Detection method for determining antigen content of inactivated vaccine of hemorrhagic fever with renal syndrome virus

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a quantitative detection method for determining the antigen content of I-type and II-type stock solutions of inactivated vaccines for hemorrhagic fever with renal syndrome. A double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) method is adopted for detection, and the determination method comprises the following steps: taking an I-type or II-type hemorrhagic fever inactivated virus purified antibody as a coating antibody, taking an HRP (horse radish peroxidase) labeled I-type or II-type hemorrhagic fever inactivated virus polyclonal antibody as a detection antibody, taking high-sensitivity TMB (tetramethylbenzidine) as a chromogenic substrate, and determining an absorbance A value at 450nm / 630nm by using a microplate reader. The method provided by the invention can quantitatively detect the antigen content of the I-type stock solution and the II-type stock solution of the inactivated vaccine against hemorrhagic fever with renal syndrome, is good in accuracy, repeatability and intermediate precision, and provides technical support for preparation of semi-finished products of the inactivated vaccine against hemorrhagic fever.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a detection method for determining the antigen content of an inactivated vaccine of hemorrhagic fever with renal syndrome virus. Background Art

[0002] Epidemic hemorrhagic fever (EHF) is a viral disease with a high mortality rate and a severe disease that is prevalent worldwide and frequently occurs in China. Hemorrhagic fever with renal syndrome (HFRS), one of the four major types of viral hemorrhagic fever, is a natural epidemic disease caused by Hantavirus (HV) and spread by rodents. In recent years, my country has made remarkable achievements in the development of HFRS vaccines. Currently, five different inactivated vaccines have obtained new drug certificates and production licenses, and have been proven to be safe and effective in large-scale applications.

[0003] The production of the bivalent inactivated vaccine for hemorrhagic fever with renal syndrome includes the preparation of stock solution and semi-finished product. The semi-finished product is made by diluting the monovalent stock solutions of type I and type II hemorrhagic fever viruses at an antigen content of 1:128, mixing them in equal amounts, and adding suitable stabilizers, appropriate amounts of thimerosal and aluminum hydroxide adjuvants. The Chinese Pharmacopoeia (2020 edition) stipulates that the antigen content of single virus harvest liquid and monovalent stock solution must be tested, and some companies have also controlled the antigen content of virus concentrates. Therefore, the antigen content is a key quality attribute of hemorrhagic fever vaccine intermediates and stock solutions, and the accuracy and effectiveness of the test results are crucial.

[0004] However, the current methods for detecting antigen content in hemorrhagic fever vaccines are all semi-quantitative methods, and the lowest dilution of semi-quantitative positive results is used as the control point of intermediate products and the preparation point of semi-finished products, and the robustness of the process cannot be guaranteed. Therefore, establishing a quantitative detection method for antigen content is of great significance to the stability of the hemorrhagic fever inactivated vaccine preparation process. Summary of the invention

[0005] Purpose of the Invention

[0006] The invention provides a double antibody sandwich ELISA detection method for quantitatively detecting the antigen content of type I and type II stock solutions of inactivated hemorrhagic fever with renal syndrome virus vaccines.

[0007] Technical Solution

[0008] A quantitative detection method for determining the antigen content of type I or type II stock solution of inactivated hemorrhagic fever with renal syndrome virus vaccine, characterized in that it is achieved according to the following steps: using a double antibody sandwich ELISA method for detection, the determination conditions are: coating an ELISA plate with purified antibodies of type I or type II inactivated hemorrhagic fever with renal syndrome virus, adding samples after washing the plate, incubating at 37°C, 100rpm for 1 hour, and washing the plate. Add type I or type II enzyme-labeled antibodies to the ELISA plate, incubating at 37°C, 100rpm for 1 hour, and washing the plate. Add TMB colorimetric solution, place in a dark place at room temperature for color development, color development time: 15 minutes (type I), 7 minutes (type II), and add stop solution to terminate the reaction.

[0009] As an optimization method, the method is implemented in the following steps:

[0010] (1) A method for detecting the antigen content of the ELISA kit of the inactivated hemorrhagic fever with renal syndrome virus vaccine type I stock solution:

[0011] The rabbit anti-type I hemorrhagic fever inactivated virus purified antibody was coated overnight, washed 4 times with washing solution, and the sample was added to the ELISA plate at 50μl / well, incubated at 37℃ for 1 hour, and washed 6 times with washing solution. Type I enzyme-labeled polyclonal antibody was added to the ELISA plate, incubated at 37℃ for 1 hour, and washed 6 times with washing solution. TMB colorimetric solution was added, placed in the dark at room temperature for 15 minutes, and the reaction was terminated by adding stop solution. The absorbance A value was measured at 450nm / 630nm using an ELISA reader, and the antigen content was calculated using a four-parameter fitting curve.

[0012] (2) A method for detecting the antigen content of the ELISA kit of the inactivated vaccine type II of hemorrhagic fever with renal syndrome virus:

[0013] The rabbit anti-type II hemorrhagic fever inactivated virus purified antibody was coated overnight, washed 4 times with washing solution, and the sample was added to the ELISA plate at 50μl / well, incubated at 37℃, 100rpm for 1 hour, and washed 6 times with washing solution. Type II enzyme-labeled antibody was added to the ELISA plate, incubated at 37℃ for 1 hour, and washed 6 times with washing solution. TMB colorimetric solution was added, placed in the dark at room temperature for 7 minutes, and the reaction was terminated by adding stop solution. The absorbance A value was measured at 450nm / 630nm using an ELISA reader, and the antigen content was calculated using a four-parameter fitting curve.

[0014] (3) Determination of antigen content in inactivated hemorrhagic fever with renal syndrome virus vaccine type I stock solution:

[0015] The sample diluent dilutes the type I stock solution to within the range of the standard curve, and the test solution, positive control, and negative control are tested according to the above parameters.

[0016] (4) Determination of antigen content in the inactivated hemorrhagic fever with renal syndrome virus vaccine type II stock solution:

[0017] The sample diluent dilutes the type II stock solution to within the range of the standard curve, and the test solution, positive control, and negative control are tested according to the above parameters.

[0018] The sample to be tested in step (3) is a monovalent stock solution of type I inactivated virus vaccine for hemorrhagic fever with renal syndrome (VERO cells) produced by a certain enterprise. The sample to be tested in step (4) is a monovalent stock solution of type II inactivated virus vaccine for hemorrhagic fever with renal syndrome (VERO cells) produced by a certain enterprise.

[0019] Detailed description of the type I and type II double antibody sandwich ELISA detection method of the present invention:

[0020] Reagents included:

[0021] 1) Coating buffer: 0.05M sodium carbonate solution, 1.59g / L Na2CO3 and 2.94g / L NaHCO3, the balance is deionized water, pH 9.6;

[0022] 2) Type I coating antibody: rabbit anti-type I hemorrhagic fever inactivated virus purified antibody (commissioned by Bioss ANTIBODIES), 0.5 mg / tube, lyophilized powder, stock solution preparation: dilute with water to 0.5 mg / ml, working concentration: 10 μg / mL, i.e. dilute 50 times before use;

[0023] 3) Type II coating antibody: rabbit anti-type II hemorrhagic fever inactivated virus purified antibody (commissioned by Bioss ANTIBODIES), 0.5 mg / tube, lyophilized powder, stock solution preparation: dilute with water to 0.5 mg / ml, working concentration: 10 μg / mL, i.e. dilute 50 times before use;

[0024] 4) The washing solution is PBS (0.8 g / L NaCl, 0.2 g / L KCl, 0.2 g / L Na2HPO4, 1.16 g / L KH2PO4, the balance is deionized water) containing 0.05% (V / V) Tween-20, called PBST, which consists of PBS and 0.05% Tween-20;

[0025] 5) Sample diluent (negative control): PBST containing 10% (W / V) BSA;

[0026] 6) Type I enzyme-labeled polyclonal antibody: HRP-labeled rabbit anti-type I hemorrhagic fever inactivated virus polyclonal antibody (commissioned by BiossANTIBODIES), the stock concentration is 2 mg / mL, the working concentration is 0.5 μg / mL, that is, diluted 4000 times before use;

[0027] 7) Type II enzyme-labeled polyclonal antibody: HRP-labeled rabbit anti-type II hemorrhagic fever inactivated virus polyclonal antibody (commissioned by BiossANTIBODIES), the stock concentration is 2 mg / mL, the working concentration is 0.5 μg / mL, that is, diluted 4000 times before use;

[0028] 8) Secondary antibody diluent: Milk Diluent / Blocking Concentrate Kit (Seracare 5140-0010), diluted 20 times;

[0029] 9) Type I standard: Type I hemorrhagic fever with renal syndrome inactivated virus standard, antigen content: 1:1024 / vial, lyophilized powder, reconstitution volume 0.5 ml;

[0030] 10) Type I positive control: Type I hemorrhagic fever with renal syndrome inactivated virus positive control, lyophilized powder, reconstituted volume 1.0 ml;

[0031] 11) Type II standard: Type II hemorrhagic fever with renal syndrome inactivated virus standard, antigen content: 1:1024 / vial, lyophilized powder, reconstitution volume 0.35 ml;

[0032] 12) Type II positive control: Type II hemorrhagic fever with renal syndrome inactivated virus positive control, lyophilized powder, reconstituted volume 1.0 ml;

[0033] 13) Color developing solution: TMB single-component color developing solution (Solarbio: PR1200);

[0034] 14) Stop solution: 1 mol / L sulfuric acid solution.

[0035] Steps included:

[0036] Type I Antigen Detection ELISA Kit:

[0037] 1) All reagents were equilibrated at room temperature for 30 minutes;

[0038] 2) Add the working concentration of type I coating antibody diluted with coating buffer to the ELISA plate, 100 μL per well, and coat overnight at 4°C;

[0039] 2) Discard the liquid in the wells and wash with washing solution 4 times, 20 seconds each time, and pat dry the residual liquid after each wash;

[0040] 3) Sample incubation: dilute the type I standard, type I positive control, type I negative control and the sample to be tested to the appropriate concentration, add 50 μL of sample solution to each well, and incubate at 37°C and 100 rpm for 1 hour;

[0041] 4) Discard the liquid in the wells and wash 6 times with washing solution, 20 seconds each time, and pat the plate dry;

[0042] 5) Add secondary antibody diluent to the working concentration of type I enzyme-labeled polyclonal antibody to each well of the ELISA plate, incubate at 37°C.

[0043] Incubate with shaking at 100 rpm for 1 hour;

[0044] 6) Discard the liquid in the wells and wash with washing solution 6 times, 20 seconds each time, and pat dry the residual liquid after each wash;

[0045] 7) Prepare the color developing solution, add 100 μL to each well, and develop the color in the dark at room temperature for 15 minutes;

[0046] 8) Add 50 μL of reaction stop solution to each well to terminate the reaction;

[0047] 9) The absorbance was measured by an enzyme-labeled instrument at wavelengths of 450 nm and 630 nm;

[0048] 10) The reciprocal of the antigen content of the standard divided by the dilution factor is used as the horizontal axis, and the absorbance (OD450-OD630) is used as the vertical axis.

[0049] Perform four-parameter fitting on the points of the type standard and calculate the standard curve equation;

[0050] 11) Calculate the antigen content of the sample to be tested using the standard curve equation.

[0051] Type II Antigen Detection ELISA Kit:

[0052] 1) All reagents were equilibrated at room temperature for 30 minutes;

[0053] 2) Add the working concentration of type II coating antibody diluted with coating buffer to the ELISA plate, 100 μL per well, and coat overnight at 4°C;

[0054] 2) Discard the liquid in the wells and wash with washing solution 4 times, 20 seconds each time, and pat dry the residual liquid after each wash;

[0055] 3) Sample incubation: dilute the type II standard, type II positive control, type II negative control, and the sample to be tested to an appropriate concentration, add 50 μL of the sample solution to each well, and incubate at 37°C, 100 rpm, and shake for 1 hour;

[0056] 4) Discard the liquid in the wells and wash with washing solution 6 times, 20 seconds each time, and pat dry the residual liquid after each wash;

[0057] 5) Add the secondary antibody diluent to the working concentration of type II enzyme-labeled polyclonal antibody to each well of the ELISA plate, incubate at 37°C.

[0058] Incubate with shaking at 100 rpm for 1 hour;

[0059] 6) Discard the liquid in the wells and wash with washing solution 6 times, 20 seconds each time, and pat dry the residual liquid after each wash;

[0060] 7) Prepare the color developing solution, add 100 μL to each well, and develop the color in the dark at room temperature for 7 minutes;

[0061] 8) Add 50 μL of reaction stop solution to each well to terminate the reaction;

[0062] 9) The absorbance was measured by an enzyme-labeled instrument at wavelengths of 450 nm and 630 nm;

[0063] 10) With the inverse of the antigen content of the standard as the horizontal axis and the absorbance (OD450-OD630) as the vertical axis, the points of the type II standard

[0064] Perform four-parameter fitting and calculate the standard curve equation;

[0065] 11) Calculate the antigen content of the sample to be tested using the standard curve equation.

[0066] The method described can quantitatively detect the antigen content of type I and type II raw liquid of inactivated hemorrhagic fever with renal syndrome virus vaccine through double antibody sandwich ELISA method, providing technical support for the preparation of hemorrhagic fever inactivated vaccine semi-finished products.

[0067] Beneficial Effects

[0068] 1. The present invention provides for the first time a method for quantitatively detecting antigens in the inactivated hemorrhagic fever with renal syndrome virus vaccine type I and type II stock solution, and there is no literature report on the quantitative detection method.

[0069] 2. The present invention uses type I or type II hemorrhagic fever inactivated virus purified antibody as coating antibody, type I or type II hemorrhagic fever inactivated virus polyclonal antibody labeled with HRP as detection antibody, and highly sensitive TMB as color substrate, and uses an enzyme marker to measure the absorbance A value at 450nm / 630nm. The present invention is simple and fast to measure, and fully complies with the guiding principles of Chinese Pharmacopoeia methodology in terms of accuracy, repeatability, and intermediate precision, and can be used to determine the antigen content of type I and type II stock solutions of hemorrhagic fever with renal syndrome virus inactivated vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 , the four-parameter fitting curve of the inverse of the antigen content of the inactivated vaccine type I standard sample of hemorrhagic fever with renal syndrome and the measured value;

[0071] Figure 2 , four-parameter fitting curve of the inverse of the antigen content of the inactivated vaccine type II standard of hemorrhagic fever with renal syndrome and the measured value. DETAILED DESCRIPTION

[0072] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0073] Example 1 Construction of a quantitative detection method for antigen in the inactivated hemorrhagic fever with renal syndrome virus vaccine type I stock solution

[0074] (1) Instrument consumables: ELISA reader, constant temperature microplate fast oscillator, ELISA plate

[0075] (2) Type I double antibody sandwich ELISA

[0076] Add the working concentration of type I coating antibody diluted with coating buffer to each well of the ELISA plate, 100 μL, and coat overnight at 4°C; discard the liquid in the wells and wash 4 times with washing buffer, 20 seconds each time, and pat dry the residual liquid after each wash; sample incubation: type I standard (gradient dilution after reconstitution: 2, 4, 8, 16, 32, 64, 128 times), type I negative control, type I positive control (diluted 4 times after reconstitution), type I stock solution sample (diluted to the standard curve range, 32 or 64 times), add 50 μL of sample solution to each well, 37°C, 1 00rpm shaking and incubating for 1 hour; discard the liquid in the wells and wash 6 times with washing solution, each time for 20 seconds; add the secondary antibody diluent to the working concentration of type I enzyme-labeled polyclonal antibody to each well, add 100μL, incubate at 37℃, 100rpm shaking for 1 hour; discard the liquid in the wells and wash 6 times with washing solution, each time for 20 seconds; add 100μL of color development solution to each well, and color at room temperature in the dark for 15 minutes; add 50μL of reaction stop solution to each well to terminate the reaction; measure the absorbance with an enzyme marker, and the detection wavelength is 450nm and 630nm;

[0077] Calculation: With the inverse of the antigen content of the standard as the horizontal coordinate and the absorbance (OD450-OD630) as the vertical coordinate, perform four-parameter fitting on the points of type I standard to calculate the standard curve equation; calculate the antigen content of the sample to be tested by multiplying the standard curve equation by the dilution factor.

[0078] Test establishment conditions:

[0079] Negative control OD450-OD630 should be <0.1, positive control OD450-OD630 should be >1.0; fitting curve R 2 ≥0.99; CV value of duplicate wells of the same sample should be <10%.

[0080] Example 2 Construction of a quantitative detection method for antigen in the inactivated vaccine type II stock solution of hemorrhagic fever with renal syndrome virus

[0081] (1) Instrument consumables: ELISA reader, constant temperature microplate fast oscillator, ELISA plate

[0082] (2) Type II double antibody sandwich ELISA

[0083] Add the working concentration of type II coating antibody diluted with coating buffer to each well of the ELISA plate, 100 μL, and coat overnight at 4°C; discard the liquid in the wells and wash 4 times with washing solution, 20 seconds each time, and pat dry the residual liquid after each wash; sample incubation: type I standard (gradient dilution after reconstitution: 16, 32, 64, 128, 256, 512, 1024 times), type II positive control (diluted 8 times after reconstitution), type I stock solution sample (diluted to within the standard curve range, 32 or 64 times), add 50 μL of sample solution to each well, 37°C, Incubate at 100 rpm for 1 hour; discard the liquid in the wells and wash with washing solution for 6 times, 20 seconds each time; add the secondary antibody diluent to the working concentration of type I enzyme-labeled polyclonal antibody to each well, add 100 μL, incubate at 37°C, 100 rpm for 1 hour; discard the liquid in the wells and wash with washing solution for 6 times, 20 seconds each time; add 100 μL of color development solution to each well, and color at room temperature in the dark for 15 minutes; add 50 μL of reaction stop solution to each well to terminate the reaction; measure the absorbance with an enzyme-labeled instrument, and the detection wavelength is 450nm and 630nm;

[0084] Calculation: With the inverse of the antigen content of the standard as the horizontal coordinate and the absorbance (OD450-OD630) as the vertical coordinate, perform four-parameter fitting on the points of type I standard to calculate the standard curve equation; calculate the antigen content of the sample to be tested by multiplying the standard curve equation by the dilution factor.

[0085] Test establishment conditions:

[0086] Negative control OD450-OD630 should be <0.1, positive control OD450-OD630 should be >1.0; fitting curve R 2 ≥0.99; CV value of duplicate wells of the same sample should be <10%.

[0087] Example 3 Methodological verification of antigen content in inactivated hemorrhagic fever with renal syndrome virus vaccine type I stock solution

[0088] The accuracy, precision, standard curve and range of the method for determining the antigen content of the inactivated hemorrhagic fever with renal syndrome virus vaccine type I stock solution developed by the present invention were verified, and the results are as follows, which proves that the method described in the present invention has good feasibility.

[0089] (1) Accuracy verification

[0090] Experimental operation: The sample was subjected to sample addition recovery determination using standard substances, i.e., a Type I stock solution sample was taken and diluted 32 times for antigen content detection. Another batch of samples and a Type I standard solution (antigen content: 1:1024) were mixed in equal volumes and diluted 32 times as a recovery spiked sample for antigen content detection; 6 portions of the sample and the spiked sample were processed, and the recovery was calculated according to the following formula: Recovery (%) = (spiked sample antigen-sample antigen*0.5) / 512*100%, and the recovery results are shown in Table 1. The recoveries of the 6 samples were all within the range of 80%-120%, with a mean recovery of 102% and RSD<10%.

[0091] Table 1 Recovery determination of type I spiked samples

[0092]

[0093] (2) Precision verification

[0094] Repeatability (within-assay precision)

[0095] The antigen content of the type II test sample was determined according to Example 1. The type II stock solution was diluted 32, 64, and 128 times, and 6 samples of each dilution factor were prepared. The test results are shown in Table 2. The intra-test precision was good, and the RSD was less than <5%

[0096] Table 2 Internal precision verification of type II stock solution test

[0097]

[0098] Intermediate precision (inter-assay precision)

[0099] Experimental operation: Different instruments were used to determine the antigen content of type I stock solution and positive control at different test times. Type I stock solution and positive control were diluted 64 and 4 times, respectively. Three samples of each dilution factor were prepared and tested with two ELISA instruments. The results are shown in Table 3. The precision of the stock solution and positive control was good between the tests, and the RSD was less than 10%.

[0100] Table 3 Inter-assay precision verification of type I stock solution and positive control

[0101]

[0102] (3) Standard curve verification

[0103] With the inverse of the antigen content of the standard as the abscissa and the absorbance (OD450-OD630) as the ordinate, a four-parameter fit was performed on the points of the type I standard. The curve is as follows: Figure 1 As shown, the regression equation is as follows, and the correlation coefficient R 2 =1.000.

[0104] Y=D+((AD) / (1+(x / C) B )

[0105]

[0106] (4) Range Verification

[0107] According to the standard curve, accuracy and precision results, the antigen content that can be detected by this assay is 1:8:~1:512. The sample to be tested is diluted to within the range and calculated by multiplying the dilution factor.

[0108] Example 4 Methodological verification of antigen content in inactivated hemorrhagic fever with renal syndrome virus vaccine type II stock solution

[0109] The accuracy, precision, standard curve and range of the method for determining the antigen content of the inactivated vaccine type II stock solution of hemorrhagic fever with renal syndrome developed by the present invention were verified, and the results are as follows, which proves that the method described in the present invention has good feasibility.

[0110] (1) Accuracy verification

[0111] Experimental operation: The sample was subjected to sample addition recovery determination using standard substances, namely, a Type II stock solution sample was taken and diluted 64 times for antigen content detection, and another batch of samples and a Type I standard solution (antigen content: 1:1024) were mixed in equal volumes and diluted 64 times as recovery spiked samples for antigen content detection; 6 portions of the sample and the spiked sample were processed, and the recovery was calculated according to the following formula: Recovery (%) = (specified sample antigen-sample antigen*0.5) / 512*100%, and the recovery results are shown in Table 4. The recoveries of the 6 samples were all within the range of 80%-120%, with a recovery mean of 102% and RSD<10%.

[0112] Table 4 Recovery rate determination of type II spiked samples

[0113]

[0114] (2) Precision verification

[0115] Repeatability (within-assay precision)

[0116] The antigen content of the type I test sample was determined according to Example 1. The type I stock solution was diluted 32, 64, and 128 times, and 6 samples were prepared for each dilution factor. The test results are shown in Table 5. The intra-test precision was good, and the RSD was less than <5%

[0117] Table 5 Internal precision verification of type I stock solution test

[0118]

[0119] Intermediate precision (inter-assay precision)

[0120] Experimental operation: Different instruments were used to determine the antigen content of type I stock solution and positive control at different test times. Type I stock solution and positive control were diluted 64 and 4 times, respectively. Three samples were prepared for each dilution factor and tested with two ELISA instruments. The results are shown in Table 6. The precision of the stock solution and positive control was good between the tests, and the RSD was less than 10%.

[0121] Table 6 Inter-assay precision verification of Type II stock solution and positive control

[0122]

[0123] (3) Standard curve verification

[0124] With the inverse of the antigen content of the standard as the abscissa and the absorbance (OD450-OD630) as the ordinate, a four-parameter fit was performed on the points of the type I standard. The curve is as follows: Figure 1 As shown, the fit is good, the correlation coefficient R2 = 1.000, and the regression equation is shown below.

[0125] Y=D+((AD) / (1+(x / C) B )

[0126]

[0127] (4) Range Verification

[0128] According to the standard curve, accuracy and precision results, the antigen content range that can be detected by this assay is 1:1 to 1:64. The sample to be tested is diluted to within the range and the dilution factor is calculated and multiplied.

[0129] Example 5 Determination of Type I and Type II Stock Solutions of Inactivated Vaccine for Hemorrhagic Fever with Renal Syndrome (Vero Cells)

[0130] Experimental operation: Three batches of type I stock solution and type II stock solution produced by a certain enterprise were respectively taken, and the antigen content was detected using the method described in Example 1 and Example 2. The type I stock solution was diluted 32 times, the type II stock solution was diluted 64 times, and 3 samples were prepared.

[0131] Table 7 Antigen content detection of three batches of type I and type II stock solutions

[0132]

[0133]

[0134] The results are shown in Table 7: the antigen content of the stock solution can be effectively detected.

Claims

1. A method for determining the antigen content of inactivated vaccine for hemorrhagic fever with renal syndrome virus, characterized in that: The method is implemented according to the following steps: the double antibody sandwich ELISA method is used for detection, and the determination conditions are: the purified antibody of inactivated hemorrhagic fever with renal syndrome type I or type II virus is coated on the ELISA plate, and the sample is added after washing the plate, and the plate is incubated with shaking at 37°C and then washed; type I or type II enzyme-labeled polyclonal antibody is added to the ELISA plate, and the plate is incubated with shaking at 37°C and then washed; TMB color developing solution is added, and the color is developed in the dark at room temperature, and the color development time is 15 minutes for type I and 7 minutes for type II, and the reaction is terminated by adding the stop solution.

2. The method according to claim 1, characterized in that Follow these steps to achieve this: (1) Antigen detection of inactivated hemorrhagic fever with renal syndrome virus vaccine type I stock solution: The purified rabbit anti-type I hemorrhagic fever inactivated virus antibody was coated overnight, washed 4 times with washing solution, and the sample was added to the ELISA plate at 50 μl / well, incubated at 37°C, 100 rpm for 1 hour, and washed 6 times with washing solution; the type I enzyme-labeled polyclonal antibody was added to the ELISA plate, incubated at 37°C, 100 rpm for 1 hour, and washed 6 times with washing solution; TMB colorimetric solution was added, and the plate was placed in the dark at room temperature for 15 minutes, and the reaction was terminated by adding the stop solution; the absorbance A value was measured at 450nm / 630nm with an ELISA reader, and the antigen content was calculated using a four-parameter fitting curve; or (2) Antigen test of inactivated hemorrhagic fever with renal syndrome virus vaccine type II stock solution: The purified rabbit anti-type II hemorrhagic fever inactivated virus antibody was coated overnight, washed 4 times with washing solution, and the sample was added to the ELISA plate at 50 μl / well, incubated at 37°C, 100 rpm for 1 hour, and washed 6 times with washing solution; the type II enzyme-labeled antibody was added to the ELISA plate, incubated at 37°C, 100 rpm for 1 hour, and washed 6 times with washing solution; TMB colorimetric solution was added, and the plate was placed in the dark at room temperature for 7 minutes, and the reaction was terminated by adding the stop solution; the absorbance A value was measured at 450nm / 630nm with an ELISA reader, and the antigen content was calculated using a four-parameter fitting curve; (3) Determination of antigen content in inactivated hemorrhagic fever with renal syndrome virus vaccine type I stock solution: The sample diluent dilutes the type I stock solution to within the range of the standard curve, and the test solution, positive control, and negative control are tested according to the above parameters; or (4) Determination of antigen content in the inactivated vaccine for hemorrhagic fever with renal syndrome type II stock solution: The sample diluent dilutes the type II stock solution to within the range of the standard curve, and the test solution, positive control, and negative control are tested according to the above parameters.

3. The method according to claim 2, characterized in that The sample to be tested in step (3) is a VERO cell type I monovalent stock solution of an inactivated vaccine for hemorrhagic fever with renal syndrome virus; and the sample to be tested in step (4) is a VERO cell type II monovalent stock solution of an inactivated vaccine for hemorrhagic fever with renal syndrome virus.

4. An ELISA detection kit, characterized in that: Contains purified antibodies against inactivated hemorrhagic fever with renal syndrome type I or type II virus, and enzyme-labeled polyclonal antibodies type I or type II.

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