Method for testing in-vitro efficacy of feline panleucopenia inactivated vaccine based on ELISA method

Abstract

The invention provides a method for detecting the in-vitro efficacy of a feline panleucopenia inactivated vaccine based on an ELISA (Enzyme-Linked Immunosorbent Assay) method, which is used for detecting the in-vitro efficacy of the feline panleucopenia inactivated vaccine in triple inactivated vaccines of feline panleucopenia, rhinotracheitis and calicivirus diseases based on a double-antibody sandwich ELISA detection method. The detection method has the advantages of simplicity in operation, good specificity, high sensitivity and short time consumption. The traditional in-vivo potency animal test can be replaced, the adverse factors of many influence factors, poor repeatability, long verification period and the like caused by the tested animal are solved, and high-throughput vaccine potency detection can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal biological product inspection, and particularly relates to a method for detecting the in vitro potency of inactivated feline panleukopenia vaccine in a feline panleukopenia, rhinotracheitis, and calicivirus triple inactivated vaccine based on a double antibody sandwich ELISA detection method. Background Art

[0002] Feline panleukopenia (FP), also known as feline distemper and infectious feline enteritis, is a highly contagious infectious disease caused by feline parvovirus (FPV) infecting feline animals. After a cat is infected with FPV, it will show clinical symptoms such as typical biphasic fever, mental depression, anorexia, diarrhea, vomiting, dehydration, etc. A significant decrease in white blood cells is a specific indicator of this disease. According to the clinical manifestations and severity at the time of onset, the fatality rate of diseased cats is between 25% and 100%. Currently, vaccination is the most effective method for preventing and treating this disease.

[0003] Currently, there are many vaccine companies in the market producing and selling feline panleukopenia, rhinotracheitis, and calicivirus triple inactivated vaccines (referred to as feline triple inactivated vaccines for short). At present, the potency test methods for feline triple inactivated vaccines approved in China are immunization challenge method and serological method respectively. Although these two methods can intuitively evaluate the in vitro potency of the vaccine, the test process involves screening animals, animal breeding, immunization, blood collection, challenge, etc., with high costs. The results are affected by many factors, and there is a biosafety risk during the test as challenge is required. More importantly, the test cycle is nearly 2 months long, which not only prolongs the R & D cycle of new vaccines but also indirectly shortens the shelf life of vaccines, increasing the pressure on vaccine inventory, seriously affecting the production and sales of vaccines. Therefore, there is an urgent need for a new method for detecting the in vitro relative potency of feline triple inactivated vaccines. Using the ELISA method for relative potency testing can be completed in vitro, with a short test cycle, low cost, few interfering factors, good result repeatability, further ensuring product quality, and having positive significance. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for detecting the in vitro potency of inactivated feline panleukopenia vaccine in a feline panleukopenia, rhinotracheitis, and calicivirus triple inactivated vaccine based on a double antibody sandwich ELISA detection method, which has the advantages of simple operation, good specificity, high sensitivity, and short time consumption.

[0005] Specifically, the present invention first provides a double-antibody sandwich ELISA detection method, and the ELISA detection method is as follows:

[0006] The coating concentration of the capture antibody is 8 μg / mL, 5% skim milk is used as the blocking solution, and it is blocked at 37°C for 120 minutes. The incubation time of the antigen to be detected is 60 minutes at 37°C. The enzyme-labeled antibody is diluted 100-fold and incubated at 37°C for 30 minutes. The single-component TMB chromogenic solution is used for color development at room temperature for 10 minutes.

[0007] Take the antigen to be detected and incubate it at 37°C for 60 minutes, wash the plate 5 times, with an interval of 3 minutes each time, and finally pat it dry on the absorbent paper; then add the HRP-labeled enzyme-labeled antibody (1:100), 100 μl / well, and incubate it at 37°C for 30 minutes, wash the plate 5 times, with an interval of 3 minutes each time, and finally pat it dry on the absorbent paper; take the chromogenic solution restored to room temperature, 100 μl / well, and incubate it in the dark at room temperature for 10 minutes; add the termination solution, 50 μl / well. Place it in an enzyme-linked immunosorbent assay (ELISA) reader and measure the absorbance at a wavelength of 450 nm, and record the measurement results.

[0008] Preferably, the capture antibody is the feline parvovirus monoclonal antibody 3E8; the enzyme-labeled antibody is the feline parvovirus polyclonal antibody.

[0009] Furthermore, the present invention provides an application of the above double-antibody sandwich ELISA detection method in detecting the in vitro potency of the inactivated feline panleukopenia vaccine in the triple inactivated vaccine against feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease. The application is as follows:

[0010] (1) Dilute the capture antibody with the coating solution to 8 μg / ml, add it to the ELISA plate, 100 μl / well, and coat it at 4°C for 16 - 20 hours; after restoring to room temperature, wash the plate 5 times with the washing solution, with an interval of 3 minutes each time, and finally pat it dry on the absorbent paper; then, add the blocking solution, 200 μl / well, and incubate it in a 37°C constant temperature incubator for 120 minutes, wash the plate 5 times, with an interval of 3 minutes each time, and finally pat it dry on the absorbent paper.

[0011] (2) Dilute the vaccine to be tested in the dilution plate, and add the reference vaccine and the vaccine to be tested to the 96-well plate respectively; add 75 μl of the washing solution to the remaining wells respectively, and serially dilute the reference vaccine and the vaccine to be tested 2-fold (horizontally), and transfer 50 μl of the liquid in each well to the corresponding wells of the ELISA plate coated with the capture antibody.

[0012] (3) The ELISA plate was incubated in a 37°C constant temperature incubator for 60 minutes, then washed 5 times, each time with an interval of 3 minutes, and patted dry on absorbent paper for the last time; then HRP-labeled ELISA antibody (1:100) was added, 100 μl / well, and incubated in a 37°C constant temperature incubator for 30 minutes, and the plate was washed 5 times, each time with an interval of 3 minutes, and patted dry on absorbent paper for the last time. The color development solution restored to room temperature was added, and the amount of liquid added was controlled to 100 μl / well, and the above ELISA plate was added, and incubated at room temperature in the dark for 10 minutes; the stop solution was added, 50 μl / well.

[0013] (4) Place the sample in an ELISA instrument and measure the absorbance at a wavelength of 450 nm. Record the measurement results and perform the following calculations:

[0014] Blank control vaccine OD 450nm Average value: blank control vaccine OD 450nm The values ​​are averaged;

[0015] The OD values ​​of 11 different dilutions of the reference vaccine and the test vaccine were measured in triplicate. 450nm The blank control vaccine OD 450nm Average value: the corrected OD of each well 450nm The values ​​calculated for the reference vaccine and the cat triple inactivated vaccine were processed according to the double parallel line bioassay principle and then input into the RelPot 4.0 software for slope and relative potency RP value calculation.

[0016] Furthermore, the method further comprises a result judgment step, specifically:

[0017] The effectiveness of the trial includes:

[0018] A: Blank control vaccine OD 450nm The average value should be less than 0.302;

[0019] B: Reference vaccine original OD 450nm The average value should not be less than 0.559;

[0020] C: Test invalidation criteria: If any of the above criteria is not met, the test is invalid.

[0021] Vaccine qualification criteria:

[0022] 1. Meet the test validity standards;

[0023] 2. Set the RP value of the reference vaccine to 1.0. The RP value of the cat vaccine to be tested should be no less than 1.0. The relative efficacy of the feline panleukopenia antigen in the cat vaccine to be tested is qualified.

[0024] 3. If the RP value of the vaccine to be tested for cats is less than 1.0, it should be retested; if the RP value of the retest result is not less than 1.0, it is judged that the relative potency of feline panleukopenia antigen in the vaccine to be tested for cats is qualified; otherwise, it is judged that the relative potency of feline panleukopenia antigen in the vaccine to be tested for cats is unqualified.

[0025] Preferably, the capture antibody is feline parvovirus monoclonal antibody 3E8; the enzyme-labeled antibody is feline parvovirus polyclonal antibody.

[0026] Beneficial effects

[0027] The method provided by the present invention for detecting the in vitro potency of feline panleukopenia inactivated vaccine in a triple inactivated vaccine against feline panleukopenia, feline rhinotracheitis, and feline calicivirus disease based on a double-antibody sandwich ELISA detection method has the advantages of simple operation, good specificity, high sensitivity, and short time consumption. It can replace the traditional in vivo potency animal experiment, solve the disadvantages of many influencing factors, poor repeatability, and a long verification period of up to 2 months brought by the test animals. At the same time, it can also reduce the large inventory of vaccines on the production line, extend the validity period of the vaccine, ensure the quality of the vaccine, reduce the verification cost and labor intensity, improve the detection efficiency, quickly complete all in vitro relative potency tests of the vaccine and data collation and judgment results, and achieve high-throughput vaccine potency detection. Specific embodiments

[0028] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0029] Main solutions and reagents

[0030] 0.05 mol / L carbonate buffer solution (CBS): Weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, dissolve them in ultrapure water, make up the volume to 1 L, adjust the pH value to 9.6, filter and sterilize, and store at 2 - 8 °C for later use.

[0031] 0.01 mol / L phosphate buffer solution (PBST): Weigh PBS buffer solution (dry powder), dissolve it in ultrapure water, make up the volume to 1 L, adjust the pH value to 7.4, add Tween-20 with a final concentration of 0.05%, mix well, filter and sterilize, and store at 2 - 8 °C for later use.

[0032] Enzyme-labeled antibody diluent: Weigh 5 g of skim milk powder and dissolve it in PBST, make up the volume to 100 mL, mix well, filter and sterilize, and store at 2 - 8 °C for later use.

[0033] Blank control vaccine: Take an appropriate amount of PBS and IMS1313 adjuvant and mix them thoroughly to make the final concentration of the adjuvant 30%.

[0034] FPV inactivated vaccine: Take an appropriate amount of FPV inactivated antigen and mix it with IMS1313 adjuvant, and mix them thoroughly.

[0035] FHV inactivated vaccine: Take an appropriate amount of FHV inactivated antigen and mix it with IMS1313 adjuvant, and mix them thoroughly.

[0036] FCV inactivated vaccine: Take an appropriate amount of FCV inactivated antigen and mix it with IMS1313 adjuvant, and mix them thoroughly.

[0037] The coating solution (CBS buffer at 0.05 mol / L pH 9.6), 0.01 mol / L PBST buffer at pH 7.4, PBST buffer containing 5% skim milk, enzyme-labeled antibody diluent (PBST buffer containing 5% skim milk), chromogenic solution (single-component TMB chromogenic solution), and termination solution are all conventional solutions in the prior art. Among them, the feline panleukopenia, rhinotracheitis, and calicivirus triple inactivated vaccine (strain BTL23 + strain BJS01 + strain BJH13, suspension culture) and the reference vaccine are all commercially available products of Taizhou Boleideli Biotechnology Co., Ltd.

[0038] Example 1 Preparation of Antibody

[0039] Preparation of Capture Antibody

[0040] According to the conventional technical means in the art, BALB / c mice were immunized with purified feline parvovirus (strain HBX05, preserved in our laboratory (GenBank: M38246.1)). The spleen cells of the immunized mice were fused with SP2 / 0 myeloma cells. After cloning and screening, the hybridoma cell line 3E8 that can stably secrete FPV monoclonal antibody was obtained. The hybridoma cell line 3E8 was inoculated into a bioreactor for culture, and the supernatant of the culture solution was harvested and purified to prepare the capture antibody for the double-antibody sandwich ELISA method for detecting the in vitro potency of the feline panleukopenia inactivated vaccine in the feline panleukopenia, rhinotracheitis, and calicivirus triple inactivated vaccine.

[0041] Among them, the feline parvovirus monoclonal antibody 3E8 was deposited on April 21, 2022, at the China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 45149.

[0042] Quality control of capture antibody (feline parvovirus monoclonal antibody):

[0043] Appearance: Colorless or light yellow clear liquid, and turbidity should not occur.

[0044] Sterility test: It shall be carried out according to the appendixes of Part III of the current Chinese Veterinary Pharmacopoeia, and no growth of microorganisms shall be found.

[0045] Determination of neutralizing antibody titer: It shall be determined according to the appendixes of Part III of the current Chinese Veterinary Pharmacopoeia, and the neutralizing antibody titer shall not be lower than 1:64.

[0046] Specificity test: Take the virus solutions of feline parvovirus (HBX05 strain), feline herpesvirus (BJS01 strain) and feline calicivirus (BJH13 strain), and dilute them with DMEM cell culture medium containing 4% bovine serum to 200 TCID 50 / 0.1 mL respectively, then mix them with the same volume of this product, neutralize at 37°C for 1 hour, and inoculate them into 96-well cell culture plates respectively (① Samples neutralized with FPV: Add 100 μL of F81 cell suspension containing 2% bovine serum to each well; ② Samples neutralized with FHV: Inoculate onto the monolayer CRFK cells that have grown; ③ Samples neutralized with FCV: Inoculate onto the monolayer F81 cells that have grown). Inoculate 4 wells for each sample, 100 μL per well; at the same time, set normal cell control and virus control (virus (200 TCID 50 / 0.1 mL) is mixed with the same volume of water for injection and neutralized at 37°C for 1 hour) with 4 wells each; place them in an incubator at 37°C with 5% CO2 and culture and observe for 4 - 7 days. No CPE should appear in the normal cell control wells, and CPE should appear in the virus control wells; no CPE should appear in the wells neutralized with FPV, CPE should appear in the wells neutralized with FHV, and CPE should appear in the wells neutralized with FCV.

[0047] Storage and validity period: Store below -70°C, and the validity period is 24 months.

[0048] Preparation of feline parvovirus polyclonal antibody

[0049] After inactivating feline parvovirus (the same as above), mix it evenly with an appropriate amount of adjuvant and immunize the test cats. After a certain period of time, collect blood. After the blood coagulates and separates the serum, centrifuge at 3000 r / min for 5 minutes, collect the supernatant, inactivate it at 56°C for 30 minutes, and filter and sterilize it with a 0.22 μm filter and then aliquot. The enzyme-labeled antibody used in the double antibody sandwich ELISA method for testing the in vitro potency of feline panleukopenia inactivated vaccine in the feline panleukopenia, rhinotracheitis, calicivirus triple inactivated vaccine is feline parvovirus polyclonal antibody.

[0050] Quality control of feline parvovirus polyclonal antibody:

[0051] Appearance: Colorless or light yellow liquid, and turbidity shall not occur.

[0052] Sterility test: It shall be carried out according to the appendixes of Part III of the current Chinese Veterinary Pharmacopoeia, and no growth of microorganisms shall be found.

[0053] Determination of neutralizing antibody titer: It is determined according to the appendix of Part III of the current Chinese Veterinary Pharmacopoeia. The neutralizing antibody titer should not be lower than 1:64.

[0054] Storage and validity period: Store at below -15°C, and the validity period is 24 months.

[0055] Example 2 Establishment and optimization of double antibody sandwich ELISA method

[0056] I. Preparation of enzyme-labeled plate for detecting feline panleukopenia virus antigen

[0057] Prepare a feline panleukopenia virus monoclonal antibody solution with a concentration of 8 μg / mL using the coating solution (0.05 mol / L carbonate buffer) as the coating solution. Add 100 μl of the coating solution to the enzyme-labeled plate for coating incubation. The coating incubation conditions are 16 hours of coating at 4°C. Discard the coating solution, wash 5 times with PBST buffer, and gently pat dry on the absorbent paper after the last wash. Use 5% skim milk by mass as the blocking solution, add 200 μl of the blocking solution to each well, and block at 37°C for 2 hours. Discard the blocking solution, wash 5 times with PBST buffer, and pat dry; store at 2 - 8°C.

[0058] II. Preparation of enzyme-labeled antibody

[0059] Weigh 5 mg of HRP and dissolve it in 0.4 mL of carbonate buffer. Add 0.1 mL of 25% glutaraldehyde and mix well, then incubate at 37°C for 2 hours; after incubation, first add 0.1 mL of 20% NaCl buffer, then add 2.5 mL of pre-cooled absolute ethanol and immediately mix well; centrifuge at 1000 r / min for 10 minutes, discard the supernatant, add 5 mL of 80% absolute ethanol again, mix well; centrifuge at 1000 r / min for 10 minutes again, discard the supernatant; let it stand at room temperature for 2 minutes and then add 0.5 mL of carbonate buffer to dissolve, add 0.5 mL of 2 mg / mL feline panleukopenia virus polyclonal antibody and mix well at room temperature, add dipotassium hydrogen phosphate to make the pH neutral, dialyze with PBS buffer at 4°C overnight for 8 hours, add 50% glycerol, aliquot; store at below -15°C.

[0060] III. Establishment of double antibody sandwich ELISA method for FPV

[0061] 1 Optimization of capture antibody coating concentration

[0062] To determine the optimal reaction conditions, in this example, the effects of different concentrations of the capture antibody on the detection results were detected under different reaction conditions, and the one with a higher P / N value was selected as the optimal concentration for use. The results of the determination of the coating concentration of the capture antibody 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 when the coating concentration of the capture antibody was 8 μg / mL, the P / N value was relatively large. Therefore, the optimal coating concentration of the capture antibody was determined to be 8 μg / mL.

[0063] Table 1 Optimization of the Coating Concentration of the Capture Antibody

[0064]

[0065] 2 Optimization of the Dilution Factor of the Enzyme-Labeled Antibody

[0066] To determine the optimal reaction conditions, in this example, the effects of different dilution factors of the enzyme-labeled antibody on the detection results were detected under different reaction conditions, and the one with a higher P / N value was selected as the optimal dilution factor. The results of the determination of the dilution factor of the enzyme-labeled antibody are shown in Table 2. The dilution factors of the enzyme-labeled antibody were diluted 100-fold, 200-fold, 400-fold, and 800-fold respectively. The results showed that when the enzyme-labeled antibody was diluted 100-fold, the P / N value was the largest. Therefore, the optimal dilution factor of the enzyme-labeled antibody was determined to be 100-fold.

[0067] Table 2 Optimization of the Dilution Factor of the Enzyme-Labeled Antibody

[0068]

[0069] 3 Optimization of the Blocking Solution

[0070] To determine the optimal reaction conditions, in this example, the effects of different blocking solutions on the detection results were detected under different reaction conditions, and the one with a higher P / N value was selected as the optimal blocking solution. The results of the determination of the blocking solution are shown in Table 3. Different blocking solutions (PBST buffer, 5% skim milk powder, 10% chicken serum, 5% chicken serum, 5% BSA, 3% BSA, 1% BSA, 3% fish gelatin, and 1% fish gelatin) were selected for testing. The results showed that when the blocking solution was 5% skim milk powder, the P / N value was the largest. Therefore, the optimal blocking solution was determined to be 5% skim milk powder.

[0071] Table 3 Optimization of the Blocking Solution

[0072]

[0073] 4 Optimization of the Blocking Time

[0074] To determine the optimal reaction conditions, in this example, under different reaction conditions, the effects of different blocking times on the detection results were detected, and the one with a higher P / N value was selected as the optimal blocking time. The results of the blocking time determination are shown in Table 4. The blocking solution was used to block for 30 minutes, 45 minutes, 60 minutes, 90 minutes, and 120 minutes respectively. The results showed that when blocking for 120 minutes, the P / N value was the largest. Therefore, the optimal blocking time was determined to be 120 minutes.

[0075] Table 4 Optimization of Blocking Time

[0076]

[0077] 5 Optimization of Incubation Time of Antigen to be Detected

[0078] To determine the optimal reaction conditions, in this example, under different reaction conditions, the effects of different incubation times of the antigen to be detected on the detection results were detected, and the one with a higher P / N value was selected as the optimal incubation time of the antigen to be detected. The results of the incubation time determination of the antigen to be detected are shown in Table 5. The antigen to be detected was incubated for 30 minutes, 45 minutes, 60 minutes, 75 minutes, and 90 minutes respectively. The results showed that when the incubation time of the antigen to be detected was 60 minutes, the P / N value was relatively large and the time used was less. Therefore, the optimal incubation time of the antigen to be detected was determined to be 60 minutes.

[0079] Table 5 Optimization of Incubation Time of Antigen to be Detected

[0080]

[0081] 6 Optimization of Incubation Time of Enzyme-Labeled Antibody

[0082] To determine the optimal reaction conditions, in this example, under different reaction conditions, the effects of different incubation times of the enzyme-labeled antibody on the detection results were detected, and the one with a higher P / N value was selected as the optimal incubation time of the enzyme-labeled antibody. The results of the incubation time determination of the enzyme-labeled antibody are shown in Table 6. The enzyme-labeled antibody was incubated for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively. The results showed that when the incubation time of the enzyme-labeled antibody was 30 minutes, the P / N value was the largest. Therefore, the optimal incubation time of the enzyme-labeled antibody was determined to be 30 minutes.

[0083] Table 6 Optimization of Incubation Time of Enzyme-Labeled Antibody

[0084]

[0085] 7 Optimization of Incubation Time of Chromogenic Solution

[0086] To determine the optimal reaction conditions, in this example, the effects of different incubation times of the chromogenic solution on the detection results were detected under different reaction conditions, and the one with a higher P / N value was selected as the optimal incubation time of the chromogenic solution. The determination results of the incubation time of the chromogenic solution are shown in Table 7, and the chromogenic solution was incubated for 5 minutes, 10 minutes, 15 minutes, 20 minutes, and 25 minutes respectively. The results show that when the incubation time of the chromogenic solution is 10 minutes, the P / N value is the largest. Therefore, the optimal incubation time of the chromogenic solution is determined to be 10 minutes.

[0087] Table 7 Optimization of Chromogenic Time

[0088]

[0089] In summary, the double antibody sandwich ELISA method is as follows:

[0090] The coating concentration of the capture antibody is 8 μg / mL, 5% skim milk is used as the blocking solution, blocked at 37 °C for 120 minutes, the incubation time of the antigen to be detected is incubated at 37 °C for 60 minutes, the dilution factor of the enzyme-labeled antibody is diluted 100-fold, incubated at 37 °C for 30 minutes, and the single-component TMB chromogenic solution is developed at room temperature for 10 minutes.

[0091] Take the antigen to be detected and incubate it at 37 °C for 60 minutes, wash the plate 5 times, with an interval of 3 minutes each time, and finally pat it dry on the absorbent paper; then add the HRP-labeled enzyme-labeled antibody (1:100), 100 μl / well, incubate at 37 °C for 30 minutes, wash the plate 5 times, with an interval of 3 minutes each time, and finally pat it dry on the absorbent paper; take the chromogenic solution restored to room temperature, 100 μl / well, incubate at room temperature in the dark for 10 minutes; add the stop solution, 50 μl / well. Put it into the microplate reader and measure the absorbance at a wavelength of 450 nm, and record the measurement results.

[0092] 8 Determination of Critical Value

[0093] 8.1 Determination of the Critical Value of the Blank Control Vaccine

[0094] The established double antibody sandwich ELISA method was used to detect 30 blank control vaccines, measure their OD 450nm values, and calculate their average value and standard deviation. The average value plus 3 times the standard deviation was used as the critical value of the method (the determination of the critical value refers to the national standard GB / T 33411-2016). The results are shown in Table 8. After calculation, the average value of the blank control vaccine is 0.185, and the standard deviation is 0.039. Therefore, the critical value of the blank control vaccine is 0.302, that is, the OD 450nm value of the blank control vaccine should be less than 0.302 for the test to be valid.

[0095] Table 8 Determination of the Critical Value of the Blank Control Vaccine

[0096]

[0097] 8.2 Reference vaccine undiluted OD 450nm value determination

[0098] The established double - antibody sandwich ELISA method was used to detect 30 reference vaccines, and their undiluted OD 450nm values were measured. The average value and standard deviation were calculated, and the average value minus 3 times the standard deviation was used as the critical value of the OD 450nm value under the condition of undiluted reference vaccine (the critical value of the OD 450nm value of the reference vaccine is determined with reference to the national standard GB / T 33411 - 2016). The results showed (see Table 9) that the OD 450nm value of the blank control vaccine was less than 0.302, and the test was valid. After calculation, the average value of the reference vaccine was 0.649, and the standard deviation was 0.030. Therefore, the critical value under the condition of undiluted reference vaccine was 0.559; that is, the OD 450nm value of the undiluted reference vaccine should not be less than 0.559 for the test to be valid.

[0099] Table 9 Determination of the critical value of undiluted reference vaccine

[0100]

[0101] To sum up, the criteria for judging the validity of the double - antibody sandwich ELISA method test are as follows:

[0102] The OD 450nm value of the blank control vaccine should be less than 0.302, and the OD 450nm value of the undiluted reference vaccine should not be less than 0.559.

[0103] 9 Specificity test

[0104] The established double - antibody sandwich ELISA method was used to test feline triple - inactivated vaccine, FPV inactivated vaccine, blank control vaccine, FHV inactivated vaccine and FCV inactivated vaccine. The results showed (Table 10) that according to the criteria for judging the validity of the double - antibody sandwich ELISA method test, the OD 450nm value of the blank control vaccine was less than 0.302, and the test was valid. The test results met the requirements, and the established double - antibody sandwich ELISA method had good specificity and could be used to detect the relative potency of feline panleukopenia antigen in feline triple - inactivated vaccine.

[0105] Table 10 Specificity test

[0106]

[0107] Example 3 Detection of the in vitro potency of feline panleukopenia antigen in vaccines by double - antibody sandwich ELISA method

[0108] (1) Dilute the capture antibody to 8 μg / ml with the coating solution, add it to the ELISA plate, 100 μl per well, and coat at 4°C for 16 - 20 hours; after restoring to room temperature, wash the plate 5 times with the washing solution, with a 3-minute interval each time, and finally pat dry on the absorbent paper; then, add the blocking solution, 200 μl per well, incubate in a 37°C incubator for 120 minutes, wash the plate 5 times, with a 3-minute interval each time, and finally pat dry on the absorbent paper.

[0109] (2) Dilute the prepared reference vaccine and the feline triple-inactivated vaccine in the dilution plate, add 150 μl of the reference vaccine to each of the wells B1, C1, and D1 on the 96-well plate, and add 150 μl of the feline triple-inactivated vaccine to the wells E1, F1, and G1;

[0110] Add 75 μl of the washing solution to the remaining wells (i.e., B2 - B11 to G2 - G11) respectively, serially dilute the reference vaccine and the feline triple-inactivated vaccine 2-fold (horizontally), transfer 50 μl of the liquid in each well to the corresponding wells of the ELISA plate coated with the capture antibody. The sample addition schematic diagram for each well is shown in Table 11.

[0111] Table 11 OD values for the determination of the reference vaccine and the feline triple-inactivated vaccine 450nm Sample addition schematic diagram

[0112] Number 1 2 3 4 5 6 7 8 9 10 11 12 A / / / / / / / / / / / Blank control vaccine B Reference vaccine 1∶2 1∶4 1∶8 1∶16 1∶32 1∶64 1∶128 1∶256 1∶512 1∶1024 Blank control vaccine C Reference vaccine 1∶2 1∶4 1∶8 1∶16 1∶32 1∶64 1∶128 1∶256 1∶512 1∶1024 Blank control vaccine D Reference vaccine 1∶2 1∶4 1∶8 1∶16 1∶32 1∶64 1∶128 1∶256 1∶512 1∶1024 Blank control vaccine E Inactivated feline triple vaccine 1∶2 1∶4 1∶8 1∶16 1∶32 1∶64 1∶128 1∶256 1∶512 1∶1024 Blank control vaccine F Inactivated feline triple vaccine 1∶2 1∶4 1∶8 1∶16 1∶32 1∶64 1∶128 1∶256 1∶512 1∶1024 Blank control vaccine G Inactivated feline triple vaccine 1∶2 1∶4 1∶8 1∶16 1∶32 1∶64 1∶128 1∶256 1∶512 1∶1024 Blank control vaccine H / / / / / / / / / / / Blank control vaccine

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

[0114] (3) Incubate the ELISA plate in a 37°C incubator for 60 minutes, then wash the plate 5 times, with a 3-minute interval each time, and finally pat dry on the absorbent paper; then add the HRP-labeled enzyme-linked antibody (1:100), 100 μl per well, incubate in a 37°C incubator for 30 minutes, wash the plate 5 times, with a 3-minute interval each time, and finally pat dry on the absorbent paper. Take the chromogenic solution restored to room temperature, control the liquid addition amount to 100 μl per well, add it to the above ELISA plate, and incubate at room temperature in the dark for 10 minutes; add the stop solution, 50 μl per well.

[0115] (4) Place it in an ELISA reader, measure the absorbance at a wavelength of 450 nm, record the measurement results, and perform the following calculations:

[0116] OD value of the blank control vaccine 450nm Average value: Take the average of the OD values of the blank control vaccine 450nm values;

[0117] Subtract the OD values of the blank control vaccine from the OD values of the 11 different dilutions of the reference vaccine and the feline triple-inactivated vaccine, each with three replicates 450nm values respectively subtract the OD of the blank control vaccine 450nmAverage value: That is, obtain the corrected OD value of each well; after processing the values calculated from the reference vaccine and the inactivated feline triple vaccine according to the principle of double parallel line biological assay, input them into the RelPot 4.0 software for slope and relative potency RP value calculation. 450nm Value; after processing the values calculated from the reference vaccine and the inactivated feline triple vaccine according to the principle of double parallel line biological assay, input them into the RelPot 4.0 software for slope and relative potency RP value calculation.

[0118] Result judgment:

[0119] The determination of test validity includes:

[0120] A: The average OD value of the blank control vaccine should be less than 0.302; 450nm The average value should be less than 0.302;

[0121] B: The original concentration OD value of the reference vaccine 450nm The average value should not be less than 0.559;

[0122] C: Standard for invalid test: If any one of the above criteria is not met, the test is invalid.

[0123] Standard for vaccine passing judgment:

[0124] 2. Meet the test validity criteria;

[0125] 2. Set the RP value of the reference vaccine to 1.0, the RP value of the inactivated feline triple vaccine should not be less than 1.0, and the relative potency of the feline panleukopenia antigen in the inactivated feline triple vaccine is qualified.

[0126] 3. If the RP value of the inactivated feline triple vaccine is less than 1.0, re - inspection should be carried out; if the RP value of the re - inspection result is not less than 1.0, it is judged that the relative potency of the feline panleukopenia antigen in the inactivated feline triple vaccine is qualified; otherwise, it is judged that the relative potency of the feline panleukopenia antigen in the inactivated feline triple vaccine is unqualified.

[0127] According to the OD values measured for the reaction solutions in each well, 450nm the data in Table 12 below are obtained. According to the calculation of the data in Table 12, the average value of the OD of the blank control vaccine is 0.174, and the average value of the original concentration OD of the reference vaccine is 0.668. The control is established and this test is valid. 450nm the average value of the OD of the blank control vaccine is 0.174, and the average value of the original concentration OD of the reference vaccine is 0.668. The control is established and this test is valid. 450nm the average value of the original concentration OD of the reference vaccine is 0.668. The control is established and this test is valid.

[0128] Table 12 OD values of the inactivated feline triple vaccine 450nm Value

[0129]

[0130] Note: " / " indicates that no operation was performed on this well.

[0131] Subtract the OD value of the blank control vaccine from the OD values of the above - mentioned reference vaccine and inactivated feline triple vaccine 450nm value and subtract the OD value of the blank control vaccine 450nmThe average value was input into the RelPot 4.0 software for slope and relative potency RP value calculation, and the results are shown in Table 13 below. According to the above results, it was determined that the slope was 0.96 and the RP value was 1.29. Taking the reference vaccine as the standard, the relative potency test of feline panleukopenia antigen in the inactivated feline triple vaccine passed the test.

[0132] Table 13 Slope and relative potency RP value of inactivated feline triple vaccine

[0133]

[0134] Example 4 Stability test of double antibody sandwich ELISA method

[0135] Using the method of Example 3 of the present invention, the relative potencies of the same batch and different batches were detected respectively. Randomly select 3 inactivated feline triple vaccines, and use the double antibody sandwich ELISA method established by the present invention to conduct relative potency tests. The test results are shown in Table 14 and Table 15 below.

[0136] The method established by the present invention was used to detect the inactivated feline triple vaccines of the same batch and different batches, and the results showed that the relative potency of feline panleukopenia antigen in the inactivated feline triple vaccine was qualified. The coefficients of variation of the slope and RP value of the same batch were 4.58% and 2.59% respectively; the coefficients of variation of the slope and RP value of different batches were 2.69% and 2.10% respectively. The coefficients of variation were all less than 10%, indicating that the test results were stable and the repeatability was good.

[0137] Table 14 Relative potency test of feline panleukopenia antigen in inactivated feline triple vaccine

[0138]

[0139] Table 15 Relative potency test of feline panleukopenia antigen in inactivated feline triple vaccines of different batches

[0140]

[0141]

[0142] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the present invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A double-antibody sandwich ELISA detection method for non-diagnostic and non-therapeutic purposes, characterized in that The described ELISA detection method includes the following steps: The concentration of the capture antibody coated is 8 μg / mL. Using 5% skim milk as the blocking solution, block at 37°C for 120 minutes. Incubate the antigen to be detected at 37°C for 60 minutes. Dilute the enzyme-labeled antibody by 100-fold and incubate at 37°C for 30 minutes. Color with the single-component TMB chromogenic solution at room temperature for 10 minutes; Incubate the antigen to be detected at 37°C for 60 minutes, wash the plate 5 times with an interval of 3 minutes each time, and finally pat dry on the absorbent paper. Subsequently, add the HRP-labeled enzyme-labeled antibody (1:100), 100 μl / well, and incubate at 37°C for 30 minutes. Wash the plate 5 times with an interval of 3 minutes each time, and finally pat dry on the absorbent paper. Take the chromogenic solution restored to room temperature, 100 μl / well, and incubate at room temperature in the dark for 10 minutes. Add the stop solution, 50 μl / well. Place it in an enzyme-linked immunosorbent assay (ELISA) reader and measure the absorbance at a wavelength of 450 nm, and record the measurement result.

2. The method according to claim 1, wherein The capture antibody is the feline parvovirus monoclonal antibody 3E8; the enzyme-labeled antibody is the feline parvovirus polyclonal antibody. Among them, the feline parvovirus monoclonal antibody 3E8 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on April 21, 2022. Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 45149.

3. Application of a double-antibody sandwich ELISA detection method in testing the in vitro potency of inactivated vaccines, characterized in that The described application is: (1) Dilute the capture antibody with the coating solution to 8 μg / ml, add it to the ELISA plate, 100 μl / well, and coat at 4°C for 16 - 20 hours. After restoring to room temperature, wash the plate 5 times with an interval of 3 minutes each time, and finally pat dry on the absorbent paper. Subsequently, add the blocking solution, 200 μl / well, and incubate in a 37°C constant temperature incubator for 120 minutes. Wash the plate 5 times with an interval of 3 minutes each time, and finally pat dry on the absorbent paper; (2) Dilute the vaccine to be tested in a dilution plate, and add the reference vaccine and the vaccine to be tested to the 96-well plate respectively. Add 75 μl of washing solution to the other wells respectively, and serially dilute the reference vaccine and the vaccine to be tested by 2-fold (horizontally). Transfer 50 μl of the liquid in each well to the corresponding well of the ELISA plate coated with the capture antibody; (3) Incubate the ELISA plate in a 37°C constant temperature incubator for 60 minutes, then wash the plate 5 times with an interval of 3 minutes each time, and finally pat dry on the absorbent paper. Subsequently, add the HRP-labeled enzyme-labeled antibody (1:100), 100 μl / well, and incubate in a 37°C constant temperature incubator for 30 minutes. Wash the plate 5 times with an interval of 3 minutes each time, and finally pat dry on the absorbent paper. Take the chromogenic solution restored to room temperature, control the liquid addition amount to 100 μl / well, add it to the above ELISA plate, and incubate at room temperature in the dark for 10 minutes. Add the stop solution, 50 μl / well; (4) Place it in an enzyme-linked immunosorbent assay (ELISA) reader, measure the absorbance at a wavelength of 450 nm, record the measurement result, and perform the following calculations: OD of blank control vaccine 450nm Average: OD of blank control vaccine 450nm Take the average value; Subtract the OD values of the blank control vaccine from the OD values of the reference vaccine and the 11 different dilutions of each triplicate well to be tested: That is, the corrected OD value of each well is obtained; After processing the values calculated for the reference vaccine and the feline triple inactivated vaccine according to the principle of parallel line bioassay, input them into the RelPot 4.0 software for slope and relative potency RP value calculation. 450nm values from the average OD of the blank control vaccine 450nm : That is, the corrected OD 450nm value of each well is obtained; After processing the values calculated for the reference vaccine and the feline triple inactivated vaccine according to the principle of parallel line bioassay, input them into the RelPot 4.0 software for slope and relative potency RP value calculation.

4. The application according to claim 3, wherein the inactivated vaccine is the feline panleukopenia inactivated vaccine or a combined vaccine or a multivalent vaccine containing the feline panleukopenia inactivated vaccine.

5. The application according to claim 3, wherein the method further comprises a result judgment step, specifically: The determination of the test validity includes: A: OD of blank control vaccine 450nm The average value should be less than 0.302; B: Reference OD of the original vaccine 450nm The average value should be not less than 0.559; C: The criteria for an invalid test: If any of the above criteria is not met, the test is invalid; The criteria for judging the vaccine to be qualified: 1) Meeting the test validity criteria; 2) Setting the RP value of the reference vaccine to 1.0, the RP value of the vaccine to be tested for cats should be not less than 1.0, and the relative potency of feline panleukopenia antigen in the vaccine to be tested for cats is qualified; 3) If the RP value of the vaccine to be tested for cats is less than 1.0, a retest should be conducted; if the RP value of the retest result is not less than 1.0, it is judged that the relative potency of feline panleukopenia antigen in the vaccine to be tested for cats is qualified; otherwise, it is judged that the relative potency of feline panleukopenia antigen in the vaccine to be tested for cats is unqualified.

6. The application according to claim 3, wherein, The capture antibody is the feline parvovirus monoclonal antibody 3E8; the enzyme-labeled antibody is the feline parvovirus polyclonal antibody.

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