Canine distemper vaccine neutralizing antibody detection method and application

Abstract

The invention discloses a canine distemper vaccine neutralizing antibody detection method and application. The detection method comprises the following steps: (1) co-incubating virus and serum; (2) infecting cells with a virus-serum mixture; and (3) judging the valence of the neutralizing antibody through immunofluorescence or enzyme-linked immunosorbent assay. The application comprises a kit, a test strip and other products prepared on the basis of the neutralizing antibody detection method. The detection method for the canine distemper vaccine neutralizing antibody can be used for accurately and effectively determining the canine distemper virus neutralizing antibody in serum. Compared with a traditional neutralizing antibody detection method, the method overcomes the problems of personal error, poor specificity, low sensitivity and long time consumption, provides a powerful tool for prevention and control of canine distemper, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting neutralizing antibodies of canine distemper vaccine and application thereof. Background Art

[0002] Canine distemper virus is a highly contagious virus that can cause serious illness in dogs and damage their immune systems, triggering secondary infections with other pathogens, posing a major threat to canine health and socioeconomics. Regular vaccination with canine distemper vaccines is an effective measure to prevent canine distemper and its secondary infections. The titer of neutralizing antibodies is an important indicator for evaluating the effectiveness and immune protection of canine distemper vaccines. After neutralizing antibodies bind to canine distemper viruses, they form virus-antibody complexes that prevent the virus from binding to cell receptors, thereby preventing the virus from entering cells. The binding of neutralizing antibodies may also activate immune responses, trigger inflammatory responses, and induce the participation of other immune cells, which helps to clear infected viruses. Neutralizing antibodies can make viruses lose their ability to infect and mark viral particles, making them easy for macrophages or other immune cells to take up and clear them. Therefore, the accuracy and sensitivity of neutralizing antibody detection methods are of great significance for the research and supervision of canine distemper vaccines.

[0003] The shortcomings and problems of the current canine distemper vaccine neutralizing antibody detection mainly include the following:

[0004] 1. Lack of standardization of methods: At present, the neutralizing antibody detection method of canine distemper vaccine lacks unified standardization. Different laboratories may use different reagents and operating procedures, resulting in differences in the comparability and accuracy of the results.

[0005] 2. Low sensitivity: The traditional method of detecting neutralizing antibodies through cytopathic effect may be insensitive to low-titer antibodies and has large human errors, thereby underestimating the antibody level.

[0006] 3. Poor specificity: Certain nonspecific reactions may also lead to misdiagnosis.

[0007] In summary, how to design a detection method that can detect antibodies and ensure the specificity and sensitivity of the results is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0008] The main purpose of the embodiments of the present invention is to propose a method for detecting neutralizing antibodies of canine distemper vaccine and its application, aiming to design a detection method that can detect antibodies and ensure the specificity and sensitivity of the results.

[0009] The technical solution of the present invention to solve the above technical problems is to provide a method for detecting neutralizing antibodies of canine distemper vaccine, comprising the following steps:

[0010] 1) Co-incubation of virus and serum:

[0011] The serum and an equal volume of canine distemper virus were mixed evenly and incubated at 34°C to 37°C for 1 to 2 hours to form a complex of virus and antibody;

[0012] 2) Virus-serum mixture infects cells:

[0013] Add the virus-antibody complex to the well plate and incubate at 34-37°C for 1-2 hours to allow the virus to infect the cells;

[0014] The virus-serum mixture was discarded, and the cells were washed twice with culture medium;

[0015] The culture medium was added to the cells, and the cells were placed in a 37°C incubator for another 24 h;

[0016] 3) Determine the neutralizing antibody titer by immunofluorescence or enzyme-linked immunosorbent assay:

[0017] Wash cells twice with buffer and add fixative;

[0018] The fixative was discarded, the product was washed twice with buffer, and a permeabilizing agent was added for transparency;

[0019] Antibodies to the nucleoprotein of canine distemper virus were added for immunofluorescence and / or enzyme-linked immunosorbent assay.

[0020] Furthermore, in the step of uniformly mixing the serum with an equal volume of canine distemper virus and incubating at 34° C. to 37° C. for 1 to 2 hours to form a complex of the virus and the antibody,

[0021] The titer of the canine distemper virus is 200×TCID 50 .

[0022] Furthermore, in the step of washing the cells twice with a buffer and adding a fixative,

[0023] The well plate is a 96-well plate with Vero or MDCK cells attached to the wall in advance; the cell density is 70% to 90%.

[0024] Furthermore, in the step of discarding the fixative, washing twice with a buffer solution, and adding a permeabilizing agent for transparent treatment,

[0025] The fixing agent is 4% paraformaldehyde, and the fixation is carried out at room temperature for 10 to 20 minutes; or the fixing agent is acetone, and the fixation is carried out at 4°C for 5 to 10 minutes.

[0026] Furthermore, in the step of discarding the fixative, washing twice with a buffer solution, and adding a permeabilizing agent for transparent treatment, the permeabilizing agent for transparent treatment includes 0.1% to 2% Triton X-100 or 0.1% to 1% Tween20 permeating the membrane at room temperature for 5 minutes to 10 minutes.

[0027] Furthermore, in the step of adding canine distemper virus nucleoprotein antibody for immunofluorescence detection, the primary antibody is an antibody against canine distemper virus nucleoprotein, and the secondary antibody is a fluorescently labeled secondary antibody; or the primary antibody is a fluorescently labeled antibody against canine distemper virus nucleoprotein.

[0028] Furthermore, in the step of adding canine distemper virus nucleoprotein antibodies for enzyme-linked immunosorbent assay detection, the primary antibody is an antibody against canine distemper virus nucleoprotein, and the secondary antibody is a secondary antibody labeled with alkaline phosphatase or peroxidase; or the primary antibody is an antibody against canine distemper virus nucleoprotein labeled with alkaline phosphatase or peroxidase.

[0029] The present invention also proposes an application of the canine distemper vaccine neutralizing antibody detection method in preparing a kit.

[0030] The present invention also proposes an application of the canine distemper vaccine neutralizing antibody detection method in preparing a test strip.

[0031] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0032] First, the present invention provides a method for detecting neutralizing antibodies of canine distemper vaccine. By formulating a unified operating procedure and quality control standards, this method can ensure the comparability and accuracy of the detection results of neutralizing antibodies of canine distemper vaccine; adopting more sensitive detection methods, such as immunofluorescence or enzyme-linked immunosorbent assay, can improve the detection effect of low-titer antibodies and reduce the influence of human errors; introducing specific antibodies against canine distemper virus nucleoprotein can avoid cross-reactions with other pathogens and improve the specificity of the detection results; in addition, the whole process of this method takes no more than 36 hours, which effectively reduces the cost and time consumption. This method overcomes the problems of human errors, poor specificity, low sensitivity and long time consumption in traditional methods, and provides an effective tool for the prevention and control of canine distemper.

[0033] Second, considering the technical solution as a whole or from the perspective of the product, the technical effects and advantages of the technical solution to be protected by the present invention are described in detail as follows:

[0034] The present invention provides a method for detecting neutralizing antibodies of canine distemper vaccine. The method introduces specific detection antibodies against canine distemper virus nucleoprotein by formulating a unified operation process and quality control standard, and uses a more sensitive immunofluorescence or enzyme-linked immunosorbent assay to determine the level of canine distemper virus neutralizing antibodies in serum. This method can effectively avoid the problems of misjudgment and missed diagnosis, and has the characteristics of accuracy, effectiveness, high specificity and high sensitivity.

[0035] Third, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0036] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0037] The present invention provides a method for detecting neutralizing antibodies of canine distemper vaccine, which can accurately evaluate the level of neutralizing antibodies of canine distemper virus in dogs, help veterinarians determine the immune status of dogs, and thus guide the work of preventing and controlling canine distemper; it has high specificity and high sensitivity, can effectively avoid the problems of misjudgment and missed diagnosis, and provide reliable detection results; it can provide reliable detection means for veterinary drug research, development and supervision, and help evaluate the effect and quality of vaccines. The technical solution of the present invention has broad application prospects and commercial value, and is of great significance in the fields of prevention and control of canine distemper, veterinary drug research and development, and vaccine supervision.

[0038] (2) Whether the technical solution of the present invention solves the technical problems that people have been eager to solve but have never been able to solve successfully:

[0039] The present invention provides a method and application for detecting neutralizing antibodies of canine distemper vaccine, formulates a unified operation process and quality control standard, ensures the comparability and accuracy of neutralizing antibody detection results; adopts immunofluorescence, enzyme-linked immunosorbent assay and detection antibodies against canine distemper virus nucleoprotein to ensure the specificity and sensitivity of the results, which solves the problems existing in traditional methods.

[0040] (3) Whether the technical solution of the present invention overcomes technical prejudice:

[0041] Compared with traditional neutralizing antibody detection methods, this technical solution has indeed overcome the problems of human error, poor specificity, low sensitivity and long time consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0043] Figure 1 This is a flow chart of the steps of the canine distemper vaccine neutralizing antibody detection method of the present invention;

[0044] Figure 2 This is a flow chart of one embodiment of the method for detecting neutralizing antibodies of canine distemper vaccine of the present invention;

[0045] Figure 3 This is a flow chart of one embodiment of the method for detecting neutralizing antibodies of canine distemper vaccine of the present invention;

[0046] Figure 4 This is a graph showing the neutralizing antibody titer induced by the DNA vaccine of the present invention. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the accompanying drawings of the present invention specification to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] It should be noted that all directional indications in the embodiments of the present invention (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0049] In addition, in the present invention, descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "several" or "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0050] In the present invention, unless otherwise clearly specified and limited, the terms "connection", "fixation", etc. should be understood in a broad sense. For example, "fixation" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0051] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0052] The present invention provides a canine distemper vaccine neutralizing antibody detection method and application, aiming to design a detection method capable of detecting antibodies and ensuring the specificity and sensitivity of the results.

[0053] The canine distemper vaccine neutralizing antibody detection method proposed by the present invention will be described in the following specific embodiments:

[0054] In the technical solution of this embodiment, Figure 1 , Figure 2 , Figure 3 As shown, a method for detecting neutralizing antibodies of canine distemper vaccine comprises the following steps:

[0055] Step 1: Incubate the virus with serum:

[0056] S11: The serum and an equal volume of canine distemper virus were mixed evenly and incubated at 34°C to 37°C for 1 to 2 hours to form a virus-antibody complex;

[0057] It can be understood that by incubating the serum with an equal volume of canine distemper virus, the virus is promoted to bind to the antibody in the serum to form a complex of the virus and the antibody.

[0058] Step 2: Infect cells with virus-serum mixture:

[0059] S21: Add the virus-antibody complex to the well plate and incubate at 34-37°C for 1-2 hours to allow the virus to infect the cells;

[0060] It will be appreciated that the virus and antibody complexes are added to the wells to promote virus infection of cells.

[0061] S22: discard the virus-serum mixture and wash the cells twice with culture medium;

[0062] S23: Add the culture medium to the cells, and place the cells in a 37°C incubator for another 24 h;

[0063] Step 3: Determine the neutralizing antibody titer by immunofluorescence or enzyme-linked immunosorbent assay:

[0064] S31: Wash cells twice with buffer and add fixative;

[0065] As will be appreciated, the fixative is added to fix the cells and preserve the integrity of the cell morphology and structure as well as to retain the immunological activity of the antigen.

[0066] S32: discard the fixative, wash twice with buffer, and add a permeabilizing agent for transparency treatment;

[0067] It is understood that a permeabilizing agent is added for transparentization to disrupt the cell membrane and increase cell permeability.

[0068] S331: Add canine distemper virus nucleoprotein antibody for immunofluorescence detection;

[0069] Or S332: Add canine distemper virus nucleoprotein antibody for enzyme-linked immunosorbent assay.

[0070] Furthermore, in the step of uniformly mixing the serum with an equal volume of canine distemper virus and incubating at 34° C. to 37° C. for 1 to 2 hours to form a complex of the virus and the antibody,

[0071] The titer of the canine distemper virus is 200×TCID 50 .

[0072] Furthermore, in the step of washing the cells twice with a buffer and adding a fixative,

[0073] The well plate is a 96-well plate with Vero or MDCK cells attached to the wall in advance; the cell density is 70% to 90%.

[0074] Furthermore, in the step of discarding the fixative, washing twice with a buffer solution, and adding a permeabilizing agent for transparent treatment,

[0075] The fixing agent is 4% paraformaldehyde, and the fixation is carried out at room temperature for 10 to 20 minutes; or the fixing agent is acetone, and the fixation is carried out at 4°C for 5 to 10 minutes.

[0076] Furthermore, in the step of discarding the fixative, washing twice with a buffer solution, and adding a permeabilizing agent for transparent treatment, the permeabilizing agent for transparent treatment includes 0.1% to 2% Triton X-100 or 0.1% to 1% Tween20 permeating the membrane at room temperature for 5 minutes to 10 minutes.

[0077] Furthermore, in the step of adding canine distemper virus nucleoprotein antibody for immunofluorescence detection, the primary antibody is an antibody against canine distemper virus nucleoprotein, and the secondary antibody is a fluorescently labeled secondary antibody; or the primary antibody is a fluorescently labeled antibody against canine distemper virus nucleoprotein.

[0078] Furthermore, in the step of adding canine distemper virus nucleoprotein antibodies for enzyme-linked immunosorbent assay detection, the primary antibody is an antibody against canine distemper virus nucleoprotein, and the secondary antibody is a secondary antibody labeled with alkaline phosphatase or peroxidase; or the primary antibody is an antibody against canine distemper virus nucleoprotein labeled with alkaline phosphatase or peroxidase.

[0079] The present invention also proposes an application of the canine distemper vaccine neutralizing antibody detection method in preparing a kit.

[0080] The present invention also proposes an application of the canine distemper vaccine neutralizing antibody detection method in preparing a test strip.

[0081] Example 1

[0082] A method for detecting neutralizing antibodies of canine distemper vaccine comprises the following steps:

[0083] Step 1: Incubate the virus with serum: dilute 50 μL serum in 2-fold gradients (2 1 ~2 10 ) and then mixed with 50 μL of 200×TCID 50 After mixing evenly, incubate at 37°C for 1 hour to promote the binding of the virus with the antibody in the serum to form a virus-antibody complex.

[0084] Step 2: Infect cells with the virus-serum mixture: 24 hours in advance, spread the digested Vero cells into a 96-well plate and wait until the cell confluence reaches 70%; wash the cells twice with phosphate buffer; add 100 μL of the virus-serum mixture to the cells and incubate at 37°C for 1 hour to promote virus infection of the cells; then, discard the virus-serum mixture and wash the cells twice with phosphate buffer; then, add 100 μL of culture medium to the cells and continue culturing in an incubator at 37°C for 24 hours.

[0085] Step 3: Determine the neutralizing antibody titer by immunofluorescence: First, wash the cells twice with 200 μL phosphate buffer, add 50 μL 4% paraformaldehyde and fix at room temperature for 15 minutes; then, discard the fixative, wash the cells twice with 200 μL phosphate buffer, add 0.1% TritonX-100 to permeabilize the membrane for 10 minutes at room temperature for transparency treatment to destroy the cell membrane and increase cell permeability; then wash the cells twice with 200 μL phosphate buffer, add 5% BSA and incubate at 37°C for 2 hours to block nonspecific binding; then, add FITC-labeled canine distemper virus nucleoprotein antibody and incubate at 37°C in the dark for 1 hour; finally, wash the cells 3 times with 200 μL PBST, observe under a fluorescence microscope, and detect the distribution and content of canine distemper virus by the intensity of the fluorescence signal.

[0086] Example 2

[0087] Step 1: Incubate the virus with serum: dilute 50 μL serum in 2-fold gradients (2 1 ~2 10 ) and then mixed with 50 μL of 200×TCID50 After mixing evenly, incubate at 37°C for 1 hour to promote the binding of the virus with the antibody in the serum to form a virus-antibody complex.

[0088] Step 2: Infect cells with the virus-serum mixture: 24 hours in advance, spread the digested Vero cells into a 96-well plate and wait until the cell confluence reaches 70%; wash the cells twice with phosphate buffer; add 100 μL of the virus-serum mixture to the cells and incubate at 37°C for 1 hour to promote virus infection of the cells; then, discard the virus-serum mixture and wash the cells twice with phosphate buffer; then, add 100 μL of culture medium to the cells and continue culturing in an incubator at 37°C for 24 hours.

[0089] Step 3: Determine the neutralizing antibody titer by ELISA:

[0090] The neutralizing antibody titer was determined by immunofluorescence: first, the cells were washed twice with 200 μL phosphate buffer, and 50 μL 4% paraformaldehyde was added to fix at room temperature for 15 min; then, the fixative was discarded, and the cells were washed twice with 200 μL phosphate buffer, and 0.1% Triton X-100 was added to permeabilize the membrane for 10 min at room temperature to destroy the cell membrane and increase cell permeability; then, the cells were washed twice with 200 μL phosphate buffer, and 5% BSA was added to incubate at 37°C for 2 h to block nonspecific binding; then, sheep anti-canine distemper virus nucleoprotein antibody was added and incubated at 37°C for 1 h; then, the cells were washed three times with 200 μL PBST, peroxidase-labeled rabbit anti-sheep secondary antibody was added, and incubated at 37°C for 45 min; then, the cells were washed three times with 200 μL PBST, 100 μL TMB colorimetric solution was added, and incubated at 37°C in the dark for 15 min; finally, 50 μL 2M H2SO4 was used to terminate the color development, and the absorbance at OD450 nm was measured in an enzyme reader to quantitatively detect the content of canine distemper virus.

[0091] Among them, animal immunization: the animal model is BALB / c female mice (6-8 weeks old, weighing 18-20g, SPF level), the immunogen is DNA vaccine (hemagglutinin gene of canine distemper virus Yanaka strain), the immunization dose is 100μg / 200μL / mouse, and the electric shock boost immunization method is used for three intramuscular injections, with an interval of two weeks.

[0092] Serum treatment: Two weeks after the third immunization, blood was collected from the tail vein. The blood was allowed to stand overnight at 4°C and then centrifuged at 3000 rpm for 30 min to collect the supernatant. The serum was treated at 56°C for 30 min to inactivate complement.

[0093] Enzyme-linked immunosorbent assay for neutralizing antibodies: 50 μL of serum was diluted 2-fold (21 ~2 10 ) and then mixed with 50 μL of 200×TCID 50 After mixing evenly, the canine distemper virus was incubated at 37°C for 1 hour, and 4 replicates were set for each gradient. The virus-serum mixture was added to the pre-attached Vero cells and incubated at 37°C for 1 hour; then, the virus-serum mixture was discarded and the cells were washed twice with phosphate buffer; 100 μL of culture medium was added to the cells and cultured in a 37°C incubator for 24 hours. The cells were washed twice with 200 μL phosphate buffer, and 50 μL 4% paraformaldehyde was added to fix at room temperature for 15 min; then, the fixative was discarded, and the cells were washed twice with 200 μL phosphate buffer, and 0.1% Triton X-100 was added to permeabilize at room temperature for 10 min; then, the cells were washed twice with 200 μL phosphate buffer, and 5% BSA was added to block at 37°C for 2 h; then, sheep anti-canine distemper virus nucleoprotein antibody was added and incubated at 37°C for 1 h; then, the cells were washed 3 times with 200 μL PBST, peroxidase-labeled rabbit anti-goat secondary antibody was added, and incubated at 37°C for 45 min; then, the cells were washed 3 times with 200 μL PBST, 100 μL TMB colorimetric solution was added, and incubated at 37°C in the dark for 15 min; finally, 50 μL 2M H2SO4 was added to stop the color development, and the absorbance at OD450 nm was detected in an enzyme reader to quantitatively detect the content of canine distemper virus.

[0094] The traditional method of measuring neutralizing antibodies by observing the cytopathic effect is to dilute 50 μL of serum in a 2-fold gradient (2 1 ~2 10 ) and then mixed with 50 μL of 200×TCID 50 After mixing the canine distemper virus evenly, incubate at 37°C for 1 hour, with 4 replicates per gradient. Add the virus-serum mixture to pre-attached Vero cells and incubate at 37°C for 1 hour; then discard the virus-serum mixture and wash the cells twice with phosphate buffer; add 100 μL of culture medium to the cells and continue to culture in a 37°C incubator for 5 to 7 days, observe the cytopathic effect, and calculate the titer of neutralizing antibodies by the Reed-Muench method.

[0095] The experiment was repeated three times to compare the traditional observation of cytopathic effect to determine the neutralizing antibody, the detection method of Example 1 and the detection method of Example 2. The difference and repeatability of the neutralizing antibody detection method are as follows. Figure 4 As shown, the detection results obtained by the neutralizing antibody detection method of the present invention have better repeatability and accuracy.

[0096] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for detecting neutralizing antibodies of canine distemper vaccine, characterized in that: The following steps are involved: 1) Co-incubation of virus and serum: The serum and an equal volume of canine distemper virus were mixed evenly and incubated at 34°C to 37°C for 1 to 2 hours to form a complex of virus and antibody; 2) Virus-serum mixture infects cells: Add the virus-antibody complex to the well plate and incubate at 34-37°C for 1-2 hours to allow the virus to infect the cells; The virus-serum mixture was discarded, and the cells were washed twice with culture medium; The culture medium was added to the cells, and the cells were placed in a 37°C incubator for another 24 h; 3) Determine the neutralizing antibody titer by immunofluorescence or enzyme-linked immunosorbent assay: Wash cells twice with buffer and add fixative; The fixative was discarded, the product was washed twice with buffer, and a permeabilizing agent was added for transparency; Add canine distemper virus nucleoprotein antibody for immunofluorescence or enzyme-linked immunosorbent assay.

2. The canine distemper vaccine neutralizing antibody detection method according to claim 1, characterized in that: In the step of uniformly mixing the serum and an equal volume of canine distemper virus and incubating them at 34° C. to 37° C. for 1 to 2 hours to form a complex of the virus and the antibody, The titer of the canine distemper virus is 200×TCID 50 .

3. The canine distemper vaccine neutralizing antibody detection method according to claim 1, characterized in that: In the step of washing the cells twice with a buffer and adding a fixative, The well plate is a 96-well plate with Vero or MDCK cells attached to the wall in advance; the cell density is 70% to 90%.

4. The canine distemper vaccine neutralizing antibody detection method according to claim 1, characterized in that: In the step of discarding the fixative, washing twice with a buffer solution, and adding a permeabilizing agent for transparent treatment, The fixing agent is 4% paraformaldehyde, and the fixation is carried out at room temperature for 10 to 20 minutes; or the fixing agent is acetone, and the fixation is carried out at 4°C for 5 to 10 minutes.

5. The canine distemper vaccine neutralizing antibody detection method according to claim 1, characterized in that: In the step of discarding the fixative, washing twice with a buffer solution, and adding a permeabilizing agent for transparent treatment, the permeabilizing agent for transparent treatment includes 0.1% to 2% Triton X-100 or 0.1% to 1% Tween20 permeating the membrane at room temperature for 5 minutes to 10 minutes.

6. The method for detecting neutralizing antibodies of canine distemper vaccine according to claim 1, characterized in that: In the step of adding canine distemper virus nucleoprotein antibody for immunofluorescence detection, the primary antibody is an antibody against canine distemper virus nucleoprotein, and the secondary antibody is a fluorescently labeled secondary antibody; or the primary antibody is a fluorescently labeled antibody against canine distemper virus nucleoprotein.

7. The method for detecting neutralizing antibodies of canine distemper vaccine according to claim 1, characterized in that: In the step of adding canine distemper virus nucleoprotein antibody for ELISA detection, the primary antibody is an antibody against canine distemper virus nucleoprotein, and the secondary antibody is a secondary antibody labeled with alkaline phosphatase or peroxidase; or the primary antibody is an antibody against canine distemper virus nucleoprotein labeled with alkaline phosphatase or peroxidase.

8. Use of the canine distemper vaccine neutralizing antibody detection method according to any one of claims 1 to 7 in the preparation of a kit.

9. Use of the canine distemper vaccine neutralizing antibody detection method according to any one of claims 1 to 7 in the preparation of a test strip.

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