CN114748614A - Method for immunizing animals by using needleless injector - Google Patents
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Abstract
The invention discloses a method for immunizing animals by using a needleless injector, which comprises the following steps: s1, preparing antigen mixed liquor, wherein the antigen mixed liquor comprises a DNA expression plasmid, a CPG-ODN adjuvant and normal saline, and the DNA expression plasmid is used as a specific antigen; s2, carrying out needleless injection immunization on the immunized animal, and injecting the prepared antigen mixed solution into the immunized animal by using a needleless injector to enable the immunized animal to generate immunoreaction and generate an antibody aiming at the specific antigen; s3, repeating the needleless injection immunization, and detecting the antibody titer of the specific antigen in the serum of the immunized animal after 2-6 times of needleless injection immunization. The immunization method provided by the invention greatly improves the immunoreaction of animals, provides more opportunities for the research and development of monoclonal antibody medicines, simplifies the administration process by the immunization mode of needleless injection, reduces the pain and complications of experimental animals, and can also improve the occupational safety of experimental operators.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for immunizing animals by using a needleless injector.
Background
DNA immunization is a new immunological theory and technology that was established and developed in the early 90 s. Plasmid DNA can be present in the cells of a recipient in a circular, non-integrated, non-replicating state for up to 1 month. The plasmid DNA can be directly taken up by the cells in the body of the animal of the receptor and express the protein of the gene product, thereby laying the foundation of DNA immunity. Through DNA immunization technology, the target gene DNA can directly generate antigen in a body of a receptor animal, stimulate the body of the receptor animal to generate immune response to target gene protein and generate antibody. The animals with the immune response can be further used for producing serum antibodies of the animals and can also be used for developing monoclonal antibody medicaments.
The use of DNA immunization techniques eliminates the need for in vitro production and purification of protein antigens, which has led to the implementation and implementation of many projects where it has previously been difficult to obtain purified protein antigens using conventional in vitro protein expression methods. Therefore, the work of protein expression and purification is reduced, and the cost of manpower and reagent materials is saved. The antibody produced by DNA immunization recognizes structural proteins in native conformation, and the affinity of the antibody produced by the antigen expressed in vivo is higher. Compared with the traditional protein immunization, the complex protein purification process is avoided. Furthermore, the expressed protein can maintain the native structure of the protein to the maximum extent, which is very important for the production of high affinity therapeutic antibodies that recognize the native conformation of the target antigen. Most of the existing DNA immunization methods adopt a conventional needle injection method to immunize animals, and for some antigens with lower immunogenicity, the conventional immunization cannot produce ideal antibody serum titer, so that the requirement of developing new antibody medicines cannot be met.
The principle of action of needleless injection immunization DNA is as follows: the major antigen presenting cells in the epidermis, Langerhans Cells (LCs), serve the dual functions of immune surveillance and antigen presentation, and needleless injectors deliver DNA antigens to the skin under high pressure, where they are taken up by LCs, subsequently migrate to draining lymph nodes carrying the antigens, present the antigens to T cells, activate CD4+ and CD8+ T cells, perform effector functions at peripheral and mucosal sites, and activate B cells to produce systemic and mucosal antibodies. The stimulation of the antigen given to the dermis and the subcutaneous tissue can increase the targeting effect of the APCs and improve the immune response, the antigen injected into the dermis tissue has more types and quantity than the antigen generated in the intramuscular injection, because the quantity of the APCs is more, the larger dispersion of the antigen increases the surface area contacted with the APCs when the needleless injector is used for immunizing the animal, and the antigen enters the animal body to be distributed in a block shape when the conventional needle injector is used for immunizing the animal, thereby greatly reducing the dispersion of the antigen and the surface area contacted with the APCs.
The needleless injector for immunization is a spray gun, and the antigen is made to reach a plurality of tissue parts including epidermis, dermis, subcutaneous tissue and muscle through a tiny orifice under high pressure. The basic components of the medicine spray nozzle comprise a power source (spring) piston, a medicine chamber and a spray nozzle. The action process of the needleless injection immunization comprises the following steps: when the activation device is triggered, the power source pushes the piston to act on the medicine chamber to quickly form pressure, the speed of the medicine passing through the nozzle reaches 100-200 m/s, the medicine collides with the skin and then penetrates the skin through a stream of superfine liquid jet flow containing the antigen, the whole process is quickly finished within 100 mu s, and the jet flow is dispersed in the skin in a multidirectional way and presented to a plurality of tissue parts. The process comprises 3 stages: in the peak pressure period, the administration period (dispersion period) and the dissipation period, as the jet flow forms a passage in the tissue by itself, the resistance of the passage is small, so that the antigen can be dispersed widely and presents cobweb-shaped distribution, the antigen can be dispersed in the tissue by a small force in the dispersion period, and the contact probability of the antigen and Antigen Presenting Cells (APCs) can be increased by the DNA antigen with a wide dispersion area.
The conventional animal immunization method uses a conventional needle injector, generally needs a large DNA antigen dose, is complex to operate, and has low vector delivery efficiency and in vivo expression level, so that an animal body can generate low immune reaction, and the requirement of antibody drug discovery cannot be met. The experiment process needs to use the sharp ware of syringe needle to puncture, causes the animal blooding and subcutaneous damage, produces complication such as induration to the operation process has increased the probability that the experimenter received the injury. Therefore, immunization of animals using needleless injectors has become a method of DNA immunization, preferably injection. However, the conventional needleless injector for animal use is usually large in size, expensive and cumbersome to operate, and needs additional power gas as a power source, which are not favorable for the development of experiments and cannot meet the requirement of a certain experimental throughput.
Therefore, it is necessary to develop an animal immunization method with convenient operation, low price and good immune response effect, which can avoid complications of experimental animals, ensure safety of experimenters, and simultaneously can ensure high delivery efficiency and high in vivo expression level of antigen vectors.
Disclosure of Invention
In order to solve the technical problem, the invention provides a method for immunizing animals by using a needleless injector, which comprises the following steps of:
S1, preparing an antigen mixed solution, wherein the antigen mixed solution comprises a DNA expression plasmid, a CPG-ODN adjuvant and normal saline, and the DNA expression plasmid is used as a specific antigen;
s2, carrying out needleless injection immunization on the immunized animal, and injecting the prepared antigen mixed solution into the immunized animal by using a needleless injector to enable the immunized animal to generate immunoreaction and generate an antibody aiming at the specific antigen;
s3, repeating the needleless injection immunization, and detecting the antibody titer of the immune animal serum aiming at the specific antigen after 2-6 times of needleless injection immunization.
Specifically, in S1, the amount of the CPG-ODN adjuvant is 10 μ g per experimental animal.
Specifically, in S1, the dosage of the DNA expression plasmid is 50-300 mug per experimental animal.
Specifically, in S1, the saline is a sodium chloride solution with a concentration of 0.9%, and the saline is used to dilute the antigen mixture and supplement the volume of the antigen mixture.
Specifically, in S2, the needleless injector is a human insulin needleless injector, and the human insulin needleless injector includes a pusher, a drug tube and a drug taking port, and the drug tube is used for loading the antigen mixture.
Specifically, in S2, the immune animal is an SPF-grade experimental animal, which includes but is not limited to SD rat, OMT rat and experimental-grade mouse.
Specifically, in S2, the needleless injection refers to the injection immunization of the experimental animal at a plurality of administration sites, including a back subcutaneous site, a back intradermal site and a back muscle site.
Specifically, in S3, the repeated needleless injection immunization refers to performing needleless injection immunization on the experimental animal every 7-14 days.
Preferably, in S3, the repeated needleless injection immunization refers to performing one needleless injection immunization on the experimental animal every 14 days.
Specifically, in S3, the step of detecting the antibody titer against the specific antigen in the serum of the immunized animal refers to preparing the serum from the tail vein blood of the experimental animal, and detecting the antibody titer against the specific antigen in the serum of the immunized animal by using an indirect ELISA method.
The invention uses a needleless injection mode to replace a needle injector, and uses a human-used insulin needleless injector to replace a needleless injector for animals. The human insulin needleless injector has the advantages of small and light specification, convenient operation and no need of additional power gas. The syringe was made non-invasive by making a micropore in the skin, directly through the skin and corresponding tissue layers, into a 0.05mL solution at the injection site. The immune animal is proved to be capable of greatly reducing the dosage of the antigen and obviously improving the immune response of the animal. The immune process has little damage to the skin, avoids complications such as bleeding, subcutaneous injury, induration and the like caused by repeated puncture of a needle head, and better accords with animal welfare policies. Meanwhile, the treatment of needles and harmful waste materials is avoided, and the environment is not damaged.
The human insulin needleless injector is inoculated with less antigens and can generate similar or higher immune response reaction, the dispersion degree of the antigens is improved, and the antigens can be better contacted with immune cells.
Compared with the prior art, the invention has the beneficial effects that:
1. the human-used needleless injector used in the invention has the advantages that the energy generated by the injection system can inject the antigen into tissues with different depths from dermis to muscle, the immune response of experimental animals can be obviously increased, the specific antibody titer in serum is improved, the serum titer higher than that of the conventional DNA injection immunity is obtained, more animal tissue materials are provided, more specific high-affinity monoclonal antibodies aiming at the target are obtained after cell fusion, the foundation is laid for the research and development of new antibody medicines, and more opportunities are provided for the selection of subsequent antibody medicines.
2. The invention uses the human insulin needleless injector, the required DNA immunization dose is greatly reduced compared with the conventional immunization method, and the required DNA immunization dose is 1/4-1/6 of the conventional immunization DNA expression plasmid, so the experiment cost can be greatly saved.
3. The method provided by the invention needs less DNA for animal immunization, so that the period for producing a small amount of DNA is shorter, the operation is more convenient and faster, the material preparation time is greatly shortened, and the experiment cost is saved.
4. The invention uses a needleless injector to deliver low-dose DNA to immunize experimental animals, so that a plurality of projects which are difficult to immunize by using a traditional injector to improve the antibody titer in the serum of the animals are carried out and implemented.
5. For some difficult targets, the needleless injection immunization technology provided by the invention provides more opportunities for the subsequent discovery of monoclonal antibody medicines.
6. Compared with the monoclonal antibody produced by the conventional DNA immunization, the monoclonal antibody produced by the invention has the advantages of high affinity, and especially for producing membrane protein or other antigens which are difficult to produce ideal antibodies, the DNA immunization by needle-free injection is the most ideal immunization mode.
7. The invention uses the human-used needleless injector to deliver the DNA antigen, simplifies the administration process, reduces the pain of animals, and simultaneously avoids the risk of injury of experimenters by the needle injector due to misoperation, thereby improving the occupational safety of the experimenters.
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FIG. 1 shows the third blood serum titer test results of 8 SD rats in the first experiment of the first embodiment of the present invention;
FIG. 2 shows the third blood serum titer test results of 8 SD rats in the second experiment in the first embodiment of the present invention;
FIG. 3 shows the second blood serum titer test results of 8 SD rats in the third experiment in the first embodiment of the present invention;
FIG. 4 shows the third blood serum titer test results of 4 SD rats in experiment four in the first embodiment of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention. Materials, instruments, reagents and the like used in the following examples are commercially available unless otherwise specified. The technical means used in the examples are conventional means well known to those skilled in the art, unless otherwise specified.
Example one
In a specific embodiment, the SD rat is selected as an immunized animal, and the same or different immunization doses are set for the immunoreaction test to compare the effect of the needle-injected immunoreaction with the effect of the needle-free immunoreaction provided by the invention.
Experiment one:
8 SD rats were randomly divided into 2 groups, and each four rats were divided into a group A (needle-free injection group) and a group B (needle-injection group). The immunization dose of the group A and the group B is 200 mu g/animal, and the animal numbers of each group are respectively rat 1#, rat 2#, rat 3#, and rat 4 #. After a week of observation and quarantine, the animals are adapted to a new environment and can be immunized under a healthy state.
The animals in each group were subjected to a blank whole blood (Pre-dark) in tail vein before immunization, followed by whole blood after each two immunizations, and serum was collected by centrifugation at 8000rpm for 5 min. Respectively using ELISA method to determine antibody titer of anti-specific antigen in rat serum of each group, and A in experimental result450The reading value is 3 times or more than 3 times of the reading value of the control hole, and the positive reaction is obtained.
The immunization schedule for group a was:
1) mixing the DNA, the CPG-ODN adjuvant and the normal saline to prepare a mixed solution; wherein the DNA is 200. mu.g/rat, the CPG-ODN adjuvant is 10. mu.g/rat, and the volume is supplemented with physiological saline to the required volume.
2) Performing needleless injection immunization on the back of an immunized animal by using the mixed solution, performing immunization again by using DNA antigen after 14 days, collecting mouse whole blood in a tail vein manner on 7 days after 2 times of immunization, and collecting serum, wherein the sequence is 1st-bleed， 2ndBlank … … and detecting the antibody titres against the specific antigen in the serum of the immunized animals.
The immunization schedule for group B was:
1) mixing the DNA, the CPG-ODN adjuvant and the Aluminum phospate adjuvant to prepare a mixed solution; wherein the DNA is 200 mug/rat, the CPG-ODN adjuvant is 10 mug/rat, the volume of the total volume is half of the volume of the DNA, and the volume of the dosage of the Aluminum Phosphate adjuvant is 1/2 of the total volume of the immune material.
2) Injecting the mixture solution into biceps femoris, calf gastrocnemius muscle, waist and hip of immunized animal, immunizing again with DNA antigen after 14 days, collecting whole blood of mouse in tail vein mode on 7 days after 2 times of immunization, and collecting serum 1st-bleed，2ndBlank … … and detecting the antibody titres against the specific antigen in the serum of the immunized animals.
As shown in FIG. 1, the result of the serum titer test of the third blood collection of 8 SD rats shows that the antibody titer generated in SD rats by the needleless injection for animal immunization against specific antigen is obviously superior to that of the immune reaction by the needle injection.
Experiment two:
8 SD rats were randomly divided into 2 groups, and each four rats were divided into a group A (needle-free injection group) and a group B (needle-injection group). The groups A and B were divided into a high dose immunization group and a low dose immunization group. The animal numbers of each group are respectively rat 1#, rat 2#, rat 3#, and rat 4#, wherein rat 1# and rat 2# belong to a high dose group (200 mug/rat), rat 3#, and rat 4# belong to a low dose group (50 mug/rat), and after a week of observation and quarantine period, the animal is adapted to a new environment and can be immunized under a healthy state. The animals in each group were subjected to tail vein collection of blank whole blood (Pre-fed) followed by whole blood after each two immunizations and serum was collected by centrifugation at 8000rpm for 5min before immunization. ELISA was used to determine the antibody titer against specific antigens for each rat serum, A in the experimental results 450The reading value is 3 times or more than 3 times of the reading value of the control hole, and the positive reaction is obtained.
The immunization schedule for group a was:
1) mixing the DNA, the CPG-ODN adjuvant and the normal saline to prepare a mixed solution; wherein, the rat 1#, rat 2# DNA is 200 mu g/rat, rat 3#, rat 4# DNA is 50 mu g/rat, and CPG-ODN adjuvant is 10 mu g/rat, and the required volume is supplemented by physiological saline.
2) The mixture is used for carrying out needleless injection immunization on the back of an immunized animal, DNA antigen is used for carrying out immunization again after 14 days, and after 2 times of immunization, tail silence is carried out on the 7 th dayThe pulse method comprises collecting mouse whole blood and serum, sequentially 1st-bleed， 2ndBleed … … and detecting the antibody titres against the specific antigen in the serum of the immunized animal.
The immunization schedule for group B was:
1) mixing the DNA, the CPG-ODN adjuvant and the Aluminum phospate adjuvant to prepare a mixed solution; wherein, the rat 1#, rat 2# DNA is 200 mu g/rat, rat 3#, rat 4# DNA is 50 mu g/rat, CPG-ODN adjuvant is 10 mu g/rat, the volume of the adjuvant is half of the total volume, and the volume of the adjuvant is 1/2 of the total volume of the immune material.
2) Injecting the mixture solution into biceps femoris, calf gastrocnemius muscle, waist and hip of immunized animal, immunizing again with DNA antigen after 14 days, collecting whole blood of mouse in tail vein mode on 7 days after 2 times of immunization, and collecting serum 1 st-bleed，2ndBlank … … and detecting the antibody titres against the specific antigen in the serum of the immunized animals.
As shown in FIG. 2, the serum titer test results of the third blood collection of 8 SD rats, the animals are immunized by the (low dose and high dose) needleless injection, and the antibody titer generated by the SD rats in vivo aiming at the specific antigen is obviously superior to that of the immune reaction by the needle injection. It can be seen that the use of low doses of needleless injection can reduce the amount of antigen used to a great extent.
Experiment three:
8 SD rats were randomly divided into 2 groups, and each 4 rats were assigned to one group, namely group A (needle-free low dose group, 50. mu.g/rat) and group B (needle-free high dose group, 200. mu.g/rat). The animal numbers of each group are respectively rat 1#, rat 2#, rat 3#, and rat 4#, after a week of observation and quarantine period, the animal can adapt to new environment and can implement immunity under a healthy state. The animals in each group were subjected to tail vein collection of blank whole blood (Pre-fed) followed by whole blood after each two immunizations and serum was collected by centrifugation at 8000rpm for 5min before immunization. The antibody titer against specific antigen in each rat serum was determined by ELISA method, and the A450 reading in the experimental results was 3 times or more than 3 times positive to that in the control wells.
The group a immunization schedule was:
1) mixing the DNA, the CPG-ODN adjuvant and the normal saline to prepare a mixed solution; wherein the DNA is 50. mu.g/rat, the CPG-ODN adjuvant is 10. mu.g/rat, and the volume is supplemented with physiological saline to the required volume.
2) Performing needleless injection immunization on the back of an immunized animal by using the mixed solution, performing immunization again by using DNA antigen after 14 days, collecting mouse whole blood in a tail vein manner on 7 days after 2 times of immunization, and collecting serum, wherein the sequence is 1st-bleed， 2ndBleed … … and detecting the antibody titres against specific antigens in the sera of the immunized animals.
The group B immunization protocol was:
1) mixing the DNA, the CPG-ODN adjuvant and the Aluminum phospate adjuvant to prepare a mixed solution; wherein the DNA is 200 mug/rat, the CPG-ODN adjuvant is 10 mug/rat, the volume of the total volume is half of the volume of the DNA, and the volume of the dosage of the Aluminum Phosphate adjuvant is 1/2 of the total volume of the immune material.
2) Injecting the mixture solution into biceps femoris, calf gastrocnemius muscle, waist and hip of immunized animal, immunizing again with DNA antigen after 14 days, collecting whole blood of mouse in tail vein mode on 7 days after 2 times of immunization, and collecting serum 1st-bleed，2ndBleed … … and detecting the antibody titres against specific antigens in the sera of the immunized animals.
As shown in figure 3, the result of the second blood serum titer detection of 8 SD rats is obtained, and the SD rats are immunized by low-dose needleless injection, and the antibody titer generated in vivo by the SD rats to specific antigens is obviously superior to that of the immune reaction by needle injection. It can be seen that the use of low doses of needleless injection can greatly reduce the amount of antigen used.
Experiment four:
4 SD rats were randomly divided into 2 groups, one group of 2 rats was assigned to each group, namely group A (needle-free low dose group, 50. mu.g/rat) and group B (needle-free high dose group, 300. mu.g/rat). The animal numbers of group A are rat 1#, rat 2#, and the animal numbers of group B are rat 1#, rat 2#, after a week observation and quarantine period, the animal can adapt to new environment and can implement immunity under the healthy state. The animals in each group were subjected to tail vein collection of blank whole blood (Pre-fed) followed by whole blood after each two immunizations and serum was collected by centrifugation at 8000rpm for 5min before immunization. The antibody titer against specific antigen in each rat serum was determined by ELISA method, and the A450 reading in the experimental results was 3 times or more than 3 times positive to that in the control wells.
Group a immunization protocol:
1) mixing the DNA, the CPG-ODN adjuvant and normal saline to prepare a mixed solution; wherein the DNA is 50. mu.g/rat, the CPG-ODN adjuvant is 10. mu.g/rat, and the required volume is supplemented with physiological saline.
2) The mixed solution is used for carrying out needleless injection immunization on the back of an immunized animal, DNA antigen is used for carrying out immunization again after 14 days, and mouse whole blood is collected in a tail vein mode on the 7 th day after 2 times of immunization and serum is collected, wherein the sequence is 1st-bleed， 2ndBlank … … and detecting the antibody titres against the specific antigen in the serum of the immunized animals.
Group B immunization protocol:
1) mixing the DNA, the CPG-ODN adjuvant and the Aluminum Phosphate adjuvant to prepare a mixed solution; wherein the DNA is 200 mu g/rat, the CPG-ODN adjuvant is 10 mu g/rat, the volume of the DNA is half of the total volume of the materials, and the volume of the adjuvant of Aluminum phosphor is 1/2 of the total volume of the materials.
2) Injecting the mixture solution into biceps femoris, calf gastrocnemius muscle, waist and two sides of recommended hip of immunized animal, immunizing again with DNA antigen after 14 days, collecting whole blood of mouse in tail vein manner on 7 days after 2 times of immunization, and collecting serum 1st-bleed，2ndBlank … … and detecting the antibody titres against specific antigens in the sera of the immunized animals.
As shown in FIG. 4, the serum titer detection result of the third blood collection of 4 SD rats adopts low-dose needle-free injection for animal immunization, and the antibody titer generated in SD rats to specific antigens is still obviously superior to that of the immune reaction of the injection with a needle. It can be seen that the use of low doses of needleless injection can greatly reduce the amount of antigen used.
The following table shows the serum titer data of each experimental animal group:
in the above table,/is the group in which immunization and blood collection were not performed.
In conclusion, compared with the injection with a needle, the needleless injection immunization method provided by the invention can greatly improve the immune response of animals, greatly reduce the antigen amount used by the immunized animals, and thus play a role in reducing the cost.
The results of hybridoma fusion screening of 2 rats of the needleless injection group in experiment three are shown in fig. 5, and it can be seen that the effect of hybridoma fusion screening obtained by immunoreacting SD rats through needleless injection is good, while the SD rats immunoreacting through conventional needle injection in experiment three do not produce ideal specific antibodies, and thus hybridoma fusion experiments cannot be performed.
In summary, the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A method of immunizing an animal with a needleless injector, comprising the steps of:
S1, preparing an antigen mixed solution, wherein the antigen mixed solution comprises a DNA expression plasmid, a CPG-ODN adjuvant and normal saline, and the DNA expression plasmid is used as a specific antigen;
s2, carrying out needleless injection immunization on the immunized animal, and injecting the prepared antigen mixed solution into the body of the immunized animal by using a needleless injector to enable the body of the immunized animal to generate immunoreaction and generate an antibody aiming at a specific antigen;
s3, repeating the needleless injection immunization, and detecting the antibody titer of the specific antigen in the serum of the immunized animal after 2-6 times of needleless injection immunization.
2. The method for immunizing an animal with a needleless injector according to claim 1, wherein the amount of the CPG-ODN adjuvant used in S1 is 10 μ g per experimental animal.
3. The method for immunizing an animal with a needleless injector according to claim 1, wherein in S1, the amount of the DNA expression plasmid is 50 to 300 μ g per experimental animal.
4. The method for immunizing an animal with a needleless syringe according to claim 1, wherein in S1, the physiological saline is a sodium chloride solution having a concentration of 0.9%, and the physiological saline is used to dilute the antigen mixture and supplement the volume of the antigen mixture.
5. The method for immunizing an animal with a needleless injector according to claim 1, wherein in step S2, the needleless injector is a human insulin needleless injector, and the human insulin injector comprises a pusher, a drug tube and a drug delivery port, and the drug tube is used for loading the antigen mixture.
6. The method of claim 1, wherein in S2 the immunized animal is an SPF-grade laboratory animal including but not limited to SD rats, OMT rats and laboratory-grade mice.
7. The method for immunizing an animal with a needle-free injector according to claim 1, wherein said needle-free injection is injection immunization of a plurality of administration sites including a back subcutaneous site, a back intradermal site and a back muscle site to the experimental animal in S2.
8. The method for immunizing an animal with a needle-free injector according to claim 1, wherein said repeating of the needleless injection immunization in S3 is carried out by performing the needleless injection immunization on the experimental animal every 7-14 days.
9. The method for immunizing an animal with a needleless syringe according to claim 1, wherein in step S3, the step of detecting the antibody titer against the specific antigen in the serum of the immunized animal comprises preparing the serum from the tail vein of the experimental animal and detecting the antibody titer against the specific antigen in the serum of the immunized animal by indirect ELISA.
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| CN (1) | CN114748614A (en) |
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