA16 antigen immunogenicity in inactivated vaccine, is characterized in that, containing the inactivated vaccine immune rat of CA16 antigen, will detect CA16 viral inactivation vaccine immunogenicity by the NAT measuring rat blood serum;
Wherein, described rat is Wistar rat, SD rat or BrownNorway rat; Described immunization route is lumbar injection, intramuscular injection or subcutaneous injection; Immunizing dose is at every turn the inactivated vaccine of every rat injection containing the CA16 antigen of 25 ~ 1600U;
Immune programme for children is 0 day, 14 days immune 2 times respectively, and blood sampling in the 28th day, surveys tiring of serum neutralizing antibody; Or immune programme for children is 0 day, 14 days, 28 days immune 3 times respectively, blood sampling in the 42nd day, surveys tiring of serum neutralizing antibody.
2. detection method as claimed in claim 1, it is characterized in that, the adjuvant of described inactivated vaccine is aluminium hydroxide.
3. detection method as claimed in claim 1, is characterized in that, described inactivated vaccine is CA16 inactivated vaccine or EV71-CA16 virus bivalent inactivated vaccine.
4. a detection method for CA16 viral immunogenic, is characterized in that, by CA16 virus inoculation rat, is detected the immunogenicity of CA16 virus by the NAT measuring rat blood serum;
Wherein, described rat is Wistar rat, SD rat or BrownNorway rat; Described immunization route is lumbar injection, intramuscular injection or subcutaneous injection; Immunizing dose is at every turn the inactivated vaccine of every rat injection containing the CA16 antigen of 25 ~ 1600U;
Immune programme for children is 0 day, 14 days immune 2 times respectively, and blood sampling in the 28th day, surveys tiring of serum neutralizing antibody; Or immune programme for children is 0 day, 14 days, 28 days immune 3 times respectively, blood sampling in the 42nd day, surveys tiring of serum neutralizing antibody.
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