Method and system for testing in-vitro potency of cat herpesvirus inactivated vaccine

Abstract

The invention provides a method and a system for testing in-vitro potency of a cat herpesvirus inactivated vaccine. The method comprises the following steps: respectively preparing a capture antibody, preparing a cat herpes virus polyclonal antibody and preparing an enzyme-labeled antibody, and generating a detection enzyme-labeled plate based on the cat herpes virus polyclonal antibody; in response to a first incubation operation performed on the captured antibody based on the detection elisa plate, adding a cat herpes virus inactivated vaccine and a reference vaccine to each vaccine hole site of the detection elisa plate, and performing a second incubation operation on the detection elisa plate; and determining the relative potency ratio of the corresponding feline herpesvirus inactivated vaccine based on the absorbance value of each vaccine hole site, and determining the relative potency attribute of the antigen based on the relative potency ratio. The accuracy of the test result is at least improved.

Description

Technical Field

[0001] This invention relates to data processing technology, and more particularly to a method and system for testing the in vitro efficacy of feline herpesvirus inactivated vaccines. Background Technology

[0002] Feline herpesvirus 1 (FHV-1) is a common and highly contagious virus in felines, causing feline viral rhinotracheitis. FHV-1 primarily proliferates in the nose, pharynx, trachea, bronchi, and conjunctiva of cats. It mainly affects kittens, causing upper respiratory tract inflammation, keratoconjunctivitis, and chronic sinusitis. It can also affect the trigeminal and optic nerves. Severe infections present with fever and upper respiratory symptoms, with a morbidity rate reaching 100% and a mortality rate exceeding 50%. Currently, vaccination is a crucial means of preventing and controlling feline herpesvirus disease.

[0003] Currently, several vaccine companies are producing and selling feline panleukopenia, rhinotracheitis, and calicivirus disease trivalent inactivated vaccines (hereinafter referred to as feline trivalent inactivated vaccines). At present, the efficacy testing methods for domestically approved feline trivalent inactivated vaccines are the immune challenge method and the serological method. Although both methods can directly evaluate the in vitro efficacy of the vaccine, the testing process involves animal selection, animal husbandry, immunization, blood collection, and challenge, which is costly. The results are affected by many factors, and the need for challenge during the testing period poses biosafety risks. More importantly, the testing cycle is nearly two months long, which not only prolongs the development cycle of new vaccines but also indirectly shortens the vaccine's shelf life, increases vaccine inventory pressure, and seriously affects vaccine production and sales. Therefore, a new method for testing the relative in vitro efficacy of feline trivalent inactivated vaccines is urgently needed. Summary of the Invention

[0004] In view of the above problems, the present invention is proposed to provide a method and system for testing the in vitro efficacy of feline herpesvirus inactivated vaccine that overcomes or at least partially solves the above problems.

[0005] According to one aspect of the present invention, a method for testing the in vitro potency of a feline herpesvirus inactivated vaccine is provided, comprising the following steps: Capture antibody preparation, feline herpesvirus polyclonal antibody preparation, and enzyme-labeled antibody preparation were performed separately, and a detection enzyme-labeled plate was generated based on the feline herpesvirus monoclonal antibody. The response involves completing the first incubation operation on the captured antibody based on the detection ELISA plate, adding the feline herpesvirus inactivated vaccine and the reference vaccine to each vaccine well of the detection ELISA plate, and then performing a second incubation operation on the detection ELISA plate. The relative potency ratio of the corresponding feline herpesvirus inactivated vaccine was determined based on the absorbance value of each vaccine well, and the relative potency attribute of the antigen was determined based on the relative potency ratio.

[0006] Optionally, in the method according to the invention, the preparation of the capture antibody includes: After the purified feline herpesvirus was mixed with an appropriate amount of adjuvant, experimental mice were immunized. The spleen cells of the immunized mice were then fused with pre-defined myeloma cells, subcloned, and screened to obtain hybridoma cell lines that secrete feline herpesvirus monoclonal antibodies. Hybridoma cell lines were cultured using a bioreactor, and the resulting culture supernatant was purified to obtain capture antibodies.

[0007] Optionally, in the method according to the invention, the method further includes: The capture antibody undergoes a first quality control, which includes morphological testing, sterility testing, neutralizing antibody titer determination, specificity testing, and first storage and shelf life.

[0008] Optionally, in the method according to the invention, the preparation of feline herpesvirus polyclonal antibodies includes: The feline herpesvirus after inactivation was mixed with an appropriate amount of adjuvant and immunized with experimental cats. Blood was collected from the experimental cats after a preset time period. The obtained serum sample is centrifuged at a first preset centrifugation rate, and the obtained initial supernatant sample is inactivated at a first preset temperature for a continuous first preset time to obtain the current supernatant sample. The supernatant sample is purified using salting-out, and then filtered and sterilized using a filter membrane of a pre-defined size to obtain feline herpesvirus polyclonal antibodies. Optionally, in the method according to the present invention, the method further includes: A second quality control was performed on the feline herpesvirus polyclonal antibody, which included phenotype testing, sterility testing, neutralizing antibody titer determination, and second storage and shelf life.

[0009] Optionally, in the method according to the invention, the preparation of enzyme-labeled antibodies includes: A preset weight of horseradish peroxidase is added to a carbonate buffer solution corresponding to a first preset volume, and glutaraldehyde of a first preset concentration is added to the obtained first mixture based on a second preset volume. A second preset volume of sodium chloride buffer solution with a second preset concentration and a third preset volume of pre-cooled anhydrous ethanol are sequentially added to a first mixture that is incubated for a second preset time based on a second preset temperature, and the first mixture is thoroughly mixed. The first mixture is centrifuged at a second preset centrifugation rate for a third preset time, and the first supernatant is removed. Enzyme-labeled antibodies were prepared based on the first supernatant-free solution obtained.

[0010] Optionally, in the method according to the invention, enzyme-labeled antibody preparation is performed based on the obtained first supernatant-free solution, comprising: Anhydrous ethanol of a third preset concentration is added to the first supernatant solution obtained based on the fourth preset volume and then thoroughly mixed. The mixed supernatant-free solution is centrifuged for a third preset time based on a second preset centrifugation rate, and then placed at room temperature for a fourth preset time after removing the second supernatant to obtain a second supernatant-free solution. Add the fifth preset volume of carbonate buffer and the fifth preset volume of feline herpesvirus polyclonal antibody with the fourth preset concentration to the second supernatant-free solution in sequence, and mix at room temperature. Add dipotassium hydrogen phosphate to the second mixture until the corresponding acidity or alkalinity of the second mixture is neutral; The second mixture, after being incubated overnight at a third preset temperature, was dialyzed with phosphate buffer at a fifth preset time, and glycerol with a fifth preset concentration was added to the resulting dialyzing solution. The dialysis solution is dispensed and stored at a fourth preset temperature.

[0011] Optionally, in the method according to the present invention, generating a detection ELISA plate based on feline herpesvirus monoclonal antibody includes: Based on the coating solution, feline herpesvirus monoclonal antibody was prepared into a feline herpesvirus monoclonal antibody solution with a fifth preset concentration, and the updated coating solution was obtained. Add the first preset well volume of coating solution to the initial microplate and perform coating incubation based on the first incubation conditions; The coating solution is removed, and the rinsing operation is performed with phosphate buffer based on a preset number of times. The rinsing operation is completed, the air-drying operation is performed based on absorbent paper, and the skim milk with a mass fraction of the first preset fraction is identified as the sealing liquid; Add the second preset volume of blocking solution to each vaccine well of the initial ELISA plate, and perform a blocking operation for a sixth preset time based on the second preset temperature. The blocking solution is removed, and a rinsing operation is performed based on phosphate buffer based on a first preset number of times; After the rinsing operation is completed, the plate is patted dry using absorbent paper and stored within a preset temperature range to generate a detection ELISA plate.

[0012] Optionally, in the method according to the invention, the method further includes: Based on the strategy of optimizing the coating concentration of capture antibodies, the coating concentration corresponding to the maximum P / N value is determined as the optimal usage concentration; Based on the sealing liquid optimization strategy, the sealing liquid with a corresponding P / N value greater than a preset ratio and sealing cost less than a preset cost is determined as the optimal sealing liquid; Based on the closure time optimization strategy, the closure time corresponding to the maximum P / N value is determined as the optimal closure time; Based on the strategy for optimizing the incubation time of the antigen to be tested, the incubation time of the antigen to be tested corresponding to the maximum P / N value is determined as the optimal incubation time of the antigen to be tested. Based on the enzyme-labeled antibody incubation time optimization strategy, the incubation time of the enzyme-labeled antibody corresponding to the maximum P / N value is determined as the optimal incubation time of the enzyme-labeled antibody; Based on the strategy of optimizing the incubation time of the colorimetric solution, the incubation time of the colorimetric solution corresponding to the maximum P / N value is determined as the optimal incubation time of the colorimetric solution. Based on the strategy for determining the critical value of blank control vaccines, the critical value of the blank control vaccine is determined. Based on the strategy for determining the original multiple critical value of the reference vaccine, the original multiple critical value under the original multiple condition is determined; Based on the specificity testing strategy, the specificity status is determined.

[0013] Optionally, in the method according to the invention, in response to the completion of a first incubation operation on the capture antibody based on the detection ELISA plate, feline herpesvirus inactivated vaccine and reference vaccine are added to each vaccine well of the detection ELISA plate, and a second incubation operation is performed on the detection ELISA plate, including: The capture antibody, diluted to a sixth preset concentration based on the coating solution, is added to the detection ELISA plate based on the first preset well volume, and the coating operation is performed based on the third preset temperature and preset time interval. In response to the current temperature being the same as room temperature, the detection ELISA plate is washed a first preset number of times based on the washing solution at a first preset interval. After the plate washing operation is completed, the plate is patted dry using absorbent paper. Based on the second preset well volume, the blocking solution is added to the detection ELISA plate, and the first incubation operation is performed based on the second preset temperature and the sixth preset time. The detection ELISA plate is washed a first preset number of times at a first preset interval using washing solution. After the plate washing operation is completed, the plate is patted dry using absorbent paper. The feline triple inactivated vaccine, including feline herpesvirus inactivated vaccine, and the reference vaccine were added to each vaccine well of the detection ELISA plate, and the detection ELISA plate was then subjected to a second incubation operation.

[0014] Optionally, in the method according to the present invention, a feline triple inactivated vaccine comprising a feline herpesvirus inactivated vaccine and a reference vaccine are added to each vaccine well of the detection ELISA plate, and the detection ELISA plate is subjected to a second incubation operation, including: The feline triple inactivated vaccine and the reference vaccine were diluted using a dilution plate. The reference vaccine was added to each of the first preset wells based on the volume of the third preset well, and the feline triple inactivated vaccine was added to each of the second preset wells based on the volume of the third preset well. Based on the fourth preset well volume, the washing solution is added to each vaccine well except for each first preset well and each second preset well. The feline triple inactivated vaccine and the reference vaccine were diluted according to preset ratios. Based on the fifth preset well volume, the liquid located in each vaccine well is transferred to the corresponding vaccine well of the ELISA plate coated with capture antibody, and the detection ELISA plate is subjected to a second incubation operation.

[0015] Optionally, in the method according to the invention, a second incubation operation is performed on the detection ELISA plate, including: The detection ELISA plate is subjected to a first incubation operation for a sixth preset time based on a second preset temperature. The detection ELISA plate is washed a first preset number of times at a first preset interval using washing solution. After the plate washing operation is completed, the plate is patted dry using absorbent paper, and horseradish peroxidase-labeled antibody is added based on the first preset well volume. The detection ELISA plate is subjected to a second incubation operation based on a seventh preset time at a second preset temperature. The detection ELISA plate is washed a first preset number of times at a first preset interval using washing solution. After the plate washing operation is completed, the plate is patted dry using absorbent paper. In response to the current temperature being the same as room temperature, the rewarming colorimetric solution is obtained and added to the detection ELISA plate based on the first preset well volume; Based on the eighth preset time of incubation at room temperature in the dark, and based on the sixth preset well volume, the stop solution is added to the detection ELISA plate.

[0016] Optionally, in the method according to the present invention, determining the relative potency ratio of the corresponding feline triple inactivated vaccine based on the absorbance value of each vaccine well, and determining the antigen relative potency attribute based on the relative potency ratio, includes: Place each vaccine well in the ELISA reader and determine the absorbance value of each vaccine well based on the preset wavelength; Determine the average absorbance of the blank control vaccine; The absorbance values ​​of each well site of the vaccine corresponding to different dilutions were calculated by comparing them with the average absorbance value of the blank control vaccine to obtain the corrected absorbance value of each well site of the vaccine and to determine the relative efficacy ratio. The experiment is considered valid if the average absorbance of the blank control vaccine is less than the first preset value and the original absorbance value of the reference vaccine is not less than the second preset value. The third preset value is determined as the reference relative efficacy value of the vaccine. The relative efficacy value of the feline triple inactivated vaccine should not be less than the third preset value. The relative efficacy attribute of feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be a qualified attribute. If the relative potency value of the feline triple inactivated vaccine is less than the third preset value, a retest will be performed. If the relative efficacy value of the response retest result is not less than the third preset value, the relative efficacy attribute of the feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be a qualified attribute; otherwise, the relative efficacy attribute of the feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be a unqualified attribute.

[0017] Optionally, in the method according to the invention, the method further includes: The relative antigenic potency of feline triple inactivated vaccines from the same batch and different batches was determined based on a preset number of doses.

[0018] According to another aspect of the present invention, an in vitro potency testing system for feline herpesvirus inactivated vaccines is provided, comprising: The preparation module is configured to perform capture antibody preparation, feline herpesvirus polyclonal antibody preparation, and enzyme-labeled antibody preparation, and to generate a detection enzyme-labeled plate based on feline herpesvirus monoclonal antibody. The incubation module is configured to, in response to the first incubation operation performed on the captured antibody based on the detection ELISA plate, add the feline herpesvirus inactivated vaccine and the reference vaccine to each vaccine well of the detection ELISA plate, and perform a second incubation operation on the detection ELISA plate. The determination module is configured to determine the relative potency ratio of the corresponding feline herpesvirus inactivated vaccine based on the absorbance value of each vaccine well, and to determine the antigen relative potency attribute based on the relative potency ratio.

[0019] According to the present invention, by preparing capture antibodies, polyclonal antibodies, and enzyme-labeled antibodies in a step-by-step manner, and then combining them with a dedicated detection ELISA plate for detection, the efficacy of feline herpesvirus inactivated vaccines can be accurately assessed, demonstrating significant beneficial effects. Furthermore, by preparing capture antibodies and ELISA antibodies separately, interference from other viruses can be effectively eliminated, greatly improving the specificity of the detection. Moreover, the present invention also strictly controls each incubation step, making the detection results more stable and reliable. Finally, the present invention can calculate the relative efficacy ratio based on absorbance values ​​and determine the relative efficacy attributes of the antigen, achieving a quantitative assessment of vaccine efficacy. This provides a scientific and accurate testing standard for vaccine quality control, ensuring the accuracy and standardization of the test results. Attached Figure Description

[0020] Figure 1 A flowchart is shown below illustrating a method for testing the in vitro potency of a feline herpesvirus inactivated vaccine according to an embodiment of the present invention; Figure 2 A schematic diagram of each of the first preset hole positions according to an embodiment of the present invention is shown; Figure 3 A schematic diagram of each of the second preset hole positions according to an embodiment of the present invention is shown; Figure 4 A structural block diagram of an in vitro efficacy testing system for feline herpesvirus inactivated vaccine, according to another embodiment of the present invention, is shown. Detailed Implementation

[0021] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0022] To address the problems existing in the aforementioned background art, the inventors have proposed the solution of this invention. One embodiment of this invention provides a method for testing the in vitro potency of a feline herpesvirus inactivated vaccine, which can be executed on a computing device.

[0023] Figure 1 A flowchart is shown of a method for testing the in vitro potency of an inactivated feline herpesvirus vaccine according to an embodiment of the present invention, the method being adapted to be performed in a computing device.

[0024] like Figure 1 As shown, the method for testing the in vitro potency of the feline herpesvirus inactivated vaccine proposed in this embodiment begins with step S102, which includes the following: Capture antibody preparation, feline herpesvirus polyclonal antibody preparation, and enzyme-labeled antibody preparation were carried out separately, and detection enzyme-labeled plates were generated based on feline herpesvirus monoclonal antibody. Example 1

[0025] Purified feline herpesvirus was mixed with an appropriate amount of adjuvant and then used to immunize experimental mice. The spleen cells of the immunized mice were then fused with pre-defined myeloma cells, subcloned, and screened to obtain hybridoma cell lines that secrete feline herpesvirus monoclonal antibodies. Hybridoma cell lines were cultured using a bioreactor, and the resulting culture supernatant was purified to obtain capture antibodies.

[0026] The capture antibody undergoes a first quality control, which includes morphological testing, sterility testing, neutralizing antibody titer determination, specificity testing, and first storage and shelf life.

[0027] For example, in this embodiment, the purified feline herpesvirus is first mixed with an appropriate amount of adjuvant and then used to immunize experimental mice, such as BALB / c mice. Then, spleen cells from the immunized mice are fused with predetermined myeloma cells, such as SP2 / 0 myeloma cells.

[0028] Subsequently, after subcloning and screening, the hybridoma cell line 2H12, which can stably secrete FHV monoclonal antibodies, is obtained. At this point, the hybridoma cell line 2H12 is inoculated into a bioreactor for culture, and the culture supernatant is harvested, purified, and used to produce capture antibodies.

[0029] This embodiment enables the acquisition of capture antibodies for a double-antibody sandwich ELISA method used to test the in vitro potency of feline herpesvirus inactivated vaccine in a trivalent inactivated vaccine for feline panleukopenia, rhinotracheitis, and calicivirus disease.

[0030] To ensure the final effect, the capture antibody will also undergo a first quality control, which includes appearance testing, sterility testing, neutralizing antibody titer determination, specificity testing, and first storage and shelf life.

[0031] Specifically, the pass standard for the morphological test is that the capture antibody is a colorless or pale yellow clear liquid and should not be cloudy.

[0032] The sterility test is conducted according to the appendix of the current Chinese Veterinary Pharmacopoeia, Part III. The pass standard is that the captured antibodies should grow sterilely.

[0033] The neutralizing antibody titer was determined according to the appendix of the current Chinese Veterinary Pharmacopoeia, Part III. The qualified standard is that the neutralizing antibody titer of the captured antibody should not be less than 1:16.

[0034] The specificity test involved diluting feline herpesvirus (BJS01 strain), feline parvovirus (HBX05 strain), and feline calicivirus (BJH13 strain) to 200 TCID using DMEM cell culture medium containing 4% bovine serum. 50 After adding 0.1 ml, mix with an equal volume of this product, neutralize at 37°C for 1 hour, and then seed into 96-well cell culture plates (① Samples neutralized with FHV: seeded onto CRFK cells that have formed a monolayer; ② Samples neutralized with FPV: add 100 μl of F81 cell suspension containing 2% bovine serum to each well; ③ Samples neutralized with FCV: seeded onto F81 cells that have formed a monolayer), with each sample seeded into 4 wells, 100 μl per well; normal cell control and virus control were also included (virus (200 TCID)). 50 Mix 0.1 ml of the antibody with an equal volume of DMEM and neutralize at 37°C for 1 hour. Place in 4 wells of each mixture and incubate at 37°C in a 5% CO2 incubator for 4–7 days. The acceptable criteria are: no CPE should appear in the capture antibody neutralization wells with FHV, and CPE should appear in all FPV and FCV neutralization wells; no CPE should appear in the normal cell control wells, and CPE should appear in all FHV, FPV, and FCV virus control wells.

[0035] The capture antibody must be stored below -70°C and has a shelf life of 24 months. Example 2

[0036] The feline herpesvirus after inactivation was mixed with an appropriate amount of adjuvant and immunized with experimental cats. Blood was collected from the experimental cats after a preset time period. The obtained serum sample is centrifuged at a first preset centrifugation rate, and the obtained initial supernatant sample is inactivated at a first preset temperature for a continuous first preset time to obtain the current supernatant sample. The supernatant sample was purified using salting-out, and then filtered and sterilized using a filter membrane of a pre-defined size to obtain feline herpesvirus polyclonal antibodies. A second quality control procedure was performed on the feline herpesvirus polyclonal antibodies, which included phenotypic testing, sterility testing, neutralizing antibody titer determination, and a second storage and shelf-life assessment.

[0037] For example, in this embodiment, the inactivated feline herpesvirus is first mixed with an appropriate amount of adjuvant, then used to immunize test cats, and blood is collected from the test cats after a certain period of time.

[0038] After the blood coagulates and serum is separated, the obtained serum sample will be centrifuged at a first preset centrifugation rate, for example, the first preset centrifugation rate can be 3000 r / min for 5 minutes.

[0039] Then, an initial supernatant sample is collected from the serum sample and inactivated at a first preset temperature for a first preset time to obtain the current supernatant sample. For example, the first preset temperature can be 56°C and the first preset time can be 30 minutes.

[0040] Next, the supernatant sample was purified using salting out. The purified supernatant sample was then filtered and sterilized using a filter membrane of a preset size to obtain feline herpesvirus polyclonal antibodies. For example, the preset size could be 0.22 μm.

[0041] This embodiment provides an enzyme-labeled antibody for testing the in vitro potency of a feline herpesvirus inactivated vaccine using a double-antibody sandwich ELISA method in a trivalent inactivated vaccine for feline panleukopenia, rhinotracheitis, and calicivirus disease.

[0042] To ensure the final effect, a second quality control will be performed on the feline herpesvirus polyclonal antibody. This second quality control includes phenotype detection, sterility testing, neutralizing antibody titer determination, and second storage and shelf life.

[0043] Specifically, the pass standard for morphological testing is that the feline herpesvirus polyclonal antibody should be a colorless or pale yellow liquid and should not be cloudy.

[0044] The sterility test is conducted according to the appendix of the current Chinese Veterinary Pharmacopoeia, Part III. The passing standard is that feline herpesvirus polyclonal antibodies should grow sterilely.

[0045] The neutralizing antibody titer was determined according to the appendix of the current Chinese Veterinary Pharmacopoeia, Part III, and the neutralizing antibody titer should not be lower than 1:16.

[0046] Feline herpesvirus polyclonal antibodies must be stored below -15°C and have a shelf life of 24 months. Example 3

[0047] A preset weight of horseradish peroxidase is added to a carbonate buffer solution corresponding to a first preset volume, and glutaraldehyde of a first preset concentration is added to the obtained first mixture based on a second preset volume. A second preset volume of sodium chloride buffer solution with a second preset concentration and a third preset volume of pre-cooled anhydrous ethanol are sequentially added to a first mixture that is incubated for a second preset time based on a second preset temperature, and the first mixture is thoroughly mixed. The first mixture is centrifuged at a second preset centrifugation rate for a third preset time, and the first supernatant is removed. Enzyme-labeled antibodies were prepared based on the first supernatant-free solution obtained.

[0048] For example, in this embodiment, a preset weight of horseradish peroxidase is first added to a carbonate buffer solution of a corresponding first preset volume. The preset weight can be 5 mg and the first preset volume can be 0.4 mL.

[0049] Next, glutaraldehyde of the first preset concentration will be added to the first mixture according to the second preset volume, that is, 0.1 mL of 25% glutaraldehyde will be added and mixed to obtain the first mixture.

[0050] The first mixture is incubated at a second preset temperature for a second preset time, where the second preset temperature can be 37°C and the second preset time can be 2 hours.

[0051] Then, a second preset volume of sodium chloride buffer solution with a second preset concentration and a third preset volume of pre-cooled anhydrous ethanol are sequentially added to the first mixture, and the first mixture is thoroughly mixed. The second preset volume can be 0.1 mL, the second preset concentration can be 20%, and the third preset volume can be 2.5 mL.

[0052] At this point, the first mixture is centrifuged at a second preset centrifugation rate for a third preset time, and the first supernatant is removed to obtain a first supernatant-free solution. The second preset centrifugation rate can be 1000 r / min.

[0053] Finally, enzyme-labeled antibodies were prepared based on the first solution without supernatant.

[0054] Furthermore, the aforementioned "preparation of enzyme-labeled antibodies based on the obtained first supernatant-free solution" also includes the following steps: Anhydrous ethanol of a third preset concentration is added to the first supernatant solution obtained based on the fourth preset volume and then thoroughly mixed. The mixed supernatant-free solution is centrifuged for a third preset time based on a second preset centrifugation rate, and then placed at room temperature for a fourth preset time after removing the second supernatant to obtain a second supernatant-free solution. Add the fifth preset volume of carbonate buffer and the fifth preset volume of feline herpesvirus polyclonal antibody with the fourth preset concentration to the second supernatant-free solution in sequence, and mix at room temperature. Add dipotassium hydrogen phosphate to the second mixture until the corresponding acidity or alkalinity of the second mixture is neutral; The second mixture, after being incubated overnight at a third preset temperature, was dialyzed with phosphate buffer at a fifth preset time, and glycerol with a fifth preset concentration was added to the resulting dialyzing solution. The dialysis solution is dispensed and stored at a fourth preset temperature.

[0055] For example, in this embodiment, 5 mL of 80% anhydrous ethanol is first added to the obtained first supernatant-free solution and mixed thoroughly. The mixed supernatant-free solution is then centrifuged again at a second preset centrifugation rate for a third preset time, and its supernatant is removed. After being left at room temperature for a fourth preset time, a second supernatant-free solution is obtained. The fourth preset time can be 2 minutes.

[0056] Next, a fifth preset volume of carbonate buffer and a fifth preset volume of feline herpesvirus polyclonal antibody at a fourth preset concentration were sequentially added to the second supernatant-free solution and mixed at room temperature. The fifth preset volume can be 0.5 mL, and the fourth preset concentration can be 2 mg / mL.

[0057] Then, dipotassium hydrogen phosphate is added to the second mixture until the pH of the second mixture is neutral. The second mixture, after being incubated overnight at a third preset temperature of 4°C, is then dialyzed with phosphate buffer for a fifth preset time, which can be 8 hours, to obtain the dialysate.

[0058] Next, glycerol with a fifth preset concentration (50%) is added to the dialysis solution. Finally, the dialysis solution is dispensed and stored at a fourth preset temperature (-15°C).

[0059] Furthermore, the aforementioned "ELISA plate for detecting the generation of feline herpesvirus monoclonal antibodies" also includes the following steps: Based on the coating solution, feline herpesvirus monoclonal antibody was prepared into a feline herpesvirus monoclonal antibody solution with a fifth preset concentration, and the updated coating solution was obtained. Add the first preset well volume of coating solution to the initial microplate and perform coating incubation based on the first incubation conditions; The coating solution is removed, and the rinsing operation is performed with phosphate buffer based on a preset number of times. The rinsing operation is completed, the air-drying operation is performed based on absorbent paper, and the skim milk with a mass fraction of the first preset fraction is identified as the sealing liquid; Add the second preset volume of blocking solution to each vaccine well of the initial ELISA plate, and perform a blocking operation for a sixth preset time based on the second preset temperature. The blocking solution is removed, and a rinsing operation is performed based on phosphate buffer based on a first preset number of times; After the rinsing operation is completed, the plate is patted dry using absorbent paper and stored within a preset temperature range to generate a detection ELISA plate.

[0060] For example, in this embodiment, the feline herpesvirus monoclonal antibody was first prepared into a 16 μg / ml solution using coating buffer (0.05 mol / L carbonate buffer) and used as the coating buffer. Then, 100 μl of the coating buffer was added to the initial ELISA plate for coating incubation under the first incubation condition, which was 4°C for 16 hours.

[0061] Then, after discarding the coating solution, rinse five times with phosphate buffer and gently pat dry with absorbent paper after the last wash.

[0062] Next, skim milk with a mass fraction of 5% was selected as the sealing liquid, and 200 μl of the sealing liquid was added to each well. Then, the sealing operation was carried out continuously for a sixth preset time based on the second preset temperature. The sixth preset time can be 45 minutes.

[0063] Then, remove the blocking solution, rinse five times with phosphate buffer and pat dry, store within a preset temperature range to generate a detection ELISA plate. The preset temperature range can be 2-8℃.

[0064] Furthermore, the above method also includes the following steps: Based on the strategy of optimizing the coating concentration of capture antibodies, the coating concentration corresponding to the maximum P / N value is determined as the optimal usage concentration; Based on the sealing liquid optimization strategy, the sealing liquid with a corresponding P / N value greater than a preset ratio and sealing cost less than a preset cost is determined as the optimal sealing liquid; Based on the closure time optimization strategy, the closure time corresponding to the maximum P / N value is determined as the optimal closure time; Based on the strategy for optimizing the incubation time of the antigen to be tested, the incubation time of the antigen to be tested corresponding to the maximum P / N value is determined as the optimal incubation time of the antigen to be tested. Based on the enzyme-labeled antibody incubation time optimization strategy, the incubation time of the enzyme-labeled antibody corresponding to the maximum P / N value is determined as the optimal incubation time of the enzyme-labeled antibody; Based on the strategy of optimizing the incubation time of the colorimetric solution, the incubation time of the colorimetric solution corresponding to the maximum P / N value is determined as the optimal incubation time of the colorimetric solution. Based on the strategy for determining the critical value of blank control vaccines, the critical value of the blank control vaccine is determined. Based on the strategy for determining the original multiple critical value of the reference vaccine, the original multiple critical value under the original multiple condition is determined; Based on the specificity testing strategy, the specificity status is determined.

[0065] For example, in this embodiment, to determine the optimal reaction conditions, the effect of different concentrations of the capture antibody on the detection effect under different reaction conditions was examined, and the concentration with the highest P / N value was selected as the optimal concentration. The results of the capture antibody coating concentration determination are shown in Table 1. The capture antibody was diluted to 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, respectively. The results showed that the P / N value was the highest when the capture antibody coating concentration was 16 μg / mL, therefore, the optimal coating concentration of the capture antibody was determined to be 16 μg / mL.

[0066] Table 1 Optimization of Capture Antibody Coating Concentration

[0067] To determine the optimal reaction conditions, this embodiment investigated the effect of different blocking solutions on the detection results under different reaction conditions. The blocking solution with a P / N value greater than a preset ratio and a blocking cost less than a preset cost was selected as the optimal blocking solution. The results of the blocking solution determination are shown in Table 2. Different blocking solutions (3% fish gelatin, 3% BSA, 5% skim milk powder, 1% fish gelatin, 10% chicken serum, 5% chicken serum, and PBST buffer) were tested. The results showed that although the P / N value was not the highest when using 5% skim milk powder as the blocking solution, its cost was lower, and it was soluble at room temperature. Therefore, 5% skim milk powder was determined to be the optimal blocking solution.

[0068] Table 2 Optimization of Sealing Fluid

[0069] To determine the optimal reaction conditions, this embodiment investigated the effect of different blocking times on the detection effect under different reaction conditions, selecting the time with the highest P / N value as the optimal blocking time. The blocking time results are shown in Table 3, with blocking solutions used for 30 minutes, 45 minutes, 60 minutes, 90 minutes, and 120 minutes, respectively. The results showed that the P / N value was highest at 45 minutes, therefore, the optimal blocking time was determined to be 45 minutes.

[0070] Table 3 Optimization of Closure Time

[0071] To determine the optimal reaction conditions, this embodiment investigated the effect of different incubation times on the detection effect of the test antigen under different reaction conditions, selecting the highest P / N value as the optimal incubation time. The incubation time results are shown in Table 4, with the test antigen incubated for 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, and 90 minutes, respectively. The results showed that when the incubation time was 45 minutes, the P / N value was relatively high, and the incubation time was relatively short; therefore, the optimal incubation time for the test antigen was determined to be 45 minutes.

[0072] Table 4 Optimization of incubation time for the antigen to be tested

[0073] To determine the optimal reaction conditions, this embodiment examined the effect of different incubation times of enzyme-labeled antibody on the detection effect under different reaction conditions, selecting the highest P / N value as the optimal incubation time. The results of the enzyme-labeled antibody incubation time determination are shown in Table 5, with incubation times of 15 minutes, 30 minutes, and 45 minutes. The results showed that the P / N value was highest when the enzyme-labeled antibody was incubated for 15 minutes; therefore, the optimal incubation time for the enzyme-labeled antibody was determined to be 15 minutes.

[0074] Table 5 Optimization of enzyme-labeled antibody incubation time

[0075] To determine the optimal reaction conditions, this embodiment investigated the effect of different incubation times of the chromogenic solution on the detection effect under various reaction conditions, selecting the highest P / N value as the optimal incubation time. The incubation time results are shown in Table 6, with the chromogenic solution incubated for 5 minutes, 10 minutes, 15 minutes, 20 minutes, and 25 minutes, respectively. The results showed that the P / N value was highest when the incubation time was 20 minutes; therefore, the optimal incubation time for the chromogenic solution was determined to be 20 minutes.

[0076] Table 6 Optimization of color development time

[0077] In summary, the double-antibody sandwich ELISA method is as follows: The capture antibody was coated at a concentration of 16 μg / mL, and 5% skim milk was used as the blocking solution. The mixture was blocked at 37°C for 45 minutes. The antigen to be tested was incubated at 37°C for 45 minutes. The enzyme-labeled antibody was incubated at 37°C for 15 minutes. The mixture was then developed with single-component TMB chromogenic solution at room temperature for 20 minutes.

[0078] Incubate the antigen to be tested at 37°C for 45 minutes, wash the plate 5 times with 3-minute intervals, and pat dry on absorbent paper after the last wash. Then add 100 μl of horseradish peroxidase-labeled antibody per well, incubate at 37°C for 15 minutes, wash 5 times with 3-minute intervals, and pat dry on absorbent paper after the last wash. Take 100 μl of the chromogenic solution that has been brought to room temperature and incubate at room temperature in the dark for 20 minutes. Add 50 μl of stop solution per well. Place the plate in a microplate reader and measure the absorbance at a wavelength of 450 nm, and record the results.

[0079] Based on the strategy for determining the critical value of blank control vaccines, the critical value of the blank control vaccines was determined: 30 blank control vaccine samples were tested using the established double-antibody sandwich ELISA method to measure their OD values. 450nm The values ​​were calculated, and their mean and standard deviation were determined. The mean plus three times the standard deviation was used as the critical value for the method (the determination of the critical value refers to the national standard GB / T33411-2016). The results are shown in Table 7. The calculated mean value of the blank control vaccine was 0.171, and the standard deviation was 0.041. Therefore, the critical value of the blank control vaccine was 0.294, i.e., the OD value of the blank control vaccine was... 450nm The value should be less than 0.294 for the test to be valid.

[0080] Table 7 Determination of Critical Values ​​for Blank Control Vaccine

[0081] Based on the strategy for determining the original fold critical value of the reference vaccine, the original fold critical value was determined: 30 reference vaccines were tested using the established double-antibody sandwich ELISA method to measure their OD at the original fold. 450nm The values ​​were calculated, and their mean and standard deviation were determined. The mean minus three times the standard deviation was used as the reference OD value under the original vaccine condition. 450nm The critical value of the value (refer to the original OD of the vaccine). 450nm The critical value for the value was determined with reference to the national standard GB / T 33411-2016. The results (see Table 8) show that the blank control vaccine OD... 450nm A value less than 0.294 indicates the trial is valid. The calculated mean value for the reference vaccine is 2.355, with a standard deviation of 0.067. Therefore, the critical value under the original-fold OD condition of the reference vaccine is 2.154; that is, the OD value of the original-fold OD of the reference vaccine. 450nm The value should not be less than 2.154 for the test to be valid.

[0082] Table 8. Determination of reference vaccine original multiple threshold value

[0083] In summary, the criteria for determining the validity of the double-antibody sandwich ELISA method are as follows: Blank control vaccine OD 450nm The value should be less than 0.294, referring to the original OD of the vaccine. 450nm The value should be no less than 2.154.

[0084] Based on the specificity testing strategy, the specificity status was determined: the established double-antibody sandwich ELISA method was used to test the feline triple inactivated vaccine, FHV inactivated vaccine, blank control vaccine, FPV inactivated vaccine, and FCV inactivated vaccine. The results showed (Table 9) that, according to the validity criteria of the double-antibody sandwich ELISA method, the OD of the blank control vaccine was [not specified]. 450nm A value less than 0.294 indicates the test is valid. The test results meet the requirements, and the established double-antibody sandwich ELISA method has good specificity and can be used to detect the relative potency of feline herpesvirus antigen in feline triple inactivated vaccines.

[0085] Table 9 Specificity Tests

[0086] Step S104 includes the following: The response involves completing the first incubation operation on the captured antibody based on the detection ELISA plate, adding the feline herpesvirus inactivated vaccine and the reference vaccine to each vaccine well of the detection ELISA plate, and then performing a second incubation operation on the detection ELISA plate.

[0087] Example 4 The capture antibody, diluted to a sixth preset concentration based on the coating solution, is added to the detection ELISA plate based on the first preset well volume, and the coating operation is performed based on the third preset temperature and preset time interval. In response to the current temperature being the same as room temperature, the detection ELISA plate is washed a first preset number of times based on the washing solution at a first preset interval. After the plate washing operation is completed, the plate is patted dry using absorbent paper. Based on the second preset well volume, the blocking solution is added to the detection ELISA plate, and the first incubation operation is performed based on the second preset temperature and the sixth preset time. The detection ELISA plate is washed a first preset number of times at a first preset interval using washing solution. After the plate washing operation is completed, the plate is patted dry using absorbent paper. The feline triple inactivated vaccine, including feline herpesvirus inactivated vaccine, and the reference vaccine were added to each vaccine well of the detection ELISA plate, and the detection ELISA plate was then subjected to a second incubation operation.

[0088] For example, in this embodiment, the capture antibody solution is first diluted to a sixth preset concentration using a coating solution. The sixth preset concentration can be 16 μg / ml. The antibody solution is then added to each well of the detection ELISA plate at a first preset well volume. The first preset well volume can be 100 μl / well. The coating operation is then performed at a third preset temperature and a preset time interval, which can be 16-20 hours.

[0089] When the current temperature is the same as the room temperature, wash the ELISA plate 5 times with washing solution, with a 3-minute interval between each wash, and pat dry with absorbent paper after washing.

[0090] Add blocking buffer to the detection ELISA plate according to the second preset well volume, and perform the first incubation operation based on the second preset temperature and the sixth preset time. The second preset well volume can be 200 μl / well. Wash the detection ELISA plate 5 times with washing buffer, with each wash 3 minutes apart, and pat dry with absorbent paper after washing.

[0091] Next, the feline triple inactivated vaccine, including the feline herpesvirus inactivated vaccine, and the reference vaccine were added to each vaccine well of the detection ELISA plate, and the detection ELISA plate was then subjected to a second incubation operation.

[0092] Furthermore, the aforementioned "adding the feline triple inactivated vaccine, including feline herpesvirus inactivated vaccine, and the reference vaccine to each vaccine well of the detection ELISA plate, and performing a second incubation operation on the detection ELISA plate" also includes the following steps: The feline triple inactivated vaccine and the reference vaccine were diluted using a dilution plate. The reference vaccine was added to each of the first preset wells based on the volume of the third preset well, and the feline triple inactivated vaccine was added to each of the second preset wells based on the volume of the third preset well. Based on the fourth preset well volume, the washing solution is added to each vaccine well except for each first preset well and each second preset well. The feline triple inactivated vaccine and the reference vaccine were diluted according to preset ratios. Based on the fifth preset well volume, the liquid located in each vaccine well is transferred to the corresponding vaccine well of the ELISA plate coated with capture antibody, and the detection ELISA plate is subjected to a second incubation operation.

[0093] For example, in this embodiment, the feline triple inactivated vaccine and the reference vaccine are first diluted using a dilution plate. Then, the reference vaccine is added to each of the first preset wells based on the volume of the third preset well. The volume of the third preset well can be 150 μl / well. Each of the first preset wells can be wells B1, C1, and D1, such as... Figure 2 The solid-filled circles are shown. The feline triple inactivated vaccine is added to each of the second preset wells based on the volume of the third preset well. Each second preset well can be well E1, F1, or G1, as shown. Figure 3 The diagonal lines shown are filled with circles.

[0094] Then, based on the fourth preset well volume, washing solution is added to each vaccine well except for each of the first and second preset wells. The fourth preset well volume can be 75 μl / well, and the remaining wells are wells B2-B11 to G2-G11.

[0095] Next, the feline triple inactivated vaccine and the reference vaccine were diluted at a preset ratio, which could be 2-fold. Then, the liquid from each vaccine well was transferred to the corresponding vaccine well of the ELISA plate coated with the capture antibody using the fifth preset well volume, and the ELISA plate was subjected to a second incubation. The fifth preset well volume could be 50 μl / well. A schematic diagram of sample loading for each well is shown in Table 1 below.

[0096] Table 1. OD values ​​of reference vaccine and feline triple inactivated vaccine 450nm Value sampling diagram

[0097] Note: " / " indicates that no operation was performed on this hole.

[0098] Furthermore, the aforementioned "second incubation operation on the detection ELISA plate" also includes the following steps: The detection ELISA plate is subjected to a first incubation operation for a sixth preset time based on a second preset temperature. The detection ELISA plate is washed a first preset number of times at a first preset interval using washing solution. After the plate washing operation is completed, the plate is patted dry using absorbent paper, and horseradish peroxidase-labeled antibody is added based on the first preset well volume. The detection ELISA plate is subjected to a second incubation operation based on a seventh preset time at a second preset temperature. The detection ELISA plate is washed a first preset number of times at a first preset interval using washing solution. After the plate washing operation is completed, the plate is patted dry using absorbent paper. If the current temperature is the same as room temperature, obtain the colorimetric solution and add it to the detection ELISA plate based on the first preset well volume; Based on the eighth preset time of incubation at room temperature in the dark, and based on the sixth preset well volume, the stop solution is added to the detection ELISA plate.

[0099] For example, in this embodiment, the detection ELISA plate is first incubated at a second preset temperature for a sixth preset time, i.e., incubated in a 37°C constant temperature incubator for 45 minutes. Then, the detection ELISA plate is washed 5 times with washing solution, with each wash spaced 3 minutes apart, and after washing, it is patted dry with absorbent paper.

[0100] Next, horseradish peroxidase-labeled antibody was added to the wells according to the first preset volume, and then the plate was incubated for a second time based on the seventh preset time, i.e., 15 minutes in a constant temperature incubator at 37°C, based on the second preset temperature.

[0101] After completing the second incubation step, wash the detection ELISA plate 5 times with washing buffer, with 3 minutes between each wash, and then pat dry with absorbent paper after washing.

[0102] When the current temperature is the same as room temperature, obtain the colorimetric solution and add it to the detection microplate according to the first preset well volume. Incubate for the eighth preset time at room temperature in the dark, i.e., 20 minutes. Finally, add the stop solution to the detection microplate according to the sixth preset well volume, which can be 50 μl / well.

[0103] Step S106 includes the following: The relative potency ratio of the corresponding feline herpesvirus inactivated vaccine was determined based on the absorbance value of each vaccine well, and the relative potency attribute of the antigen was determined based on the relative potency ratio.

[0104] For example, in this embodiment, the relative potency ratio of the corresponding feline herpesvirus inactivated vaccine is determined by obtaining the absorbance value of each vaccine well, and then the relative potency attribute of the antigen is determined based on the relative potency ratio.

[0105] Furthermore, the aforementioned "determining the relative potency ratio of the corresponding feline triple inactivated vaccine based on the absorbance values ​​of each vaccine well, and determining the antigen relative potency attribute based on the relative potency ratio" also includes the following steps: Place each vaccine well in the ELISA reader and determine the absorbance value of each vaccine well based on the preset wavelength; Determine the average absorbance of the blank control vaccine; The absorbance values ​​of each well site of the vaccine corresponding to different dilutions were calculated by comparing them with the average absorbance value of the blank control vaccine to obtain the corrected absorbance value of each well site of the vaccine and to determine the relative efficacy ratio. The experiment is considered valid if the average absorbance of the blank control vaccine is less than the first preset value and the original absorbance value of the reference vaccine is not less than the second preset value. The third preset value is determined as the reference relative efficacy value of the vaccine. The relative efficacy value of the feline triple inactivated vaccine should not be less than the third preset value. The relative efficacy attribute of feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be a qualified attribute. If the relative potency value of the feline triple inactivated vaccine is less than the third preset value, a retest will be performed. If the relative efficacy value of the response retest result is not less than the third preset value, the relative efficacy attribute of the feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be a qualified attribute; otherwise, the relative efficacy attribute of the feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be a unqualified attribute.

[0106] For example, in this embodiment, each vaccine well is first placed in an ELISA reader, and the absorbance value of each vaccine well is determined according to a preset wavelength, which can be 450 nm. The average absorbance of the blank control vaccine is then calculated based on the absorbance values.

[0107] Next, the absorbance values ​​of each well site of the vaccine corresponding to different dilutions were calculated with the average absorbance value of the blank control vaccine to obtain the corrected absorbance value of each well site. Then, the values ​​calculated for the reference vaccine and the feline triple inactivated vaccine were processed according to the principle of double parallel line bioassay and entered into RelPot 4.0 software to calculate the relative potency ratio, thereby determining the relative potency ratio.

[0108] The experiment is considered valid if the average absorbance of the blank control vaccine is less than the first preset value and the original absorbance value of the reference vaccine is not less than the second preset value. The first preset value can be 0.294, and the second preset value can be 2.154.

[0109] Then, the third preset value is determined as the reference relative efficacy value of the vaccine, which can be 1.0. When the relative efficacy value of the feline triple inactivated vaccine is not less than the third preset value, the relative efficacy attribute of the feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be a qualified attribute.

[0110] If the relative potency value of the feline triple inactivated vaccine is less than the third preset value, a retest is performed. If the relative potency value of the retest result is not less than the third preset value, the relative potency attribute of the feline herpesvirus antigen in the feline triple inactivated vaccine can be determined to be qualified; otherwise, the relative potency attribute of the feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be unqualified.

[0111] Based on the OD measured in the reaction solution of each well 450nm The values ​​are shown in Table 2 below. Based on the data in Table 2, the OD values ​​of the blank control vaccine are calculated. 450nm The average value was 0.177, referring to the original OD of the vaccine. 450nm The average value was 2.448, which is consistent with the control, indicating that this experiment was effective.

[0112] Table 2. OD of feline triple inactivated vaccine 450nm value

[0113] Note: " / " indicates that no operation was performed on this hole.

[0114] The OD values ​​of the above-mentioned reference vaccines and feline triple inactivated vaccines were compared. 450nm Value minus blank control vaccine OD 450nmThe average value was input into RelPot 4.0 software to calculate the relative potency (RP) value, and the results are shown in Table 3 below. Based on the above results, the RP value is determined to be 1.13. Using the reference vaccine as the standard, the relative potency test of the feline trivalent inactivated vaccine for feline herpesvirus antigen is qualified.

[0115] Table 3. Relative Potential (RP) Values ​​of Feline Trivalent Inactivated Vaccine

[0116] Furthermore, the above method also includes the following steps: The relative antigenic potency of feline triple inactivated vaccines from the same batch and different batches was determined based on a preset number of doses.

[0117] Example 5 For example, in this embodiment, the method of Embodiment 4 of the present invention is first used to detect the relative potency of the same batch and different batches, with a preset number of doses of 3. That is, 3 feline triple inactivated vaccines are randomly selected, and the relative potency is tested using the double-antibody sandwich ELISA method established in this invention. The test results are shown in Tables 1 and 2 below.

[0118] The method established in this invention was used to test feline triple inactivated vaccines from the same batch and different batches. All results showed that the relative potency of the feline herpesvirus antigen in the feline triple inactivated vaccine was satisfactory. The coefficient of variation (RP) for the same batch was 3.52%, while the RP for different batches was 3.21%. The RP values ​​were all less than 10%, indicating stable test results and good repeatability.

[0119] Table 1. Relative potency test of feline herpesvirus antigen in the same batch of feline triple inactivated vaccine

[0120] Table 2. Relative potency test of feline herpesvirus antigen in different batches of feline triple inactivated vaccine

[0121] According to the present invention, by preparing capture antibodies, polyclonal antibodies, and enzyme-labeled antibodies in a step-by-step manner, and then combining them with a dedicated detection ELISA plate for detection, the efficacy of feline herpesvirus inactivated vaccines can be accurately assessed, demonstrating significant beneficial effects. Furthermore, by preparing capture antibodies and ELISA antibodies separately, interference from other viruses can be effectively eliminated, greatly improving the specificity of the detection. Moreover, the present invention also strictly controls each incubation step, making the detection results more stable and reliable. Finally, the present invention can calculate the relative efficacy ratio based on absorbance values ​​and determine the relative efficacy attributes of the antigen, achieving a quantitative assessment of vaccine efficacy. This provides a scientific and accurate testing standard for vaccine quality control, ensuring the accuracy and standardization of the test results.

[0122] Another embodiment of the present invention provides an in vitro potency testing system for feline herpesvirus inactivated vaccines. Figure 4 Its corresponding system block diagram includes: The preparation module is configured to perform capture antibody preparation, feline herpesvirus polyclonal antibody preparation, and enzyme-labeled antibody preparation, and to generate a detection enzyme-labeled plate based on feline herpesvirus monoclonal antibody. The incubation module is configured to, in response to the first incubation operation performed on the captured antibody based on the detection ELISA plate, add the feline herpesvirus inactivated vaccine and the reference vaccine to each vaccine well of the detection ELISA plate, and perform a second incubation operation on the detection ELISA plate. The determination module is configured to determine the relative potency ratio of the corresponding feline herpesvirus inactivated vaccine based on the absorbance value of each vaccine well, and to determine the antigen relative potency attribute based on the relative potency ratio.

[0123] In the specification provided herein, the algorithms and displays are not inherently related to any particular computer, virtual system, or other device. Various general-purpose systems can also be used with the examples of this invention. The required structure for constructing such systems is apparent from the above description. Furthermore, this invention is not directed to any particular programming language. It should be understood that the contents of the invention described herein can be implemented using various programming languages, and the above description of specific languages ​​is for the purpose of disclosing preferred embodiments of the invention.

[0124] Numerous specific details are set forth in the specification provided herein. However, it will be understood that embodiments of the invention may be practiced without these specific details. In some instances, well-known methods, structures, and techniques have not been shown in detail so as not to obscure the understanding of this specification.

[0125] Similarly, it should be understood that, in order to streamline this disclosure and aid in understanding one or more of the various aspects of the invention, in the above description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof.

[0126] Those skilled in the art will understand that modules, units, or components of the devices disclosed in the examples herein can be arranged in the devices described in this embodiment, or alternatively, can be located in one or more devices different from the devices in this example. The modules in the foregoing examples can be combined into a single module or, in addition, can be divided into multiple sub-modules.

[0127] Those skilled in the art will understand that the modules in the device of the embodiment can be adaptively changed and placed in one or more devices different from that embodiment. Modules, units, or components in the embodiment can be combined into a single module, unit, or component, and further, they can be divided into multiple sub-modules, sub-units, or sub-components.

[0128] Furthermore, those skilled in the art will understand that although some embodiments described herein include certain features included in other embodiments but not others, combinations of features from different embodiments are meant to be within the scope of the invention and form different embodiments.

[0129] Furthermore, some of the embodiments described herein are methods or combinations of method elements that can be implemented by a processor of a computer system or by other means of performing the functions. Therefore, a processor having the necessary instructions for implementing the methods or method elements forms means for implementing the methods or method elements. Furthermore, the elements described herein in the apparatus embodiments are examples of means for implementing the functions performed by elements for the purposes of carrying out the invention.

[0130] As used herein, unless otherwise specified, the use of ordinal numbers such as “first,” “second,” “third,” etc., to describe ordinary objects merely indicates different instances of similar objects and is not intended to imply that the objects being described must have a given order in time, space, ordering, or any other manner.

[0131] Although the invention has been described with reference to a limited number of embodiments, those skilled in the art will understand from the foregoing description that other embodiments are conceivable within the scope of the invention described herein. Furthermore, it should be noted that the language used in this specification has been chosen primarily for readability and instructional purposes, and not for the purpose of explaining or limiting the subject matter of the invention.

Claims

1. A method for testing the in vitro potency of a feline herpesvirus inactivated vaccine, characterized in that, include: Capture antibody preparation, feline herpesvirus polyclonal antibody preparation, and enzyme-labeled antibody preparation were performed separately, and a detection enzyme-labeled plate was generated based on the feline herpesvirus monoclonal antibody. The response involves completing the first incubation operation on the captured antibody based on the detection ELISA plate, adding the feline herpesvirus inactivated vaccine and the reference vaccine to each vaccine well of the detection ELISA plate, and then performing a second incubation operation on the detection ELISA plate. The relative potency ratio of the corresponding feline herpesvirus inactivated vaccine was determined based on the absorbance value of each vaccine well, and the relative potency attribute of the antigen was determined based on the relative potency ratio.

2. The method according to claim 1, characterized in that, Capture antibody preparation includes: Purified feline herpesvirus was mixed with an appropriate amount of adjuvant and then used to immunize experimental mice. The spleen cells of the immunized mice were then fused with pre-defined myeloma cells, subcloned, and screened to obtain hybridoma cell lines that secrete feline herpesvirus monoclonal antibodies. Hybridoma cell lines were cultured using a bioreactor, and the resulting culture supernatant was purified to obtain capture antibodies.

3. The method according to claim 2, characterized in that, The method further includes: The capture antibody undergoes a first quality control, which includes morphological testing, sterility testing, neutralizing antibody titer determination, specificity testing, and first storage and shelf life.

4. The method according to claim 1, characterized in that, Preparation of polyclonal antibodies against feline herpesvirus, including: The feline herpesvirus after inactivation was mixed with an appropriate amount of adjuvant and immunized with experimental cats. Blood was collected from the experimental cats after a preset time period. The obtained serum sample is centrifuged at a first preset centrifugation rate, and the obtained initial supernatant sample is inactivated at a first preset temperature for a continuous first preset time to obtain the current supernatant sample. The supernatant sample was purified by salting out, and the purified supernatant sample was filtered, sterilized, and dispensed using a filter membrane of a corresponding preset size to obtain feline herpesvirus polyclonal antibody.

5. The method according to claim 4, characterized in that, The method further includes: A second quality control was performed on the feline herpesvirus polyclonal antibody, which included phenotype testing, sterility testing, neutralizing antibody titer determination, and second storage and shelf life.

6. The method according to claim 1, characterized in that, Preparation of enzyme-labeled antibodies includes: A preset weight of horseradish peroxidase is added to a carbonate buffer solution corresponding to a first preset volume, and glutaraldehyde of a first preset concentration is added to the obtained first mixture based on a second preset volume. A second preset volume of sodium chloride buffer solution with a second preset concentration and a third preset volume of pre-cooled anhydrous ethanol are sequentially added to a first mixture that is incubated for a second preset time based on a second preset temperature, and the first mixture is thoroughly mixed. The first mixture is centrifuged at a second preset centrifugation rate for a third preset time, and the first supernatant is removed. Enzyme-labeled antibodies were prepared based on the first supernatant-free solution obtained.

7. The method according to claim 6, characterized in that, Based on the obtained first supernatant-free solution, enzyme-labeled antibody preparation was performed, including: Anhydrous ethanol of a third preset concentration is added to the first supernatant solution obtained based on the fourth preset volume and then thoroughly mixed. The mixed supernatant-free solution is centrifuged for a third preset time based on a second preset centrifugation rate, and then placed at room temperature for a fourth preset time after removing the second supernatant to obtain a second supernatant-free solution. Add the fifth preset volume of carbonate buffer and the fifth preset volume of feline herpesvirus polyclonal antibody with the fourth preset concentration to the second supernatant-free solution in sequence, and mix at room temperature. Add dipotassium hydrogen phosphate to the second mixture until the corresponding acidity or alkalinity of the second mixture is neutral; The second mixture, after being incubated overnight at a third preset temperature, was dialyzed with phosphate buffer at a fifth preset time, and glycerol with a fifth preset concentration was added to the resulting dialyzing solution. The dialysis solution is dispensed and stored at a fourth preset temperature.

8. The method according to claim 1, characterized in that, An enzyme-linked immunosorbent assay (ELISA) plate for detecting the generation of feline herpesvirus monoclonal antibodies includes: Based on the coating solution, feline herpesvirus monoclonal antibody was prepared into a feline herpesvirus monoclonal antibody solution with a fifth preset concentration, and the updated coating solution was obtained. Add the first preset well volume of coating solution to the initial microplate and perform coating incubation based on the first incubation conditions; The coating solution is removed, and the rinsing operation is performed with phosphate buffer based on a preset number of times. The rinsing operation is completed, the air-drying operation is performed based on absorbent paper, and the skim milk with a mass fraction of the first preset fraction is identified as the sealing liquid; Add the second preset volume of blocking solution to each vaccine well in the initial ELISA plate, and perform a blocking operation for a sixth preset time based on the second preset temperature. The blocking solution is removed, and a rinsing operation is performed based on phosphate buffer based on a first preset number of times; After the rinsing operation is completed, the plate is patted dry using absorbent paper and stored within a preset temperature range to generate a detection ELISA plate.

9. The method according to claim 8, characterized in that, The method further includes: Based on the strategy of optimizing the coating concentration of capture antibodies, the coating concentration corresponding to the maximum P / N value is determined as the optimal usage concentration; Based on the sealing liquid optimization strategy, the sealing liquid with a corresponding P / N value greater than a preset ratio and sealing cost less than a preset cost is determined as the optimal sealing liquid; Based on the closure time optimization strategy, the closure time corresponding to the maximum P / N value is determined as the optimal closure time; Based on the strategy for optimizing the incubation time of the antigen to be tested, the incubation time of the antigen to be tested corresponding to the maximum P / N value is determined as the optimal incubation time of the antigen to be tested. Based on the enzyme-labeled antibody incubation time optimization strategy, the incubation time of the enzyme-labeled antibody corresponding to the maximum P / N value is determined as the optimal incubation time of the enzyme-labeled antibody; Based on the strategy of optimizing the incubation time of the colorimetric solution, the incubation time of the colorimetric solution corresponding to the maximum P / N value is determined as the optimal incubation time of the colorimetric solution. Based on the strategy for determining the critical value of blank control vaccines, the critical value of the blank control vaccine is determined. Based on the strategy for determining the original multiple critical value of the reference vaccine, the original multiple critical value under the original multiple condition is determined; Based on the specificity testing strategy, the specificity status is determined.

10. The method according to claim 1, characterized in that, In response to the first incubation operation of the capture antibody based on the detection ELISA plate, feline herpesvirus inactivated vaccine and reference vaccine are added to each vaccine well of the detection ELISA plate, and a second incubation operation is performed on the detection ELISA plate, including: The capture antibody, diluted to a sixth preset concentration based on the coating solution, is added to the detection ELISA plate based on the first preset well volume, and the coating operation is performed based on the third preset temperature and preset time interval. In response to the current temperature being the same as room temperature, the detection ELISA plate is washed a first preset number of times based on the washing solution at a first preset interval. After the plate washing operation is completed, the plate is patted dry using absorbent paper. Based on the second preset well volume, the blocking solution is added to the detection ELISA plate, and the first incubation operation is performed based on the second preset temperature and the sixth preset time. The detection ELISA plate is washed a first preset number of times at a first preset interval using washing solution. After the plate washing operation is completed, the plate is patted dry using absorbent paper. The feline triple inactivated vaccine, including feline herpesvirus inactivated vaccine, and the reference vaccine were added to each vaccine well of the detection ELISA plate, and the detection ELISA plate was then subjected to a second incubation operation.

11. The method according to claim 10, characterized in that, Add the feline triple inactivated vaccine, including feline herpesvirus inactivated vaccine, and the reference vaccine to each vaccine well of the detection ELISA plate, and perform a second incubation operation on the detection ELISA plate, including: The feline triple inactivated vaccine and the reference vaccine were diluted using a dilution plate. The reference vaccine was added to each of the first preset wells based on the volume of the third preset well, and the feline triple inactivated vaccine was added to each of the second preset wells based on the volume of the third preset well. Based on the fourth preset well volume, the washing solution is added to each vaccine well except for each first preset well and each second preset well. The feline triple inactivated vaccine and the reference vaccine were diluted according to preset ratios. Based on the fifth preset well volume, the liquid located in each vaccine well is transferred to the corresponding vaccine well of the ELISA plate coated with capture antibody, and the detection ELISA plate is subjected to a second incubation operation.

12. The method according to claim 11, characterized in that, The second incubation step for the detection ELISA plate includes: The detection ELISA plate is subjected to a first incubation operation for a sixth preset time based on a second preset temperature. The detection ELISA plate is washed a first preset number of times at a first preset interval using washing solution. After the plate washing operation is completed, the plate is patted dry using absorbent paper, and horseradish peroxidase-labeled antibody is added based on the first preset well volume. The detection ELISA plate is subjected to a second incubation operation based on a seventh preset time at a second preset temperature. The detection ELISA plate is washed a first preset number of times at a first preset interval using washing solution. After the plate washing operation is completed, the plate is patted dry using absorbent paper. If the current temperature is the same as the room temperature, obtain the rewarming colorimetric solution and add it to the detection ELISA plate based on the first preset well volume; Based on the eighth preset time of incubation at room temperature in the dark, and based on the sixth preset well volume, the stop solution is added to the detection ELISA plate.

13. The method according to claim 1, characterized in that, The relative potency ratio of the corresponding feline triple inactivated vaccine was determined based on the absorbance values ​​of each vaccine well, and the relative potency attribute of the antigen was determined based on the relative potency ratio, including: Place each vaccine well in the ELISA reader and determine the absorbance value of each vaccine well based on the preset wavelength. Determine the average absorbance of the blank control vaccine; The absorbance values ​​of each well site of the vaccine corresponding to different dilutions were calculated by comparing them with the average absorbance value of the blank control vaccine to obtain the corrected absorbance value of each well site of the vaccine and to determine the relative efficacy ratio. The experiment is considered valid if the average absorbance of the blank control vaccine is less than the first preset value and the original absorbance value of the reference vaccine is not less than the second preset value. The third preset value is determined as the reference relative efficacy value of the vaccine. The relative efficacy value of the feline triple inactivated vaccine should not be less than the third preset value. The relative efficacy attribute of feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be a qualified attribute. If the relative potency value of the feline triple inactivated vaccine is less than the third preset value, a retest will be performed. If the relative efficacy value of the response retest result is not less than the third preset value, the relative efficacy attribute of the feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be a qualified attribute; otherwise, the relative efficacy attribute of the feline herpesvirus antigen in the feline triple inactivated vaccine is determined to be a unqualified attribute.

14. The method according to claim 13, characterized in that, The method further includes: The relative antigenic potency of feline triple inactivated vaccines from the same batch and different batches was determined based on a preset number of doses.

15. A system for testing the in vitro potency of feline herpesvirus inactivated vaccines, characterized in that, include: The preparation module is configured to perform capture antibody preparation, feline herpesvirus polyclonal antibody preparation, and enzyme-labeled antibody preparation, and to generate a detection enzyme-labeled plate based on feline herpesvirus monoclonal antibody. The incubation module is configured to, in response to the first incubation operation performed on the captured antibody based on the detection ELISA plate, add the feline herpesvirus inactivated vaccine and the reference vaccine to each vaccine well of the detection ELISA plate, and perform a second incubation operation on the detection ELISA plate. The determination module is configured to determine the relative potency ratio of the corresponding feline herpesvirus inactivated vaccine based on the absorbance value of each vaccine well, and to determine the antigen relative potency attribute based on the relative potency ratio.

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