An elisa method for detecting the content of vaccine glycoprotein and a method for rapidly evaluating the efficacy of vaccine by using the method

Abstract

The application relates to the technical field of biological detection, and specifically discloses an ELISA detection method for the content of vaccine glycoprotein and a method for rapidly evaluating the efficacy of a vaccine by using the method. The detection method comprises the following steps: blocking a virus monoclonal antibody by using a blocking solution, adding a to-be-detected vaccine, respectively adding a primary antibody, an enzyme label, a chromogenic solution and a termination solution, measuring a detection result and calculating the content of the glycoprotein in the to-be-detected vaccine; the blocking solution comprises the following components: skimmed milk, sodium stearate, butyric acid geranyl ester and a buffer solution. The correlation between the content of the vaccine glycoprotein and the efficacy of the vaccine is established, so that the efficacy of the vaccine is evaluated by detecting the content of the vaccine glycoprotein. Therefore, the method for evaluating the efficacy of the vaccine can effectively reduce the test period and the detection cost of the vaccine efficacy detection, and simultaneously reduce the use of test animals in the detection process.

Description

Technical Field

[0001] This application relates to the technical field of biological detection, specifically to an ELISA method for detecting the content of vaccine glycoproteins and a method for rapidly evaluating vaccine efficacy using this method. Background Technology

[0002] Rabies is an acute zoonotic infectious disease caused by the rabies virus. It is widespread, and once symptoms appear, the mortality rate is close to 100%. Currently, the most effective way to prevent rabies is through the use of rabies vaccines, and the efficacy of rabies vaccines is a key indicator of their quality.

[0003] The current pharmacopoeia uses the NIH method as the standard method for testing rabies vaccine potency. This method involves injecting mice with the rabies vaccine, challenging them with the CVS strain, and calculating the potency of the tested rabies vaccine by comparing it to a standard vaccine. The NIH method involves subjecting the mice to suffering during the testing process, which is unethical and violates the internationally advocated "3R" principle (respect for animal health, environmental protection, and safety). Furthermore, the NIH method has a long testing cycle and cannot quickly obtain results, resulting in high costs for testing rabies vaccine potency. Summary of the Invention

[0004] To reduce the testing cycle and cost of vaccine efficacy testing, and to reduce the use of test animals during the testing process, this application provides an ELISA method for detecting vaccine glycoprotein content and a method for rapidly evaluating vaccine efficacy using this method.

[0005] Firstly, this application provides an ELISA method for detecting the content of vaccine glycoproteins.

[0006] An ELISA method for detecting the content of vaccine glycoproteins, specifically including the following steps: The viral monoclonal antibody is blocked using a blocking solution, and then the vaccine to be tested is added. Primary antibody, enzyme marker, chromogenic solution, and stop solution are added separately. The test results are measured and the content of glycoprotein in the vaccine to be tested is calculated. The blocking solution contains the following components: skim milk, sodium stearate, geraniol butyrate, and buffer solution.

[0007] In the ELISA method for detecting vaccine glycoprotein content provided in this application, skim milk, sodium stearate, and geraniol butyrate are fully dissolved in buffer solution. The resulting blocking solution effectively fills the gaps in the ELISA plate not occupied by antibodies, thereby avoiding the influence of interfering substances on the ELISA detection process. When the above blocking solution is used in the ELISA detection method, the obtained detection results have a small coefficient of variation, indicating that the blocking solution provided in this application can significantly improve the stability of the ELISA detection method.

[0008] Experimental analysis shows that, compared to using bovine serum albumin dissolved in phosphate buffer alone, or casein dissolved in phosphate buffer alone, this application selected skim milk, sodium stearate, and geraniol butyrate to fully dissolve in phosphate buffer to prepare a blocking solution for ELISA detection of vaccine glycoprotein content. The resulting detection results showed a significantly reduced coefficient of variation and a significantly improved stability of the detection method.

[0009] Furthermore, compared to using one or two of skim milk, sodium stearate, and geranyl butyrate fully dissolved in phosphate buffer, this application chooses to simultaneously use skim milk, sodium stearate, and geranyl butyrate fully dissolved in phosphate buffer to prepare a blocking solution for ELISA detection of vaccine glycoprotein content, resulting in significantly improved stability of the detection method. Therefore, this application chooses to simultaneously use skim milk, sodium stearate, and geranyl butyrate fully dissolved in phosphate buffer to prepare the blocking solution.

[0010] Further, the weight parts of each component in the blocking solution are: 2-10 parts of skim milk; 0.2-0.6 parts of sodium stearate; 0.04-0.08 parts of geraniol butyrate; and 150-200 parts of buffer solution.

[0011] Further, the weight parts of each component in the blocking solution are: 4-8 parts of skim milk; 0.3-0.5 parts of sodium stearate; 0.05-0.07 parts of geraniol butyrate; and 160-190 parts of buffer solution.

[0012] In one specific implementation, the amount of skim milk added can be the following components by weight: 2 parts, 4 parts, 6 parts, 8 parts, or 10 parts.

[0013] In some specific implementations, the amount of skim milk added may also be the following components by weight: 2-4 parts, 2-6 parts, 2-8 parts, 4-6 parts, 4-10 parts, 6-8 parts, 6-10 parts, 8-10 parts.

[0014] In one specific implementation, the amount of sodium stearate added can be the following parts by weight: 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, and 0.6 parts.

[0015] In some specific implementations, the amount of sodium stearate added may also be the following parts by weight: 0.2-0.3 parts, 0.2-0.4 parts, 0.2-0.5 parts, 0.3-0.4 parts, 0.3-0.6 parts, 0.4-0.5 parts, 0.4-0.6 parts, or 0.5-0.6 parts.

[0016] In one specific embodiment, the amount of geraniol butyrate added may be the following parts by weight: 0.04 parts, 0.05 parts, 0.06 parts, 0.07 parts, and 0.08 parts.

[0017] In some specific embodiments, the amount of geraniol butyrate added may also be the following parts by weight: 0.04-0.05 parts, 0.04-0.06 parts, 0.04-0.07 parts, 0.05-0.06 parts, 0.05-0.08 parts, 0.06-0.07 parts, 0.06-0.08 parts, or 0.07-0.08 parts.

[0018] In one specific implementation, the amount of buffer solution added may be the following components by weight: 150 parts, 160 parts, 175 parts, 190 parts, or 200 parts.

[0019] In some specific implementations, the amount of buffer solution added may also be the following components by weight: 150-160 parts, 150-175 parts, 150-190 parts, 160-175 parts, 160-200 parts, 175-190 parts, 175-200 parts, and 190-200 parts.

[0020] Experimental analysis shows that when the amount of each component added to the blocking solution is controlled within the above-mentioned range, the coefficient of variation of the detection results obtained by using the prepared blocking solution in the ELISA detection method is further reduced, and the stability of the detection method is further improved. Therefore, this application controls the amount of each component added to the blocking solution within the above-mentioned range.

[0021] Preferably, the amount of sealing liquid used is 100-150 μL / well.

[0022] In one specific implementation, the amount of sealing liquid used can be: 100 μL / well, 130 μL / well, or 150 μL / well.

[0023] In some specific implementations, the amount of sealing liquid used may also be: 100-130 μL / well or 130-150 μL / well.

[0024] Experimental analysis shows that when the amount of blocking solution used is controlled within the above-mentioned range, the coefficient of variation of the detection results obtained by the ELISA detection method is further reduced, and the stability of the detection method is further improved. Therefore, this application controls the amount of blocking solution used within the above-mentioned range.

[0025] Preferably, the sealing time of the sealing liquid is 20-40 minutes.

[0026] In one specific implementation, the sealing time of the sealing liquid can be 20 min, 30 min, or 40 min.

[0027] In some specific implementations, the sealing time of the sealing liquid can also be 20-30 min or 30-40 min.

[0028] Experiments have shown that when the blocking time of the blocking solution is controlled within the above-mentioned range, the coefficient of variation of the detection results obtained by the ELISA detection method is further reduced, and the stability of the detection method is further improved. Therefore, this application controls the blocking time of the blocking solution within the above-mentioned range.

[0029] Preferably, the buffer solution is selected from phosphate buffer or carbonate buffer solution.

[0030] Furthermore, the buffer solution is a phosphate buffer solution with a pH of 7.2.

[0031] Secondly, this application provides a method for rapidly evaluating vaccine efficacy, which utilizes the ELISA detection method for vaccine glycoprotein content to rapidly evaluate vaccine efficacy.

[0032] This application utilizes the ELISA method to detect vaccine glycoprotein content and the NIH method to detect vaccine potency. Based on the detection results, a correlation between vaccine glycoprotein content and potency can be established, enabling rapid evaluation of vaccine potency through this established correlation. Therefore, the rapid method for evaluating vaccine potency provided in this application can reduce the experimental cycle and cost of vaccine potency testing, while also reducing the use of experimental animals during the testing process.

[0033] Preferably, the vaccine is a rabies vaccine; the correlation between the vaccine glycoprotein content and the vaccine efficacy is: b = 2.5507a + 23.809; In the above relationship, b represents the glycoprotein content of the vaccine; a represents the efficacy of the vaccine.

[0034] Experimental analysis shows that, in the correlation between rabies vaccine glycoprotein content and efficacy established in this application, the detected values ​​of rabies vaccine glycoprotein content and efficacy are distributed on or around a straight line, indicating that this application has improved the correlation between rabies vaccine glycoprotein content and efficacy by using a highly stable ELISA detection method.

[0035] Experimental analysis shows that the rabies vaccine potency value obtained using the "potency-glycoprotein content" standard curve provided in this application is very close to the rabies vaccine potency value obtained by the NIH method, with significantly higher accuracy. This indicates that the method for detecting rabies vaccine potency using the "potency-glycoprotein content" standard curve provided in this application can replace the NIH method for detecting rabies vaccine potency. Therefore, the rapid method for evaluating rabies vaccine potency provided in this application can reduce the experimental cycle and cost of vaccine potency testing, while also reducing the use of experimental animals during the testing process.

[0036] Thirdly, this application provides a vaccine glycoprotein content detection reagent or kit, the reagent or kit comprising: blocking solution, viral monoclonal antibody, primary antibody, enzyme marker, chromogenic solution, stop solution, and medically acceptable auxiliary materials.

[0037] In summary, the technical solution of this application has the following specific effects: This application prepares a blocking solution by fully dissolving skim milk, sodium stearate, and geraniol butyrate in phosphate buffer. When the blocking solution is used in an ELISA method for detecting vaccine glycoprotein content, the obtained detection results have a small coefficient of variation, which can significantly improve the stability of the ELISA detection method.

[0038] This application establishes a correlation between rabies vaccine efficacy and glycoprotein content using the ELISA method and the NIH method for detecting rabies vaccine efficacy. The established correlation allows this method to replace the NIH method for rabies vaccine efficacy testing. Therefore, the detection method provided in this application can effectively reduce the testing cycle and cost of vaccine efficacy testing, while also reducing the use of experimental animals during the testing process. Attached Figure Description

[0039] Figure 1 The standard curve of "potency-glycoprotein content" of the rabies vaccine provided in this application. Detailed Implementation

[0040] This application provides an ELISA method for detecting the content of vaccine glycoproteins, specifically including the following steps: 1. Dilute mouse anti-rabies virus monoclonal antibody 1112-1C8-3A5 100-fold with 0.05 mol / L, pH 9.6 carbonate buffer to a concentration of 1 μg / mL. Add 100 μL / well to the microplate and incubate overnight at 2-8°C to coat the microplate. Wash the microplate three times with 300 μL / well of PBST to obtain the coated microplate. Add 100-150 μL / well of blocking buffer to the coated microplate and block at 37°C for 20-40 min. Wash the microplate three times with 300 μL / well of PBST to obtain the blocked microplate. The blocking solution contains the following components: skim milk, sodium stearate, geraniol butyrate, and buffer solution.

[0041] Specifically, the amounts of each component added to the blocking solution are: 2-10 parts skim milk; 0.2-0.6 parts sodium stearate; 0.04-0.08 parts geraniol butyrate; and 150-200 parts buffer solution.

[0042] Furthermore, the amounts of each component added to the blocking solution are as follows: 4-8 parts skim milk; 0.3-0.5 parts sodium stearate; 0.05-0.07 parts geraniol butyrate; and 160-190 parts buffer solution.

[0043] In addition, the buffer solution is selected from phosphate buffer or carbonate buffer solution. Further, the buffer solution is a 0.1 mol / L phosphate buffer solution with a pH of 7.2.

[0044] 2. Add 100 μL of the rabies vaccine to be tested to a closed ELISA plate and incubate at 37°C for 30-45 min. Wash the ELISA plate 5 times with 300 μL of PBST per well to obtain an ELISA plate containing the vaccine. 3. Using phosphate buffer containing 0.5% BSA and 0.05% Tween 20 at a concentration of 0.1 mol / L and a pH of 7.0 as a diluent, dilute mouse anti-rabies virus monoclonal antibody D1-25 10,000 times to a concentration of 0.01 μg / mL. Add 100 μL / well to the ELISA plate containing the vaccine and incubate at 37°C for 30-45 min. Wash the ELISA plate 5 times with 300 μL / well of PBST to obtain the ELISA plate containing the primary antibody. 4. Using phosphate buffer containing 0.5% BSA and 0.05% Tween 20 at a concentration of 0.1 mol / L and a pH of 7.0 as a diluent, the streptavidin-horseradish peroxidase conjugate was diluted 10,000 times to a concentration of 0.1 μg / mL. 100 μL / well was added to the ELISA plate containing the vaccine and incubated at 37°C for 30-45 min. The ELISA plate was then washed 5 times with 300 μL / well of PBST to obtain the ELISA plate containing the enzyme conjugate. 5. Add 100 μL of TMB chromogenic solution to each well of the microplate containing the enzyme label. Incubate at room temperature in the dark for 20-30 min. Then, add 100 μL of 2 mol / L sulfuric acid to each well to terminate the reaction and obtain the microplate to be tested. 6. Place the ELISA plate to be tested into the ELISA reader and read the OD value of the rabies vaccine to be tested at a wavelength of 450 nm. Calculate the glycoprotein content of the rabies vaccine to be tested based on the standard curve plotted using the test results of vaccines with known concentrations.

[0045] This application also provides a method for rapidly evaluating vaccine efficacy, which utilizes the aforementioned ELISA detection method for vaccine glycoprotein content to rapidly evaluate vaccine efficacy.

[0046] Specifically, the vaccine is a rabies vaccine; the relationship between vaccine glycoprotein content and vaccine efficacy is as follows: the linear regression equation of the standard curve of "efficacy-glycoprotein content" is b = 2.5507a + 23.809; where b represents vaccine glycoprotein content and a represents vaccine efficacy.

[0047] The efficacy of the vaccine was tested using the NIH method. The specific steps for testing the efficacy of the rabies vaccine using the NIH method are as follows: 1. The standard vaccine was serially diluted with PBS buffer at 1:25, 1:125, and 1:625. Each diluted sample was used to immunize mice intraperitoneally. The weight of the mice was 12±0.5g. Each mouse was injected intraperitoneally with 0.5mL. One week later, the immunized mice were immunized a second time using the same method. 2. The vaccine to be tested was serially diluted with PBS buffer at 1:25, 1:125 and 1:625. Each dilution was used to immunize mice intraperitoneally. The weight of the mice was 12±0.5g. Each mouse was injected intraperitoneally with 0.5mL. One week later, the immunized mice were immunized again in the same way. 3. Fourteen days after the first immunization of mice, the rabies virus used for testing was diluted to a challenge virus solution of 30 LD50 / 0.03 mL using PBS buffer containing 2% newborn calf serum. This virus solution was used as 100, and the mice that had been immunized a second time were challenged in the brain with 0.03 mL of the virus solution per mouse. Normal mice raised under the same conditions were used as the virus re-tipping group. The weight of the mice was 12 ± 0.5 g. The 100 challenge virus solution was serially diluted 10-fold using PBS buffer. Four dilutions of 100, 10⁻¹, 10⁻², and 10⁻³ were taken, and each dilution was used to challenge the mice in the virus re-tipping group in the brain with 0.03 mL of the virus solution per mouse. 4. Mice were observed daily for 14 days after the attack, and the mortality of mice was recorded. Starting from day 5, the number of dead mice and mice exhibiting typical rabies brain symptoms were counted daily, and the efficacy of the rabies vaccine to be tested was calculated.

[0048] The present application will be further described in detail below with reference to preparation examples 1-31, examples 1-43, comparative examples 1-8 and performance testing tests. These examples should not be construed as limiting the scope of protection claimed in this application.

[0049] Preparation Example Preparation Examples 1-25 Preparation Examples 1-25 each provide a sealing liquid.

[0050] The difference between the above preparation examples lies in the amount of each component added to the blocking solution. See Table 1 for details.

[0051] The preparation methods for the above preparation examples are as follows: according to the amount of each component added in the blocking solution in Table 1, skim milk, sodium stearate and geraniol butyrate are fully dissolved in 0.1 mol / L phosphate buffer solution with a pH of 7.2 to prepare the blocking solution.

[0052] Table 1 shows the amount of each component added to the blocking solution in Preparation Examples 1-25. Preparation Examples 26-31 Preparation Examples 26-31 each provide a sealing liquid.

[0053] The difference between the above preparation examples and Preparation Example 4 lies in the different types of components in the sealing liquid. See Table 2 for details.

[0054] Table 2 shows the types of components in the blocking solutions used in Preparation Examples 4 and 26-31. Example

[0055] Examples 1-25 Examples 1-25 provide an ELISA method for detecting the glycoprotein content of rabies vaccine.

[0056] In the detection methods of the above embodiments, the standard vaccine in step (2) is the WHO 7th generation international standard rabies vaccine.

[0057] The difference between the above embodiments and Embodiment 4 is that the type of sealing liquid in step (1) is different. See Table 3 for details.

[0058] The implementation method of the above embodiments specifically includes the following steps: 1. Dilute mouse anti-rabies virus monoclonal antibody 1112-1C8-3A5 100-fold with 0.05 mol / L, pH 9.6 carbonate buffer to a concentration of 1 μg / mL. Add 100 μL / well to the microplate and incubate overnight at 4°C to coat the microplate. Wash the microplate three times with 300 μL / well of PBST to obtain the coated microplate. Add 130 μL / well of blocking buffer to the coated microplate and block at 37°C for 30 min. Wash the microplate three times with 300 μL / well of PBST to obtain the blocked microplate. 2. Add 100 μL of the rabies vaccine to be tested to a closed ELISA plate, incubate at 37°C for 35 min, and wash the ELISA plate 5 times with 300 μL of PBST per well to obtain an ELISA plate containing the vaccine. 3. Using phosphate buffer containing 0.5% BSA and 0.05% Tween 20 at a concentration of 0.1 mol / L and a pH of 7.0 as a diluent, dilute mouse anti-rabies virus monoclonal antibody D1-25 10,000 times to a concentration of 0.01 μg / mL. Add 100 μL / well to the ELISA plate containing the vaccine and incubate at 37°C for 35 min. Wash the ELISA plate 5 times with 300 μL / well of PBST to obtain the ELISA plate containing the antibody. 4. Using phosphate buffer containing 0.5% BSA and 0.05% Tween 20 at a concentration of 0.1 mol / L and a pH of 7.0 as a dilution buffer, the streptavidin-horseradish peroxidase conjugate was diluted 10,000 times to a concentration of 0.1 μg / mL. 100 μL / well was added to the ELISA plate containing the vaccine and incubated at 37°C for 35 min. The ELISA plate was then washed 5 times with 300 μL / well of PBST to obtain the ELISA plate containing the enzyme conjugate. 5. Add 100 μL of TMB chromogenic solution to each well of the microplate containing the enzyme label. Incubate at room temperature in the dark for 25 min. Then, add 100 μL of 2 mol / L sulfuric acid to each well to terminate the reaction and obtain the microplate to be tested. Using the methods provided in steps (1)-(5), test the standard vaccine of known concentration and plot the standard curve based on the OD value of the test results; 6. Place the ELISA plate to be tested into the ELISA reader and read the OD value of the rabies vaccine to be tested at a wavelength of 450 nm. Calculate the glycoprotein content of the rabies vaccine to be tested based on the standard curve.

[0059] Table 3. Types of sealing liquids in Examples 1-25 Examples 26-29 Examples 26-30 provide an ELISA method for detecting the glycoprotein content of rabies vaccine.

[0060] The difference between the above embodiments and Embodiment 4 is that the amount of sealing liquid used in step (1) is different. See Table 4 for details.

[0061] Table 4. Amount of sealing liquid used in Examples 4 and 26-29 Examples 30-33 Examples 30-33 provide an ELISA method for detecting the glycoprotein content of rabies vaccine.

[0062] The difference between the above embodiments and Embodiment 4 is that the closing time in step (1) is different. See Table 5 for details.

[0063] Table 5. Closure times in Examples 4, 30-33 Comparative Example Comparative Examples 1-6 Comparative Examples 1-6 each provide an ELISA method for detecting the glycoprotein content of rabies vaccine.

[0064] The difference between the above comparative examples and Example 4 is that the type of sealing liquid in step (1) is different. See Table 6 for details.

[0065] Example Types of sealing liquid Example Types of sealing liquid 1 Preparation Example 26 4 Preparation Example 29 2 Preparation Example 27 5 Preparation Example 30 3 Preparation Example 28 6 Preparation Example 31 Comparative Example 7 Comparative Example 7 provides an ELISA method for detecting the glycoprotein content of rabies vaccine.

[0066] The difference between this comparative example and Example 4 is that the blocking solution in step (1) is different. The blocking solution is prepared by weighing 6.46 mg of bovine serum albumin and dissolving it completely in 175 mg of 0.1 mol / L phosphate buffer at pH 7.2. The remaining steps are the same as in Example 4.

[0067] Comparative Example 8 Comparative Example 8 provides an ELISA method for detecting the glycoprotein content of rabies vaccine.

[0068] The difference between this comparative example and Example 4 is that the blocking solution in step (1) is different. The blocking solution is prepared by weighing 6.46 mg of casein and dissolving it completely in 175 mg of 0.1 mol / L phosphate buffer at pH 7.2. The remaining steps are the same as in Example 4.

[0069] Performance testing This performance test examines the stability of the ELISA method for detecting glycoprotein content in rabies vaccines.

[0070] Using the ELISA method for detecting glycoprotein content in rabies vaccines provided in Examples 1-33 and Comparative Examples 1-8, the glycoprotein content of the vaccine to be tested was measured repeatedly 6 times. The data from the 6 measurements were statistically processed, and the coefficient of variation of the test results was calculated.

[0071] The formula for calculating the coefficient of variation of the test results is as follows: Coefficient of variation = (standard deviation / mean) × 100%.

[0072] Test results are shown in Table 7.

[0073] Table 7. Coefficients of variation of the detection results in Examples 1-33 and Comparative Examples 1-8 Referring to Table 7, by comparing the detection results of Examples 1-33 and Comparative Examples 1-8, it can be seen that the blocking solution prepared in this application is prepared by fully dissolving skim milk, sodium stearate, and geraniol butyrate in phosphate buffer. When the above blocking solution is used in the ELISA method for detecting vaccine glycoprotein content, the coefficient of variation of the obtained detection results is small, indicating that the blocking solution provided in this application can significantly improve the stability of the ELISA detection method.

[0074] Comparing the detection results of Example 4, Comparative Example 1, and Comparative Examples 7-8, it can be seen that compared to using bovine serum albumin alone fully dissolved in phosphate buffer, or casein alone fully dissolved in phosphate buffer, the blocking solution prepared by using skim milk alone fully dissolved in phosphate buffer for the ELISA detection of vaccine glycoprotein content slightly reduces the coefficient of variation and slightly improves the stability of the detection method. Furthermore, the blocking solution prepared in this application by using skim milk, sodium stearate, and geraniol butyrate fully dissolved in phosphate buffer for the ELISA detection of vaccine glycoprotein content significantly reduces the coefficient of variation and significantly improves the stability of the detection method.

[0075] Comparing the detection results of Example 4 with Comparative Examples 2-3 and 6, it is evident that compared to using sodium stearate alone, or geranyl butyrate alone, or a combination of sodium stearate and geranyl butyrate, the blocking solution prepared by fully dissolving skim milk, sodium stearate, and geranyl butyrate in phosphate buffer for ELISA detection of vaccine glycoprotein content, yields a significantly reduced coefficient of variation and improved stability. Therefore, this application selects to fully dissolve skim milk, sodium stearate, and geranyl butyrate in phosphate buffer to prepare the blocking solution.

[0076] Comparing the detection results of Example 4 with Comparative Examples 1 and 4-5, it is evident that compared to using skim milk alone dissolved in phosphate buffer, or using geraniol butyrate and skim milk fully dissolved in phosphate buffer, or using geraniol butyrate and skim milk fully dissolved in phosphate buffer, this application selects to simultaneously use sodium stearate, geraniol butyrate, and skim milk fully dissolved in phosphate buffer to prepare the blocking solution for the ELISA detection method of vaccine glycoprotein content. The resulting detection results show a significantly reduced coefficient of variation and significantly improved stability. Therefore, this application selects to simultaneously use sodium stearate, geraniol butyrate, and skim milk fully dissolved in phosphate buffer to prepare the blocking solution.

[0077] Comparing the detection results of Examples 1-25, it is evident that when the added amounts of each component in the blocking solution are controlled within the range of 2-10 parts skim milk, 0.2-0.6 parts sodium stearate, 0.04-0.08 parts geranyl butyrate, and 150-200 parts buffer, the coefficient of variation of the detection results obtained using the ELISA method for detecting vaccine glycoprotein content is further reduced, and the stability of the detection method is further improved. Therefore, this application controls the added amounts of each component in the blocking solution within the above-mentioned range. Further, this application controls the added amounts of each component in the blocking solution within the range of 4-8 parts skim milk, 0.3-0.5 parts sodium stearate, 0.05-0.07 parts geranyl butyrate, and 160-190 parts buffer.

[0078] Comparing the detection results of Examples 4 and 26-29, it is evident that in the ELISA method for detecting vaccine glycoprotein content, when the amount of blocking solution used is controlled at 100-150 μL / well, the coefficient of variation of the obtained detection results is further reduced, and the stability of the detection method is further improved. Therefore, this application controls the amount of blocking solution used within the above range.

[0079] Comparing the detection results of Examples 4 and 30-33, it is evident that in the ELISA method for detecting vaccine glycoprotein content, when the blocking time is controlled at 20-40 min, the coefficient of variation of the obtained detection results is further reduced, and the stability of the detection method is further improved. Therefore, this application controls the blocking time of the blocking solution within the above-mentioned range.

[0080] Examples 34-43 Examples 34-43 provide an ELISA method for detecting the content of vaccine glycoproteins.

[0081] The difference between the above embodiments and Embodiment 4 is that the vaccine in step (2) is the self-produced serum-free rabies vaccine to be tested; the difference between Embodiments 34-43 is that the batches of the self-produced serum-free rabies vaccine to be tested are P1-P. 10 The details are shown in Table 8.

[0082] The results of the glycoprotein content detection are shown in Table 8.

[0083] Table 8P1-P 10 Test results of glycoprotein content and potency of batches of self-produced serum-free rabies vaccine Methods for evaluating vaccine efficacy I. For P1-P respectively above 10 The efficacy of each batch of vaccines was tested.

[0084] Batch number P1-P 10 Using domestically produced serum-free rabies vaccine as the test subject, the potency test values ​​of the corresponding batches of domestically produced serum-free rabies vaccine were obtained using the NIH method. The test results are shown in Table 8. The NIH method specifically includes the following steps: 1. The standard vaccine was serially diluted with PBS buffer at 1:25, 1:125, and 1:625. Each dilution was used to immunize mice intraperitoneally. The weight of the mice was 12±0.5g. Each mouse was injected intraperitoneally with 0.5mL. One week later, the immunized mice were immunized again using the same method. 2. The self-produced serum-free rabies vaccine to be tested was serially diluted with PBS buffer at 1:25, 1:125, and 1:625. Each dilution was used to immunize mice intraperitoneally. The weight of the mice was 12±0.5g. Each mouse was injected intraperitoneally with 0.5mL. One week later, the immunized mice were immunized again using the same method. 3. Fourteen days after the first immunization of mice, the rabies virus for testing was diluted to 30 LD using PBS buffer containing 2% newborn calf serum. 50 / 0.03mL of attack virus solution, and use this virus solution as 100 Mice that had undergone secondary immunization were challenged intracerebrally, with 0.03 mL injected into the brain of each mouse. Normal mice raised under the same conditions were used as the virus re-inoculation group, with a weight of 12 ± 0.5 g. 10 mL of the virus was injected into the brain using PBS buffer. 0 The attack virus solution was serially diluted 10-fold, and 10 were taken. 0 10 -1 10 -2 and 10 -3 Four dilutions were used, and each dilution was used to challenge the brains of mice in the viral backdipation group. Each mouse was challenged with 0.03 mL of virus in the brain. 4. Mice were observed daily for 14 days after the attack, and the mortality of mice was recorded. Starting from the 5th day, the number of dead mice and mice exhibiting typical rabies brain symptoms were counted daily, and the efficacy of the self-produced serum-free rabies vaccine to be tested was calculated.

[0085] II. Establish the correlation between rabies vaccine glycoprotein content and rabies vaccine efficacy.

[0086] Based on the glycoprotein content and potency test values ​​of different batches of self-produced serum-free rabies vaccines provided in Table 8, a standard curve of "potency-glycoprotein content" for rabies vaccines was plotted with vaccine potency as the x-axis and the corresponding glycoprotein content as the y-axis. The results are as follows: Figure 1 As shown.

[0087] Depend on Figure 1 It can be seen that the test results of glycoprotein content and potency of different batches of self-produced serum-free rabies vaccines are distributed on or around a straight line. The linear regression equation of the standard curve of "potency-glycoprotein content" of rabies vaccine is b = 2.5507a + 23.809, R0 2 =0.9657, indicating a good linear relationship between the glycoprotein content and efficacy of the self-produced serum-free rabies vaccine. Therefore, the ELISA detection method for vaccine glycoprotein content provided in this application can be combined with the NIH method for detecting rabies vaccine efficacy to plot a standard curve of "efficacy-glycoprotein content" for rabies vaccines, thereby successfully establishing the correlation between rabies vaccine glycoprotein content and efficacy, and achieving the goal of rapidly evaluating vaccine efficacy through the established correlation.

[0088] III. The accuracy of evaluating vaccine efficacy using the "potency-glycoprotein content" standard curve.

[0089] (1) Let P be the batch number 11 -P 20Using the self-produced serum-free rabies vaccine as the test object, the above vaccine was tested using the ELISA method provided in Example 4 to obtain the glycoprotein content value of the corresponding batch of self-produced serum-free rabies vaccine. Then, the glycoprotein content value was substituted into the linear regression equation of the vaccine "potency-glycoprotein content" standard curve to calculate the potency value corresponding to different batches of self-produced serum-free rabies vaccine.

[0090] (2) Batch P was obtained using the NIH method. 11 -P 20 The potency value of the domestically produced serum-free rabies vaccine.

[0091] (3) The results of the vaccine efficacy test using the above two methods are shown in Table 9.

[0092] Batch 9 is P 11 -P 20 The efficacy of domestically produced serum-free rabies vaccine Based on Table 9, the test results show that for the same batch of self-produced serum-free rabies vaccine, the rabies vaccine potency value obtained using the "potency-glycoprotein content" standard curve provided in this application is very close to the rabies vaccine potency value obtained by the NIH method, with an accuracy rate of over 99%. This indicates that the "potency-glycoprotein content" standard curve method provided in this application can replace the NIH method for detecting rabies vaccine potency. Therefore, the method for evaluating rabies vaccine potency provided in this application can effectively reduce the experimental cycle and cost of vaccine potency testing, while also reducing the use of experimental animals during the testing process.

[0093] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An ELISA method for detecting the content of vaccine glycoproteins, characterized in that, Specifically, the following steps are included: The virus monoclonal antibody was blocked with blocking solution, and then the vaccine to be tested was added. Primary antibody, enzyme marker, chromogenic solution and stop solution were added respectively. The test results were measured and the content of glycoprotein in the vaccine to be tested was calculated. The components in the sealing solution are in the following weight proportions: 2-10 parts skim milk; 0.2-0.6 parts sodium stearate; Geraniol butyrate 0.04-0.08 parts; buffer solution 150-200 parts; The amount of the sealing liquid used is 100-150 μL / well; The sealing time of the sealing liquid is 20-40 minutes; The vaccine glycoprotein is a rabies vaccine glycoprotein.

2. The ELISA method for detecting vaccine glycoprotein content according to claim 1, characterized in that, The components in the blocking solution are in the following weight proportions: 4-8 parts skim milk; 0.3-0.5 parts sodium stearate; 0.05-0.07 parts geraniol butyrate; and 160-190 parts buffer solution.

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