Dog quadruple live vaccine potency detection method

Abstract

The invention discloses a method for detecting the potency of a canine quadruple live vaccine, which comprises the following steps of: specifically neutralizing corresponding three virus components in the vaccine in a targeted manner by respectively adopting three antiserum corresponding to four virus components contained in the canine quadruple live vaccine, and determining the content of a target virus after eliminating lesion interference of the three components on cells, so as to detect the potency of the canine quadruple live vaccine. And the titration result is finally judged through a direct immunofluorescence technology, so that the content of the to-be-detected virus component can be effectively detected. According to the measured content result FA-TCID50 of the four virus components, the relationship between the theoretical value of each component of the vaccine and the FA-TCID50 is established, and then the efficacy of the dog quadruple live vaccine finished product to be detected is evaluated. The method is low in detection cost and short in detection period, the whole detection period is 5 days, the detection result is not influenced by individual differences of experimental animals, interference is avoided, in addition, a virus standard substance does not need to be used, the experiment safety is improved, and meanwhile the experiment cost is further reduced.

Description

Technical Field

[0001] The invention relates to the field of vaccine quality detection, and in particular to a method for detecting the efficacy of a canine quadruple live vaccine. Background Art

[0002] Canine quadruple live vaccine is made by inoculating susceptible cells with canine distemper virus (CDV), canine parainfluenza virus (CPIV), canine adenovirus (CAV) and canine parvovirus (CPV) attenuated strains for static culture, harvesting the cell culture, adding appropriate amounts of antibiotics and stabilizers after mixing in a certain proportion, and then freeze-vacuum drying. It is used to prevent diseases caused by canine distemper, canine parainfluenza, canine adenovirus and canine parvovirus infection.

[0003] As an important quality control indicator of viral products, viral infection titer is of great significance to product quality evaluation and clinical dosage use. Current viral biological products, including viral vaccines, viral vectors for gene and tumor therapy, etc., are all evaluated through viral titration to evaluate the effects of their research and development and production processes. In seed virus preparation and semi-finished product testing, viral titer is also used as evidence and reference for the activity and effectiveness of viral vaccines in the production process. Therefore, viral titration is widely used in the manufacturing, inspection and safety verification of biological products, and it is very necessary to establish a fast, accurate and stable viral titration method.

[0004] At present, the detection methods of live vaccine efficacy can generally be divided into serological methods and virus content determination methods. Serological method: Use susceptible animals of appropriate age, collect blood after a certain period of immunization, and determine the neutralizing antibody titer of serum against vaccine virus. This method has the following shortcomings: ① The detection cost is high, and experimental animals need to be used (blood needs to be collected from experimental animals before use, and neutralizing antibody detection and screening are required), experimental animal breeding environment (animal house, cages, feed, drinking water and other conditions), and experimental animal breeding personnel are required to feed (staff hourly cost); ② The detection cycle is long, and antibodies can only be produced in animals after a period of immunization; ③ Because it is an in vivo experiment, the test results are easily affected by individual differences in experimental animals, and the influence cannot be eliminated; ④ Virus standards (control viruses) need to be calibrated and used. The virus content determination method cannot be realized due to the reasons of the multi-unit vaccine itself (during the detection process, the components of the vaccine interfere with each other). Summary of the invention

[0005] The purpose of the present invention is to establish a new canine quadruple live vaccine efficacy detection method, which solves the problems of high economic and time costs, easy interference of results, and the need to use virus standards in the existing canine quadruple live vaccine efficacy detection methods. Thus, an excellent detection method with low detection cost, results that are not easily interfered by individual differences in experimental animals, and no need to use virus standards is obtained.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The method for detecting the efficacy of canine quadruple live vaccine proposed by the present invention comprises the following steps:

[0008] S1, respectively using anti-canine distemper virus serum, anti-canine parainfluenza virus serum, anti-canine adenovirus serum and anti-canine parvovirus serum to mix equal volumes of three antisera to obtain four mixed antisera;

[0009] S2, using one of the mixed antisera to neutralize the virus component in the anti-canine quadruple live vaccine, and using the neutralization product as the test sample for titration and culture;

[0010] S3. Determine the virus content of the virus components in the neutralization product that are not neutralized by the mixed antiserum by direct immunofluorescence antibody staining (FA-TCID) 50 ;

[0011] S4, use the other three mixed antisera in turn to process according to steps S2 and S3 to obtain the virus content FA-TCID of the four viruses respectively. 50 The results should be higher than the theoretical value of the virus content of the corresponding component, FA-TCID 50 The calculations were all done using the Reed-Muench method;

[0012] S5. Based on the theoretical values ​​and FA-TCID of four viruses 50 , establish the theoretical value and FA-TCID of each vaccine component 50 The relationship between the virus content test results of each component of the canine quadruple live vaccine is as follows: FA-TCID 50 Subtract the theoretical value of the virus content of the corresponding component, and the minimum value obtained is named A value, which is the direct immunofluorescence test result of the component FA-TCID 50 The difference in the theoretical values ​​of the virus content of the corresponding components is the B value, which is the qualified standard of the virus content of each component plus the A value;

[0013] S6. Evaluate the efficacy of the canine quadruple live vaccine to be tested. The criteria are as follows: When the detection value of each component virus in the canine quadruple live vaccine to be tested is FA-TCID 50 If both are not lower than the B value, it is judged as qualified, otherwise it is judged as unqualified.

[0014] Furthermore, the preparation of the four antisera in step S1 includes the following steps: taking canine distemper virus 10 6.0 TCID 50 / ml, canine parainfluenza virus 10 6.0 TCID 50 / ml, canine adenovirus 10 7.0 TCID 50 / ml and canine parvovirus 10 7.0 TCID 50 / ml four kinds of attenuated viruses were used to immunize rabbits respectively, and the immunization procedure adopted four immunizations; 14 days after the fourth immunization, a large amount of blood was collected, the serum was separated and inactivated and packaged, antiserum was prepared and the serum neutralizing antibodies were determined; among them, the neutralizing antibodies against canine distemper virus serum were greater than 1:256, the neutralizing antibodies against canine parainfluenza virus serum were greater than 1:32, the neutralizing antibodies against canine adenovirus serum were greater than 1:512, and the neutralizing antibodies against canine parvovirus serum were greater than 1:256.

[0015] Furthermore, the neutralization step of step S2 is as follows: the mixed antiserum is inoculated into the test canine quadruple freeze-dried live vaccine, and after being fully mixed, it is neutralized at 37° C. for 1 hour.

[0016] Further, the titration and incubation steps of step S2 are as follows: take the neutralization product, make a 10-fold serial dilution, take 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Five dilutions were inoculated simultaneously in 96-well cell plates with the corresponding cells at a cell density of 1×10 5 / ml, 8 wells were inoculated for each dilution, 100 μl was added to each well, and 4 wells with normal cells as controls were set up at the same time, and the cells were continuously cultured at 37°C and 5% CO2 for 5 days; wherein, a mixed antiserum containing anti-canine distemper virus serum, anti-canine parainfluenza virus serum and anti-canine adenovirus serum was neutralized with the virus components in the canine quadruple live vaccine and the neutralization product was inoculated into F81 cells; a mixed antiserum containing anti-canine distemper virus serum, anti-canine parainfluenza virus serum and anti-canine parvovirus serum was neutralized with the virus components in the canine quadruple live vaccine and the neutralization product was inoculated into MDCK cells; a mixed antiserum containing anti-canine distemper virus serum, anti-canine adenovirus serum and anti-canine parvovirus serum was neutralized with the virus components in the canine quadruple live vaccine and the neutralization product was inoculated into MDCK cells; a mixed antiserum containing anti-canine parainfluenza virus serum, anti-canine adenovirus serum and anti-canine parvovirus serum was neutralized with the virus components in the canine quadruple live vaccine and the neutralization product was inoculated into Vero cells.

[0017] Furthermore, step S3 uses direct immunofluorescence antibodies corresponding to the target components to be measured for staining, and the specific operation steps are as follows:

[0018] ①Discard the liquid in the culture plate, add pre-cooled 80% acetone, and fix in a 2-8℃ refrigerator for 30 minutes;

[0019] ②After fixation, wash the plate three times with PBS and pat dry any remaining liquid in the wells for the last wash;

[0020] ③Add the corresponding direct immunofluorescence antibody, 50 μl per well, and incubate at 37°C in the dark for 30 minutes;

[0021] ④After the reaction, wash the plate 3 times with PBS and pat dry the remaining liquid in the wells for the last time;

[0022] ⑤ Observe the fluorescence of each well under an inverted fluorescence microscope; judge the result: the normal cell control well should not show specific fluorescence, and the experiment is established; record the luminescence of each well, and calculate the FA-TCID according to the recorded results using the Reed-Muench method 50 .

[0023] Furthermore, the antiserum is rabbit antiserum, dog antiserum or sheep antiserum.

[0024] Furthermore, in step S5, the value of A is 10. 0.09 .

[0025] Compared with the prior art, the novelty of the method of the present invention is as follows:

[0026] 1. The present invention proposes a method for detecting the efficacy of a canine quadruple live vaccine, which uses mixed antisera corresponding to the four virus components contained in the canine quadruple vaccine to specifically neutralize the corresponding three virus components in the vaccine, and then determines the target virus content after eliminating the pathological interference of the three components on the cells, so as to effectively detect the content of the virus components to be tested.

[0027] 2. The present invention proposes a method for detecting the potency of a canine quadruple live vaccine, which measures the FA-TCID of the four virus components. 50 Results After data statistics and analysis, the theoretical values ​​of each vaccine component and FA-TCID 50 The relationship between FA-TCID 50 The results were used to evaluate the efficacy of the test sample (finished product of canine quadruple live vaccine).

[0028] Compared with the prior art, the canine quadruple live vaccine efficacy detection method proposed in the present invention has the following significant technical effects:

[0029] 1. Low testing cost. No need to purchase a large number of experimental animals and supporting environmental facilities (animal houses, cages, feed, drinking water, etc.) and feeding staff (staff hour costs). Only antiserum or a small number of experimental animals for preparing positive serum at a time are needed, and only short-term feeding is performed. The positive serum prepared once can be stored for a long time and used for testing a large number of batches of the same product.

[0030] 2. The detection cycle is short, the entire detection cycle is 5 days.

[0031] 3. The method of the present invention is an in vitro experiment, and the detection results are not affected by individual differences of experimental animals, thus avoiding interference.

[0032] 4. The method of the present invention does not require the use of virus standards, thereby improving experimental safety and further reducing experimental costs. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention.

[0034] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0035] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0036] The method for testing the potency of the canine quadruple live vaccine established in the present invention is as follows: the finished canine quadruple live vaccine is taken and neutralized with a mixed antiserum against the three virus components in the canine quadruple live vaccine. The neutralized product is then titrated with the test product, and finally the virus content of the target virus component in the neutralized product that is not neutralized by the mixed antiserum is determined by direct immunofluorescence antibody staining (FA-TCID 50 ), and then evaluate the efficacy of each component of the canine quadruple live vaccine product, and the efficacy detection of the other three target virus components is carried out according to this method. The antiserum against the four virus components of the canine quadruple live vaccine can be prepared from rabbits, dogs, sheep or other immunized animals, or it can be purchased.

[0037] Example 1 Preparation of four mixed antisera

[0038] Take canine distemper virus (10 6.0 TCID 50 / ml), canine parainfluenza virus (10 6.0 TCID 50 / ml), canine adenovirus (10 7.0 TCID 50 / ml) and canine parvovirus (10 7.0 TCID 50 / ml) attenuated virus to immunize rabbits, and the immunization procedure is shown in Table 1.

[0039] Table 1 Antiserum preparation immunization procedure

[0040] Immunization times Antigen composition Immunization dose Immune site First immunization Antigen + Freund's complete adjuvant 5ml Back subcutaneous Second immunization Antigen + Freund's incomplete adjuvant 5ml Back subcutaneous The third immunization antigen 5ml Back subcutaneous Fourth immunization antigen 5ml Back muscles

[0041] 14 days after the fourth immunization, a large amount of blood was collected, the serum was separated, inactivated and packaged, antiserum was prepared and serum neutralizing antibodies were measured. The results are shown in Table 2.

[0042] Table 2 Antiserum neutralizing antibody determination results

[0043] Antiserum type Neutralizing antibody results Antiserum Standard Anti-canine distemper virus serum 1:512 Should be > 1:256 Anti-canine parainfluenza virus serum 1:645 Response>1:32 Anti-canine adenovirus serum 1:1290 Should be>1:512 Anti-canine parvovirus serum 1:1448 Should be > 1:256

[0044] Take equal volumes of the above three antisera and mix them. The specific mixing method and the target components to be determined are shown in Table 3.

[0045] Table 3 Antiviral serum mixing method and target components

[0046]

[0047] The four mixed antisera were stored at -80°C for future use.

[0048] Example 2: Efficacy test of canine quadruple live vaccine

[0049] Vaccine neutralization: Take four mixed antisera (as shown in Table 3), and use one of the mixed antisera (containing three antisera) in turn to neutralize the virus components in the anti-canine quadruple live vaccine; each bottle of vaccine mixed antiserum is 1 ml, mixed thoroughly and neutralized at 37°C for 1 hour.

[0050] Titration and culture: Take four mixed antisera and the neutralization product of anti-canine quadruple live vaccine respectively, make 10-fold serial dilutions, and take 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Five dilutions were inoculated simultaneously in 96-well cell plates containing the corresponding cells (Table 4) at a cell density of 1×10 5 Each dilution was inoculated in 8 wells, 100 μl per well, and 4 wells were set up as normal cell controls. The cells were cultured continuously for 5 days at 37°C and 5% CO2. The corresponding inoculated cells of the four mixed antisera and the neutralization products of the anti-canine quadruple live vaccine are shown in Table 4.

[0051] Table 4 Inoculated cells of neutralization products corresponding to mixed antisera

[0052]

[0053] Staining: After the incubation, the virus content of the virus components not neutralized by the mixed antiserum in the neutralization product was determined by staining with the direct immunofluorescent antibody corresponding to the target component (FITC labeled, CDV, CAV, CPIV, CPV fluorescent antibodies were purchased from VMRD) 50 , the specific steps are as follows:

[0054] ①Discard the liquid in the culture plate, add pre-cooled 80% acetone, and fix in a 2-8℃ refrigerator for 30 minutes.

[0055] ②After fixation, wash the plate 3 times with PBS. Try to pat dry any remaining liquid in the wells the last time.

[0056] ③ Add the corresponding direct immunofluorescence antibody (FITC-labeled), 50 μl per well, and incubate at 37°C in the dark for 30 minutes.

[0057] ④After the reaction, wash the plate 3 times with PBS. The last time, try to pat dry any remaining liquid in the wells.

[0058] ⑤ Observe the fluorescence of each well under an inverted fluorescence microscope. Judgment: The normal cell control well should not show specific fluorescence, and the experiment is established; record the luminescence of each well, and calculate the FA-TCID according to the recorded results using the Reed-Muench method. 50 .

[0059] The principle of direct immunofluorescence antibody staining (FITC labeling) is to label known antibodies with fluorescein according to the law of antigen-antibody reaction to make fluorescent antibodies, and then use them as probes to detect corresponding substances in tissues or cells. Specific types of methods include direct method, indirect method, sandwich method and complement method, among which the direct method is the earliest method. Under a fluorescence microscope, the source, nature and location of the detected object are determined based on the fluorescence emitted by the complex formed. This method is simple, easy to use, highly specific, fast and convenient, and is often used for virus-specific staining. The present invention uses direct immunofluorescence antibody staining to respectively determine the virus content FA-TCID of four viruses 50 The results should be higher than the theoretical value of the virus content of the corresponding component TCID 50 / ml, FA-TCID 50 The Reed-Muench method was used for calculation.

[0060] According to the theoretical values ​​and FA-TCID of the four viruses 50 Establish theoretical values ​​and FA-TCID of vaccine components 50 The relationship between the virus content test results of each component of the canine quadruple live vaccine is as follows: FA-TCID 50 Subtract the theoretical value of the virus content of the corresponding component, and the minimum value obtained is named A value, which is the direct immunofluorescence test result of the component FA-TCID 50 The difference in the theoretical value of the virus content of the corresponding component is the qualified standard of the virus content of each component plus the A value, which is the B value. After multiple batches of experiments, the A value is taken to be 10 0.09 .

[0061] The above established relationship can be used to evaluate the efficacy of the canine quadruple live vaccine to be tested. The judgment criteria are as follows: when the test value FA-TCID 50 If it is not lower than the B value, it is judged as qualified, otherwise it is judged as unqualified.

[0062] Application Examples

[0063] Three batches of canine quadruple freeze-dried live vaccine products (batch numbers: 20240801, 20240802, 20240803) were prepared in the laboratory using virus liquids with different virus contents (high, medium, and low, see Table 6 for details). The specific preparation method is as follows:

[0064] After the cells for seedling preparation are revived and appropriately proliferated in static culture in culture flasks, the virus solutions of each component are prepared as follows.

[0065] Preparation of canine distemper virus liquid: After Vero cells are expanded to the scale of T225 cell culture flasks, 5 ml of canine distemper virus liquid is inoculated by synchronous inoculation, and cultured at 37°C and 5% CO2. The cells can be harvested when 80% of the cells are diseased. The harvested virus liquid is the canine distemper virus liquid.

[0066] Preparation of canine parainfluenza virus liquid: After MDCK cells are expanded to the scale of T225 cell culture flasks, 5 ml of canine parainfluenza virus liquid is inoculated by synchronous inoculation, and cultured at 37°C and 5% CO2. The cells can be harvested when 80% of the cells are diseased, which is the canine parainfluenza virus liquid.

[0067] Preparation of canine adenovirus fluid: After MDCK cells are expanded to the scale of T225 cell culture flasks, 2 ml of canine adenovirus fluid is inoculated by synchronous inoculation, and cultured at 37°C and 5% CO2. The cells can be harvested when 80% of the cells are diseased, which is the canine adenovirus fluid.

[0068] Preparation of canine parvovirus fluid: After the F81 cells are expanded to the scale of T225 cell culture flasks, 2 ml of canine parvovirus fluid is inoculated by synchronous inoculation, and cultured at 37°C and 5% CO2. The cells can be harvested when 80% of the cells are diseased, which is the canine parvovirus fluid.

[0069] The virus content of the harvested virus liquid is determined after being fully mixed (according to the current version of the "Chinese Veterinary Pharmacopoeia" Appendix III). The virus culture that meets the following standards can be used to prepare the vaccine: The virus content of canine distemper virus liquid should be ≥10 7.0 TCID 50 / ml, the virus content of canine parainfluenza virus liquid should be ≥

[0070] 10 7.5 TCID 50 / ml, the virus content of canine adenovirus fluid should be ≥10 7.5 TCID50 / ml, the virus content of canine parvovirus fluid should be ≥10 8.0 TCID 50 / ml. After testing, the four prepared virus solutions all met the requirements.

[0071] The prepared virus solutions were diluted using MEM cell culture medium. The virus contents after dilution are shown in Table 5.

[0072] Table 5 Theoretical values ​​of virus content in virus solution of each batch of canine quadruple freeze-dried live vaccine

[0073] batch number CDV virus liquid CPIV virus fluid CAV virus liquid CPV virus liquid 20240801 <![CDATA[10 6.0 TCID 50 / ml]]> <![CDATA[10 6.5 TCID 50 / ml]]> <![CDATA[10 6.5 TCID 50 / ml]]> <![CDATA[10 7.0 TCID 50 / ml]]> 20240802 <![CDATA[10 6.5 TCID 50 / ml]]> <![CDATA[10 7.0 TCID 50 / ml]]> <![CDATA[10 7.0 TCID 50 / ml]]> <![CDATA[10 7.5 TCID 50 / ml]]> 20240803 <![CDATA[10 7.0 TCID 50 / ml]]> <![CDATA[10 7.5 TCID 50 / ml]]> <![CDATA[10 7.5 TCID 50 / ml]]> <![CDATA[10 8.0 TCID 50 / ml]]>

[0074] Vaccines were prepared in the ratio of 1 part of each virus liquid to 6 parts of stabilizer, and after being fully shaken, they were quantitatively packaged in 1 ml (1 head) bottles. After packaging, freeze vacuum drying was quickly performed, and the cap was rolled to obtain the finished product of canine quadruple freeze-dried live vaccine. The theoretical value of virus content in the finished product is shown in Table 6.

[0075] Table 6 Theoretical values ​​of virus content in finished products of each batch of canine quadruple freeze-dried live vaccine

[0076] batch number CDV components CPIV components CAV components CPV components 20240801 <![CDATA[10 5.0 TCID 50 / head portion]]> <![CDATA[10 5.5 TCID 50 / head portion]]> <![CDATA[10 5.5 TCID 50 / head portion]]> <![CDATA[10 6.0 TCID 50 / head portion]]> 20240802 <![CDATA[10 5.5 TCID 50 / head portion]]> <![CDATA[10 6.0 TCID 50 / head portion]]> <![CDATA[10 6.0 TCID 50 / head portion]]> <![CDATA[10 6.5 TCID 50 / head portion]]> 20240803 <![CDATA[10 6.0 TCID 50 / head portion]]> <![CDATA[10 6.5 TCID 50 / head portion]]> <![CDATA[10 6.5 TCID 50 / head portion]]> <![CDATA[10 7.0 TCID 50 / head portion]]>

[0077] According to the experimental method and steps in the invention scheme, each batch of vaccine and each component were tested three times. The results are as follows (Tables 7 to 9):

[0078] Table 7 Virus content results of the finished product of the 20240801 batch of canine quadruple freeze-dried live vaccine

[0079]

[0080] Table 8 Virus content results of the finished product of the 20240802 batch of canine quadruple freeze-dried live vaccine

[0081]

[0082] Table 9 Analysis of virus content in the finished product of the 20240803 batch of canine quadruple freeze-dried live vaccine: The test data showed that the direct immunofluorescence test results of the four virus components in the finished vaccine product

[0083]

[0084] (FA-TCID 50 ) are higher than the theoretical values ​​of virus content in the corresponding components.

[0085] TCID 50 and FA-TCID 50 The calculation of LgTCID is based on the Reed-Muench method, and the formula is as follows: 50= logarithm of sample dilution greater than or equal to 50% + distance ratio × logarithm of dilution factor Example:

[0086]

[0087] When the infection rate is higher than or equal to 50% (75% in the above example), the virus dilution (10 -6 The logarithm of the dilution factor (1 / 10) is -1.

[0088]

[0089] lgTCID 50 =-6+0.33×(-1)=-6.33

[0090] Then: TCID 50 =10 -6.33 / 0.1ml, indicating that the virus suspension is 10 -6.33 After dilution, 0.1 ml is inoculated into each well of cells, which can make 50% of the wells produce CPE. The virus content can be expressed as 10 6.33 TCID 50 or 10 7.33 TCID 50 / ml.

[0091] The virus content test results of each component of canine quadruple live vaccine (FA-TCID 50 ) minus the theoretical value of the virus content of the corresponding component, and the minimum value (named A value) is the direct immunofluorescence test result of the component (FA-TCID 50 ) corresponds to the difference in the theoretical value of the virus content of the corresponding component (see Table 10 for details). The qualified standard of the virus content of each component plus the A value is the B value.

[0092] Table 10 Virus content A value of each batch of canine quadruple freeze-dried live vaccine finished product

[0093] batch number CDV Component A Value CPIV Component A Value CAV Component A Value CPV Component A Value 20240801 <![CDATA[10 0.20 ]]> <![CDATA[10 0.09 ]]> <![CDATA[10 0.21 ]]> <![CDATA[10 0.20 ]]> 20240802 <![CDATA[10 0.09 ]]> <![CDATA[10 0.20 ]]> <![CDATA[10 0.20 ]]> <![CDATA[10 0.09 ]]> 20240803 <![CDATA[10 0.29 ]]> <![CDATA[10 0.09 ]]> <![CDATA[10 0.09 ]]> <![CDATA[10 0.12 ]]> Minimum A value for each batch <![CDATA[10 0.09 ]]> <![CDATA[10 0.09 ]]> <![CDATA[10 0.09 ]]> <![CDATA[10 0.09 ]]>

[0094] The calculation formula is as follows:

[0095] Test results (FA-TCID 50 )-theoretical virus content of corresponding components = A value

[0096] Component virus content qualified standard + A value = B value

[0097] Where A value = 10 0.09 .

[0098] The test results of the samples to be tested using the newly established method (FA-TCID 50) is compared with the B value. When the detection value (FA-TCID 50 ) is not lower than the B value, it is judged as qualified; otherwise it is judged as unqualified.

[0099] The method of the present invention has low detection cost and short detection cycle (the entire detection cycle is 5 days). The detection result is not affected by individual differences of experimental animals and avoids interference. In addition, there is no need to use virus standards, which improves experimental safety and further reduces experimental costs.

[0100] The above description is only by way of illustration of certain exemplary embodiments of the present invention. It is undoubted that, for those skilled in the art, the described embodiments can be modified in various ways without departing from the spirit and scope of the present invention. Therefore, the above description is illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A method for detecting the efficacy of a canine quadruple live vaccine, characterized in that: The following steps are involved: S1, respectively using anti-canine distemper virus serum, anti-canine parainfluenza virus serum, anti-canine adenovirus serum and anti-canine parvovirus serum to mix equal volumes of three antisera to obtain four mixed antisera; S2, using one of the mixed antisera to neutralize the virus component in the anti-canine quadruple live vaccine, and using the neutralization product as the test sample for titration and culture; S3. Determine the virus content of the virus components in the neutralization product that are not neutralized by the mixed antiserum by direct immunofluorescence antibody staining (FA-TCID) 50 ; S4, use the other three mixed antisera in turn to process according to steps S2 and S3 to obtain the virus content FA-TCID of the four viruses respectively. 50 The results were higher than the theoretical values ​​of virus content of the corresponding components. 50 The calculation of is carried out using the Reed-Muench method; S5. Based on the theoretical values ​​and FA-TCID of four viruses 50 , establish the theoretical value and FA-TCID of each vaccine component 50 The relationship between the virus content test results of each component of the canine quadruple live vaccine is as follows: FA-TCID 50 Subtract the theoretical value of the virus content of the corresponding component, and the minimum value obtained is named A value, which is the direct immunofluorescence test result of the component FA-TCID 50 The difference in the theoretical values ​​of the virus content of the corresponding components is the B value, which is the qualified standard of the virus content of each component plus the A value; S6. Evaluate the efficacy of the canine quadruple live vaccine to be tested. The criteria are as follows: When the detection value of each component virus in the canine quadruple live vaccine to be tested is FA-TCID 50 If both are not lower than the B value, it is judged as qualified, otherwise it is judged as unqualified.

2. A canine quadruple live vaccine efficacy detection method according to claim 1, characterized in that: The preparation of the four antisera in step S1 comprises the following steps: Take canine distemper virus 10 6.0 TCID 50 / ml, canine parainfluenza virus 10 6.0 TCID 50 / ml, canine adenovirus 10 7.0 TCID 50 / ml and canine parvovirus 10 7.0 TCID 50 / ml four kinds of attenuated viruses were used to immunize rabbits respectively, and the immunization procedure adopted four immunizations; 14 days after the fourth immunization, a large amount of blood was collected, the serum was separated and inactivated and packaged, antiserum was prepared and the serum neutralizing antibodies were determined; among them, the neutralizing antibodies against canine distemper virus serum were greater than 1:256, the neutralizing antibodies against canine parainfluenza virus serum were greater than 1:32, the neutralizing antibodies against canine adenovirus serum were greater than 1:512, and the neutralizing antibodies against canine parvovirus serum were greater than 1:

256.

3. A canine quadruple live vaccine efficacy detection method according to claim 1, characterized in that: The neutralization step of step S2 is as follows: the mixed antiserum is inoculated into the test canine quadruple freeze-dried live vaccine, and after being fully mixed, it is neutralized at 37° C. for 1 hour.

4. A canine quadruple live vaccine efficacy detection method according to claim 1, characterized in that: The titration and incubation steps of step S2 are as follows: take the neutralized product, make a 10-fold serial dilution, take 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 Five dilutions were inoculated simultaneously in 96-well cell plates with the corresponding cells at a cell density of 1×10 5 / ml, 8 wells were inoculated for each dilution, 100 μl was added to each well, and 4 wells with normal cells as controls were set up at the same time, and the cells were continuously cultured at 37°C and 5% CO2 for 5 days; wherein, a mixed antiserum containing anti-canine distemper virus serum, anti-canine parainfluenza virus serum and anti-canine adenovirus serum was neutralized with the virus components in the canine quadruple live vaccine and the neutralization product was inoculated into F81 cells; a mixed antiserum containing anti-canine distemper virus serum, anti-canine parainfluenza virus serum and anti-canine parvovirus serum was neutralized with the virus components in the canine quadruple live vaccine and the neutralization product was inoculated into MDCK cells; a mixed antiserum containing anti-canine distemper virus serum, anti-canine adenovirus serum and anti-canine parvovirus serum was neutralized with the virus components in the canine quadruple live vaccine and the neutralization product was inoculated into MDCK cells; a mixed antiserum containing anti-canine parainfluenza virus serum, anti-canine adenovirus serum and anti-canine parvovirus serum was neutralized with the virus components in the canine quadruple live vaccine and the neutralization product was inoculated into Vero cells.

5. A canine quadruple live vaccine efficacy detection method according to claim 1, characterized in that: Step S3 uses direct immunofluorescence antibodies corresponding to the target components to be measured for staining. The specific operation steps are as follows: ①Discard the liquid in the culture plate, add pre-cooled 80% acetone, and fix in a 2-8℃ refrigerator for 30 minutes; ②After fixation, wash the plate three times with PBS and pat dry any remaining liquid in the wells for the last wash; ③Add the corresponding direct immunofluorescence antibody, 50 μl per well, and incubate at 37°C in the dark for 30 minutes; ④After the reaction, wash the plate 3 times with PBS and pat dry the remaining liquid in the wells for the last time; ⑤ Observe the fluorescence of each well under an inverted fluorescence microscope; judge the result: the normal cell control well should not show specific fluorescence, and the experiment is established; record the luminescence of each well, and calculate the FA-TCID according to the recorded results using the Reed-Muench method 50 .

6. A canine quadruple live vaccine efficacy detection method according to claim 1, characterized in that: The antiserum is rabbit antiserum, dog antiserum or sheep antiserum.

7. A canine quadruple live vaccine efficacy detection method according to claim 1, characterized in that: In step S5, the value of A is 10. 0.09 .

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