A method for detecting the content of an antigen

Abstract

This invention discloses a method for detecting antigen content, comprising the following steps: diluting the test sample and a reference sample to specific concentrations; serially diluting the test sample and reference sample to specific concentrations using a diluent on a 96-well hemagglutination plate; adding sensitized red blood cell suspension to wells of different dilutions of the test sample and reference sample, mixing, allowing to stand, and then determining the agglutination results of each well; scoring and recording the agglutination results of each well; summing the scores of each test sample and recording it as A, and summing the total scores of each reference sample and recording it as B; calculating the antigen content of the test sample according to the formula: antigen content of the reference sample × 2. (A‑B) / 16 This method is convenient and fast. The test results are calculated using the self-developed method and compared with the test results of the reference sample, which can obtain relatively accurate test results and solve the problem of inconsistent test results of different batches of sensitized red blood cell suspensions.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and specifically to a method for detecting antigen content. Background Technology

[0002] Rabies is an important zoonotic infectious disease caused by the rabies virus (RV). Once symptoms appear, it is 100% fatal, and there is currently no effective treatment. The development and production of inactivated rabies vaccines require a quantitative amount of rabies antigen. Live rabies virus can be detected by measuring the tissue median lethal dose (TCID). 50 The antigen content is determined using [method / method]. Inactivated rabies virus has no activity to infect tissues, therefore the tissue median lethal dose (TCID50) cannot be used. 50 Rabies antigen content needs to be assessed. Inactivated rabies antigen must undergo concentration and purification processes before it can be used for vaccine preparation. However, these processes can lead to antigen loss, meaning antigen is lost with the waste liquid. It is necessary to test the antigen content before and after concentration, before and after purification, and in the waste liquid to ensure that the antigen is effectively concentrated and purified, and that the antigen lost with the waste liquid is within acceptable limits. This necessitates sensitive and specific antigen content assay methods. The BCA protein quantification method requires sufficiently pure rabies antigen for quantification, limiting its application range; the ELISA method has low accuracy in determining rabies content, and the median lethal dose (TCID) in the same tissue is low. 50 The results of ELISA for detecting rabies antigen vary greatly; the agar amplification method has low sensitivity and cannot be used for quantitative detection of rabies antigen; the real-time PCR method has many steps and high reagent costs.

[0003] Porcine circovirus (PCV) belongs to the genus *Circovirus* in the family Circoviridae. The virus particles are 14–17 nm in diameter, exhibit icosahedral symmetry, and lack an envelope. Currently, three serotypes of PCV are known: PCV1, PCV2, and PCV3. The PCV2 viral genome mainly includes two open reading frames (ORF1 and ORF2). ORF1 encodes the viral protein replicase (Replicin protein, Rep), and ORF2 encodes the viral capsid protein (Cap). Cap is the main structural protein of PCV2. Vaccination against this virus is an important means of controlling PCV2 infection and related diseases. Currently, there are inactivated circovirus vaccines prepared by inactivating the whole virus, recombinant inactivated E. coli circovirus vaccines prepared using the PCV2-Cap antigen expressed by the *E. coli* expression system, and recombinant baculovirus circovirus vaccines prepared using the PCV2-Cap antigen expressed by the insect cell baculovirus expression system. Among these, the recombinant baculovirus circovirus vaccine is widely used due to its high Cap protein content and good immunogenicity.

[0004] The development and production of recombinant baculovirus circovirus inactivated vaccines require quantitative determination of PCV2-Cap antigen. The antigen must be purified before use in vaccine preparation, but the purification process may result in antigen loss, i.e., antigen is lost with the waste liquid. It is necessary to test the antigen content before and after purification, as well as the antigen content in the waste liquid, to ensure that the antigen is effectively purified and that the antigen lost with the waste liquid is within acceptable limits. This necessitates sensitive and specific antigen content determination methods. Among these methods, ELISA for PCV2-Cap antigen content determination is time-consuming, involves multiple steps, and requires expensive consumables and reagents; gel electrophoresis or Western blotting methods combined with grayscale scanning have low accuracy and are also time-consuming and involve multiple steps.

[0005] Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the genus Arteritisvirus in the family Arteritisviridae. This virus can cause respiratory disorders in pigs of all ages, leading to cyanosis due to hypoxia, exhibiting symptoms known as "blue ear disease" or "blue ear syndrome." The development and production of inactivated PRRSV vaccines require a quantitative amount of PRRSV antigen. In recent years, with advancements in vaccine manufacturing processes, using concentrated and purified antigens in vaccine preparation has become a primary method for vaccine manufacturers to improve vaccine quality. However, the purification process can lead to antigen loss, i.e., antigen is lost with the waste liquid. It is necessary to test the antigen content before and after purification, as well as the antigen content in the waste liquid, to ensure effective purification and that the antigen lost with the waste liquid is within acceptable limits. This necessitates sensitive and specific antigen content determination methods. Commonly used ELISA methods for determining PRRSV antigen content are time-consuming, involve multiple steps, and require expensive consumables and reagents.

[0006] Reverse indirect hemagglutination assay (RIHA) is an indirect agglutination test using red blood cells as carriers. Red blood cells are sensitized with known antibodies and then reacted with a sample under suitable conditions. If the sample contains the target antigen, the antigen and antibody will specifically bind, resulting in visible agglutination of the red blood cells. RIHA is a commonly used method for qualitative and quantitative detection of pathogens. It is frequently used for qualitative pathogen detection, thus serving as a diagnostic method for infectious diseases. However, this method has some drawbacks that limit its application in antigen quantification. For example, the results are inconsistent between sensitized red blood cells prepared by different laboratories and between different batches of sensitized red blood cells. Due to significant batch-to-batch variability in sensitized red blood cells, the results of testing the same sample using different batches of sensitized red blood cells vary considerably. Therefore, the reverse indirect hemagglutination method has not been widely used for antigen quantification.

[0007] Therefore, it is necessary to develop accurate quantitative methods for detecting the content of rabies, porcine circovirus type 2 (PCV2), and PRRSV antigens to solve the problems of inconsistent results, unstable and inaccurate results, and unsuitability for quantitative detection of inactivated rabies virus and porcine reproductive and respiratory syndrome virus (PRRSV) by different batches of sensitized red blood cells using the reverse indirect hemagglutination method. This will enable the control of antigen quality in the production of rabies, PCV2, and PRRSV. Summary of the Invention

[0008] The main objective of this invention is to propose a method for detecting antigen content, which aims to stably and quantitatively detect the content of rabies virus, PCV2-Cap antigen, and PRRSV antigen.

[0009] To achieve the above objectives, the present invention provides a method for detecting antigen content, the method comprising the following steps:

[0010] S10. Prepare four concentrations of test samples and reference samples. Use diluent to serially dilute the four concentrations of test samples and reference samples in a 96-well blood coagulation plate to obtain test samples and reference samples with different dilutions.

[0011] S20. Add sensitized red blood cell suspension to the wells of the test samples and reference samples with different dilutions, mix well, let stand, and then determine the agglutination results of each well. Score and record the agglutination results of each well. Sum the scores of each test sample and record them as A. Sum the total scores of each reference sample and record them as B.

[0012] S30. According to the formula, the antigen content of the tested sample = antigen content of the reference sample × 2 (A-B) / 16

[0013] Optionally, before step S10, the preparation of a sensitized red blood cell suspension is further included, and the steps for preparing the sensitized red blood cell suspension are as follows:

[0014] S101. After washing, aldehyde-modifying, and tanning sheep red blood cells, resuspend them in buffer solution to obtain a resuspended solution.

[0015] S102. A monoclonal antibody is added to the resuspended solution to sensitize it, and then a preservative is added to obtain a sensitized red blood cell suspension.

[0016] Optionally, in step S101, the buffer solution is an acetate buffer, wherein the pH value of the acetate buffer solution is 4.6–5.4; and / or,

[0017] The concentration of the acetate buffer solution is 0.01–0.1 mol / L.

[0018] Optionally, in step S102, the monoclonal antibody includes any one of the following monoclonal antibodies:

[0019] Monoclonal antibodies against rabies N protein or monoclonal antibodies against rabies G protein;

[0020] Monoclonal antibodies targeting the PCV2-Cap protein;

[0021] Monoclonal antibody against PRRSV.

[0022] Optionally, in step S10, the four concentrations of the tested samples include: samples containing 100% of the tested sample, samples containing 80% of the tested sample, samples containing 64% of the tested sample, and samples containing 51% of the tested sample; and / or,

[0023] The four reference samples include: 100% reference sample, 80% reference sample, 64% reference sample, and 51% reference sample.

[0024] Optionally, in step S10, the reference sample includes any one of the following antigens: a solution or lyophilized product containing inactivated rabies virus, a solution containing PCV2-Cap antigen, and a solution containing PRRSV antigen.

[0025] Optionally, in step S10, the diluent is a phosphate buffer containing 1% healthy rabbit serum, wherein the pH of the phosphate buffer is 7–7.4; and / or,

[0026] The volume concentration of the phosphate buffer solution is 0.01–0.1 mol / L.

[0027] Optionally, step S20 includes: scoring the agglutination results of the sensitized red blood cell suspension in each well, wherein the agglutination results include any one of 100% sensitized red blood cell agglutination, 75% sensitized red blood cell agglutination, 50% sensitized red blood cell agglutination, 25% sensitized red blood cell agglutination, and 0% sensitized red blood cell agglutination, and then recording the 100% sensitized red blood cell agglutination, 75% sensitized red blood cell agglutination, 50% sensitized red blood cell agglutination, 25% sensitized red blood cell agglutination, and 0% sensitized red blood cell agglutination results as 4, 3, 2, 1, and 0 points respectively.

[0028] Optionally, the antigen includes any one of rabies virus antigen, PCV2-Cap antigen, and PRRSV antigen.

[0029] Optionally, the rabies virus antigen includes at least one of live rabies virus, inactivated rabies antigen, purified rabies antigen, and antigen from an inactivated rabies vaccine; and / or,

[0030] The PCV2-Cap antigen includes either the PCV2-Cap antigen expressed by an insect cell baculovirus expression system or the purified antigen of the PCV2-Cap antigen; and / or,

[0031] The PRRSV antigen is either the PRRSV antigen or the purified antigen of the PRRSV antigen.

[0032] This invention discloses a reverse indirect hemagglutination method for accurately determining antigen content. This method offers high detection results and is highly sensitive. It utilizes common, low-cost reagents, equipment, and consumables commonly used in laboratories, resulting in low testing costs per sample. Sensitized red blood cells and reference samples are easily preserved for extended periods, and the diluent formulation is simple and easy to prepare. The method is simple, convenient, and rapid, requiring no special equipment or instruments, and can be completed within 2-3 hours. It is suitable for application in R&D, production, and quality control stages, quickly obtaining content results. Furthermore, the method incorporates a reference sample; the test results of the tested sample are compared with those of the reference sample using a formula, providing a relatively accurate content result for the tested sample and resolving the issue of inconsistent test results between different batches of sensitized red blood cells. The resulting reverse indirect hemagglutination method for detecting antigen levels is stable and accurate, and can be used to quantify inactivated rabies antigen, PCV2-Cap antigen, and PRRSV antigen, thereby controlling antigen quality in vaccine production. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is an example illustration of standard sensitized red blood cell agglutination at different degrees in embodiments of the present invention;

[0035] Figure 2 This is a result diagram of Embodiment 3 of the present invention;

[0036] Figure 3 This is a result diagram of Embodiment 7 of the present invention;

[0037] Figure 4 This is a result diagram of Embodiment 11 of the present invention. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0039] Furthermore, the technical solutions of the various embodiments can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0041] Rabies is an important zoonotic infectious disease caused by the rabies virus (RV). Research on the rabies virus and detection of viral load are of great significance for the effective prevention and control of rabies.

[0042] Porcine circovirus type 2 (PCV2) can cause multisystemic wasting syndrome in weaned piglets. Vaccination is an important means of controlling PCV2 infection and related diseases. Among them, recombinant baculovirus inactivated circovirus vaccine is widely used due to its high Cap protein content and good immunogenicity. Rapid and accurate detection of PCV2-Cap content can monitor antigen production and control vaccine quality.

[0043] Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the genus Arteritisvirus in the family Arteritisviridae. Vaccination is a crucial means of controlling PRRSV infection and related diseases. Currently, live and inactivated PRRSV vaccines are widely used. Rapid and accurate detection of PRRSV levels can monitor antigen production and control vaccine quality.

[0044] The reverse indirect hemagglutination assay is a commonly used method for qualitative and quantitative detection of pathogens. However, this method has some drawbacks that limit its widespread application, such as inconsistent test results for sensitized red blood cells prepared in different laboratories and inconsistent test results for different batches of sensitized red blood cells.

[0045] In view of this, the present invention proposes a method for detecting antigen content, which aims to effectively solve the problem of rapid and accurate detection of rabies antigen, PCV2-Cap antigen, and PRRSV antigen.

[0046] In the accompanying drawings of this invention, Figure 1 This is an example illustration of standard sensitized red blood cell agglutination at different degrees in embodiments of the present invention; Figure 2 This is a result diagram of Embodiment 3 of the present invention; Figure 3 This is a result diagram of Embodiment 7 of the present invention; Figure 4This is a result diagram of Embodiment 11 of the present invention.

[0047] This invention proposes a method for detecting antigen content, wherein the antigen content detection method employs a reverse indirect hemagglutination assay, and the method includes the following steps:

[0048] S10. Prepare four concentrations of test samples and reference samples. Use diluent to serially dilute the four concentrations of test samples and reference samples in a 96-well blood coagulation plate to obtain test samples and reference samples with different dilutions.

[0049] S20. Add sensitized red blood cell suspension to the wells of the test samples and reference samples with different dilutions, mix well, let stand, and then determine the agglutination results of each well. Score and record the agglutination results of each well. Sum the scores of each test sample and record them as A. Sum the total scores of each reference sample and record them as B.

[0050] S30. According to the formula, the antigen content of the tested sample = antigen content of the reference sample × 2 (A-B) / 16

[0051] It should be noted that "antigen content" mainly refers to the amount of virus or virus-like particles capable of producing immunogenicity, including live viruses, inactivated viruses, and virus-like particles. This invention employs a reverse indirect hemagglutination method, which essentially measures the amount of antigen through immunological methods.

[0052] This invention discloses a reverse indirect hemagglutination method for accurately determining antigen content. A reference sample is used, and the test results of the tested sample are calculated using a self-developed method, resulting in relatively accurate test results. This solves the problem of inconsistent test results for sensitized red blood cells from different batches. The reverse indirect hemagglutination method for detecting antigen content established in this way provides stable and accurate results, and can be used to control antigen quality in vaccine production.

[0053] Before step S10, the process also includes preparing a sensitized red blood cell suspension. The steps for preparing the sensitized red blood cell suspension include:

[0054] S101. After washing, aldehyde-modifying, and tanning sheep red blood cells, resuspend them in buffer solution to obtain a resuspended solution.

[0055] S102. A monoclonal antibody is added to the resuspended solution to sensitize it, and then a preservative is added to obtain a sensitized red blood cell suspension.

[0056] Specifically, the steps of step S101 are as follows: sheep red blood cells are washed with washing solution 1, aldehyde-treated with glutaraldehyde and / or formaldehyde, washed with washing solution 1, tanned with tannic acid, and washed with washing solution 2 to obtain aldehyde-tanned red blood cells with a red blood cell volume concentration of 1% to 5%.

[0057] The aldehyde-tanned red blood cells were centrifuged, and the precipitated red blood cells were resuspended in buffer to obtain a resuspended solution. An equal volume of monoclonal antibody diluted in buffer was added to the resuspended solution, and the solution was incubated at 37°C for 20-60 minutes. The solution was washed with diluent and resuspended in diluent to obtain a sensitized red blood cell suspension with a red blood cell volume concentration of 1%.

[0058] Subsequently, 1% by volume of preservative was added to the resuspension to obtain the final sensitized red blood cell suspension.

[0059] It should be noted that the reagents used above are specifically: the washing solution 1 is a phosphate buffer solution with a pH of 7.0 to 7.4 and a concentration of 0.01 to 0.1 mol / L;

[0060] The washing solution 2 is a phosphate buffer solution with a pH of 6.2 to 6.6 and a concentration of 0.01 to 0.1 mol / L.

[0061] The buffer solution is an acetate buffer solution with a pH of 4.6 to 5.4 and a concentration of 0.01 to 0.1 mol / L.

[0062] The diluent is a phosphate buffer solution with a pH of 7.0–7.4 and a volume concentration of 0.01–0.1 mol / L, containing 1% healthy rabbit serum.

[0063] The preservative is sodium azide or thimerosal with a mass-volume concentration of 1%.

[0064] The specific operation of the tannic acid tanning is as follows: a tannic acid solution with a mass-volume concentration of 1:10000 to 1:50000 is prepared with washing solution 1, and an equal volume of aldehyde-treated red blood cells with a volume concentration of 1% to 5% is mixed and incubated at 37°C for 10 to 30 minutes.

[0065] The glutaraldehyde and / or formaldehyde aldehyde conversion includes three methods, as detailed below:

[0066] Method 1: Prepare 0.5%–2.5% glutaraldehyde by washing buffer 1, mix it with an equal volume of 5%–10% red blood cells, incubate at 2–8°C with stirring for 2–10 hours, wash 2–4 times with washing buffer 1, and resuspend the red blood cells in washing buffer 1 to obtain 5%–10% red blood cells, which are aldehyde-treated red blood cells.

[0067] Method 2: Prepare a 1%–5% (w / v) concentration of formaldehyde using washing buffer 1, mix it with an equal volume of 5%–10% (v / v) concentration of red blood cells, incubate at room temperature with stirring for 16–24 hours, wash with washing buffer 1 2–4 times, and resuspend the red blood cells in washing buffer 1 to obtain a 5%–10% (v / v) concentration of red blood cells, which are aldehyde-treated red blood cells.

[0068] Method 3: Prepare 0.5%–2.5% glutaraldehyde using washing buffer 1, mix it with an equal volume of 5%–10% red blood cells, incubate at 2–8°C with stirring for 2–10 hours, wash 2–4 times with washing buffer 1, and resuspend the red blood cells in washing buffer 1 to obtain a 5%–10% concentration. Then prepare 1%–5% formaldehyde using washing buffer 1, mix it with an equal volume of 5%–10% red blood cells, incubate at room temperature with stirring for 16–24 hours, wash 2–4 times with washing buffer 1, and resuspend the red blood cells in washing buffer 1 to obtain a 5%–10% concentration. These are aldehyde-treated red blood cells.

[0069] It is particularly important to note that in step S102, the monoclonal antibody includes one of the following monoclonal antibodies:

[0070] Monoclonal antibodies against rabies N protein or monoclonal antibodies against rabies G protein;

[0071] Monoclonal antibodies against PCV2-Cap antigen;

[0072] Monoclonal antibody against PRRSV.

[0073] Here, monoclonal antibodies are used, unlike the commonly used polyclonal antibodies, because theoretically, the following advantages have been found during the experimental process:

[0074] 1) Rabies monoclonal antibodies only react with the rabies virus and do not react with other pathogens or with components in the antigen culture medium; rabies polyclonal antibodies may contain antibodies against other pathogens and antibodies against components of the rabies antigen culture medium; therefore, rabies monoclonal antibodies sensitize red blood cells with high specificity.

[0075] 2) Rabies monoclonal antibodies are prepared using hybridoma cells and Balb / c mice. The hybridoma cells are from the same strain, and the Balb / c mice are standardized laboratory animals, ensuring uniform performance across batches of rabies monoclonal antibodies. Rabies polyclonal antibodies, on the other hand, use purified rabies virus as an immunogen. After adding adjuvants, rabbits are immunized multiple times, and the resulting rabbit serum is the polyclonal antibody. Different batches of immunogens have varying purity, and individual rabbits also exhibit differences, leading to inconsistent quality in the polyclonal antibodies prepared in this way. Therefore, the batch-to-batch variation in monoclonal antibody-sensitized red blood cells is small, resulting in more stable antigen detection results.

[0076] 3) Some rabies monoclonal antibodies can sensitize red blood cells with low dosage, and the cost is low.

[0077] 4) PCV2-Cap monoclonal antibodies only react with PCV2-Cap and do not react with other pathogens or with components in the antigen culture medium; PCV2-Cap polyclonal antibodies may contain antibodies against other pathogens and antibodies against components of the PCV2-Cap antigen culture medium; therefore, PCV2-Cap monoclonal antibodies sensitize red blood cells with high specificity.

[0078] 5) PCV2-Cap monoclonal antibodies are prepared using hybridoma cells and Balb / c mice. The hybridoma cells are of the same strain, and the Balb / c mice are standardized laboratory animals, ensuring uniform performance across batches of PCV2-Cap monoclonal antibodies. PCV2-Cap polyclonal antibodies, on the other hand, are prepared by immunizing rabbits multiple times with purified PCV2-Cap antigen as the immunogen and adding adjuvants. The resulting rabbit serum is the polyclonal antibody. However, the purity of the immunogen varies between batches, and there are individual differences among the rabbits, leading to inconsistent quality in the polyclonal antibodies prepared in this way. Therefore, the batch-to-batch variation in monoclonal antibody-sensitized erythrocytes results in more stable antigen detection results.

[0079] 6) PCV2-Cap monoclonal antibody can sensitize red blood cells with a low dosage, and the cost is low.

[0080] 7) PRRSV monoclonal antibodies only react with PRRSV and do not react with other pathogens or with components in the antigen culture medium; PRRSV polyclonal antibodies may contain antibodies against other pathogens and antibodies against components of the PRRSV antigen culture medium; therefore, red blood cells sensitized with PRRSV monoclonal antibodies have high specificity.

[0081] 8) PRRSV monoclonal antibodies are prepared using hybridoma cells and Balb / c mice. The hybridoma cells are of the same strain, and the Balb / c mice are standardized laboratory animals, ensuring uniform performance across batches of PRRSV monoclonal antibodies. PRRSV polyclonal antibodies, on the other hand, are prepared by immunizing rabbits multiple times with purified PRRSV antigen as the immunogen and adding adjuvants. The resulting rabbit serum is the polyclonal antibody. However, the purity of the immunogen varies between batches, and there are individual differences among the rabbits, leading to inconsistent quality in the polyclonal antibodies prepared in this way. Therefore, the batch-to-batch variation in monoclonal antibody-sensitized erythrocytes is small, resulting in more stable antigen detection results.

[0082] Specifically, in step S10, the four concentrations of the tested samples include: samples containing 100% of the tested sample, samples containing 80% of the tested sample, samples containing 64% of the tested sample, and samples containing 51% of the tested sample; and / or,

[0083] The four reference samples include: 100% reference sample, 80% reference sample, 64% reference sample, and 51% reference sample.

[0084] It should be noted that using these four concentrations of the tested sample and the reference sample ensures a more accurate calculation result. Alternatively, other concentration series can be selected, such as 100%, 70%, and 49%, with the corresponding calculation becoming: antigen content of the tested sample = known antigen content of the reference sample × 2. (A-B) / 12 For seven concentrations (100%, 90%, 81%, 73%, 65.6%, 59%, and 53%), calculate the corresponding antigen content of the tested sample: (Antigen content of the known reference sample × 2) (A -B) / 28 However, compared to the concentrations in this application, these series of concentrations are either less accurate or require too much work. They are not as accurate as the calculation method for the four concentrations of the tested sample and the reference sample used in this application. The calculation method for the four concentrations used in this application is more accurate and requires less work.

[0085] The specific operating steps are as follows:

[0086] Add 0.2 ml of the test sample to 0.05 ml of diluent and mix well. Add 0.2 ml of the test sample to 0.05 ml of diluent and mix well. Add 0.2 ml of the test sample to 0.05 ml of diluent and mix well. This will yield samples containing 80% of the test sample, samples containing 64% of the test sample, and samples containing 51% of the test sample.

[0087] Add 0.2 ml of the reference sample to 0.05 ml of diluent and mix well. Add 0.2 ml of the reference sample to 0.05 ml of diluent and mix well. Add 0.2 ml of the reference sample to 0.05 ml of diluent and mix well to obtain samples containing 80% reference sample, 64% reference sample, and 51% reference sample.

[0088] Add 0.025 ml of diluent to each well of a 96-well blood coagulation plate. For the first well of rows 1 through 8, add 0.025 ml of the following solutions respectively: 100% test sample, 80% test sample, 64% test sample, 51% test sample, 100% reference sample, 80% reference sample, 64% reference sample, and 51% reference sample. After mixing the solution in the first well, add 0.025 ml to the second well. Continue this serial dilution from the first well (2-fold) up to the eleventh well. After mixing the solution in the eleventh well, discard the 0.025 ml solution. This yields test samples and reference samples at different dilutions. The twelfth well is the diluent well (i.e., the negative control well). This procedure facilitates observation of agglutination results and easy scoring.

[0089] It should be noted that: the diluent is a phosphate buffer solution with a pH of 7.0-7.4 and a volume concentration of 0.01-0.1 mol / L containing 1% healthy rabbit serum; the pH value of the phosphate buffer solution is 7-7.4; and / or, the volume concentration of the phosphate buffer solution is 0.01-0.1 mol / L; the reference sample includes any one of the following antigens: a solution or lyophilized product containing inactivated rabies virus; a solution containing PCV2-Cap antigen; a solution containing PRRSV antigen.

[0090] Preferably, in step S20, sensitized red blood cell suspension is added to the wells of test samples and reference samples with different dilutions, respectively. After mixing and standing, the agglutination results of each well are determined. The agglutination results of each well are scored and recorded. The scores of each test sample are summed and recorded as A, and the total scores of each reference sample are summed and recorded as B.

[0091] The specific operation of step S20 is as follows:

[0092] Add 0.025 ml of the sensitized red blood cell suspension to the wells of the test samples, reference samples, and negative control samples at different dilutions. Place the 96-well blood coagulation plate on a shaker and shake at high speed for 10-20 seconds to mix. After the 96-well blood coagulation plate has been left to stand at room temperature, when the negative control wells show clear "dots", determine the agglutination results of each well and record them.

[0093] The methods for determining and recording sensitized erythrocyte agglutination are as follows:

[0094] The results of 100% sensitized red blood cell agglutination, 75% sensitized red blood cell agglutination, 50% sensitized red blood cell agglutination, 25% sensitized red blood cell agglutination, and 0% sensitized red blood cell agglutination were recorded as 4, 3, 2, 1, and 0 points, respectively.

[0095] It should be noted that the criteria for 50% agglutination in the literature are: red blood cells form a thin film at the bottom of the well with loose, serrated edges. [埃博拉病毒抗体间接血凝检测方法的建立_张颖；绵羊肺炎支原体间接血凝诊断方法的建立_赵萍] Or, a small number of red blood cells are deposited in the center of the well bottom, surrounded by unevenly scattered red blood cells. [鸭病毒性肝炎间接血凝诊断抗原的制备_陶海静] These are all difficult to observe, and the results are greatly affected by the operator's subjective factors. The detection method of this invention determines 75% agglutination, 50% agglutination, and 25% agglutination based on a comprehensive assessment of the area, diameter, and color of the "dots" formed by red blood cells at the bottom of the well. In particular, the criteria for determining 50% agglutination are clear, objective, and easy to judge.

[0096] 100% agglutination, i.e. complete agglutination, with red blood cells appearing as fine sand grains evenly spread at the bottom of the well, is recorded as ++++ or #;

[0097] 75% agglutination, red blood cells are evenly distributed at the bottom of the well in the form of fine sand grains, but a very small number of red blood cells are deposited at the bottom of the well, forming "dots", recorded as +++;

[0098] 50% aggregation means the area of ​​the "dot" is less than or equal to 50% of the area of ​​the "dot" with 0% aggregation, and the diameter of the "dot" is less than or equal to 7 / 10 of the diameter of the "dot" with 0% aggregation; the color of the "dot" is significantly lighter than the color of the "dot" with 0% aggregation, and it is recorded as ++.

[0099] 25% aggregation: The area of ​​the "dot" is slightly smaller than that of the "dot" in 0% aggregation; the diameter of the "dot" is slightly smaller than that of the "dot" in 0% aggregation; the color of the "dot" is slightly lighter than that of the "dot" in 0% aggregation, and it is recorded as +.

[0100] 0% agglutination, i.e. no agglutination, red blood cells are deposited at the bottom of the well, forming "dots". The area, diameter and color of the "dots" are the same as the area, diameter and color of the "dots" in the negative control well, and are recorded as -.

[0101] Specifically, the images showing the patterns of 100% sensitized erythrocyte agglutination, 75% sensitized erythrocyte agglutination, 50% sensitized erythrocyte agglutination, 25% sensitized erythrocyte agglutination, and 0% sensitized erythrocyte agglutination are as follows: Figure 1 As shown.

[0102] Preferably, in step S30, the antigen content of the reference sample is known, and can be TCID. 50 Content can refer to IU content or a specific weight; the determination method differs for each content, and no specific requirements are specified. The antigen content of the tested sample is calculated. The sum of the scores of all tested samples is denoted as A, and the sum of the total scores of all reference samples is denoted as B. The antigen content of the tested sample = the known antigen content of the reference sample × 2. (A-B) / 16 .

[0103] It should be noted that: In this invention, the antigen includes any one of rabies virus antigen, PCV2-Cap antigen, and PRRSV antigen. The rabies virus antigen includes at least one of live rabies virus, inactivated rabies antigen, purified rabies antigen, and antigen from an inactivated rabies vaccine; the PCV2-Cap antigen is at least one of the PCV2-Cap antigen expressed by an insect cell baculovirus expression system and its purified antigen; the PRRSV antigen is at least one of the PRRSV antigen and its purified antigen.

[0104] In summary, this invention reduces batch-to-batch variation in sensitized red blood cells by optimizing monoclonal antibody-sensitized red blood cells; it stabilizes antigen quantification results by setting up a reference sample; and it measures and statistically analyzes the reverse indirect hemagglutination of reference and test samples at different concentrations, calculating accurate antigen titers, thus solving the problem of inconsistent detection results for different batches of sensitized red blood cells. The reverse indirect hemagglutination method for detecting antigen levels established in this way provides stable and accurate results and can be used to quantify inactivated rabies antigen, PCV2-Cap antigen, and PRRSV antigen, thereby controlling antigen quality in vaccine production.

[0105] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention. The following embodiments are for better understanding of the present invention, but do not limit the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.

[0106] Regarding the explanation of "reverse indirect hemagglutination titer" or "titer" in the embodiments, that is, the method for determining the reverse indirect hemagglutination titer in the conventional reverse indirect hemagglutination detection method: the titer of the tested sample is determined when button-like deposits of red blood cells appear in the negative control well, i.e., 0% red blood cell agglutination, no red blood cell agglutination, red blood cells deposited at the bottom of the well, or red blood cells forming "dots" at the bottom of the well. The highest dilution of the sample that causes 50% agglutination of sensitized red blood cells is taken as the reverse indirect hemagglutination titer of the test sample. If there is no 50% agglutination, then 75% is used; if there is also no 75% agglutination, then 100% agglutination is used as the highest dilution of the test sample. For example, if a 32-fold dilution of the sample causes 50% agglutination of sensitized red blood cells, and 32 is the highest dilution factor that causes 50% agglutination of sensitized red blood cells, then the titer of the sample is 32, recorded as 32 or 2. 5 Alternatively, 5log2, where 5log2 is a commonly used recording method in the industry.

[0107] Example 1: Study on the optimal preparation conditions of rabies sensitized red blood cells

[0108] 1. Antibody selection

[0109] 1.1 Materials: 1% aldehyde-tannic acid-treated sheep red blood cells prepared by conventional methods.

[0110] Rabies monoclonal antibody 3H3 (4 mg / ml) and rabies monoclonal antibody C11 (3 mg / ml) were purified by ammonium octanoate sulfate method and protein G chromatography column purification. Rabies rabbit polyclonal antibody (4.4 mg / ml) was obtained by immunizing healthy rabbits with purified rabies virus.

[0111] 0.1 mol / L pH 5.0 acetate buffer (abbreviated as pH 5.0 buffer).

[0112] Diluent: 0.1 mol / L phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0113] Rabies virus inactivated antigen (hereinafter referred to as the test sample).

[0114] 1.2 Methods: 3H3 rabies monoclonal antibody, C11 rabies monoclonal antibody, and rabies rabbit polyclonal antibody were diluted with pH 5.0 buffer to a dilution of 10-1. -2 10 -3 10 -4 ,

[0115] 1% aldehyde-tannic acidified sheep red blood cells were mixed with antibodies of various dilutions at a 1:1 ratio, incubated at 37°C for 30 min, centrifuged to deposit the red blood cells, and resuspended in diluent to prepare a 1% sensitized red blood cell suspension.

[0116] (1) Add 25 μl of diluent to each well of a 96-well V-bottom blood coagulation plate;

[0117] (2) Add 25 μl of the test sample to the first well of each row, and perform serial dilution from the first well to the eleventh well. After mixing, discard the 25 μl. The twelfth well is the negative control well.

[0118] (3) Add 25 μl of red blood cells sensitized with different antibodies and different dilutions to each row. Shake the blood coagulation plate to mix well, let it stand at room temperature for 1.5 to 2 hours, and determine the titer when button-shaped deposits of red blood cells appear at the bottom of the 12th well;

[0119] (4) The highest dilution of the antibody corresponding to the sensitized red blood cells with the highest titer of the tested sample (a difference of 1log2 or 2log2 from the highest titer is also considered the highest titer) is the optimal antibody and dilution.

[0120] 1.3 Results: C11 monoclonal antibody can sensitize red blood cells at a relatively low dosage, making it the optimal antibody for sensitization. The titer results are shown in Table 1-1.

[0121] Table 1-1 Reverse indirect hemagglutination titers (log2) of the tested samples

[0122]

[0123]

[0124] Example 2: Determination of the optimal pH value for sensitization in rabies

[0125] 2.1 Materials: 1% aldehyde-tannic acid-treated sheep red blood cells prepared by conventional methods.

[0126] The rabies monoclonal antibody, designated C11 (3 mg / ml), was purified by the ammonium octanoate sulfate method and protein G chromatography.

[0127] 0.1 mol / L acetate buffer solutions at pH 3.8, pH 4.2, pH 4.6, pH 5.0, pH 5.4, and pH 5.8 (abbreviated as pH 3.8, pH 4.2, pH 4.6, pH 5.0, pH 5.4, and pH 5.8 buffers).

[0128] Diluent: 0.1 mol / L phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0129] Rabies virus inactivated antigen (hereinafter referred to as the test sample).

[0130] 2.2 Methods

[0131] C11 rabies monoclonal antibody was diluted 1000-fold with buffer solutions at pH 3.8, pH 4.2, pH 4.6, pH 5.0, pH 5.4, and pH 5.8, respectively.

[0132] 1% aldehyde-tannic acidified sheep red blood cells were mixed with antibody diluted in buffer at a 1:1 ratio, incubated at 37°C for 30 min, centrifuged to deposit red blood cells, and resuspended in diluent to prepare a 1% sensitized red blood cell suspension.

[0133] (1) Add 25 μl of diluent to each well of a 96-well V-bottom blood coagulation plate;

[0134] (2) Add 25 μl of the test sample to the first well of each row, and perform serial dilution from the first well to the eleventh well. After mixing, discard the 25 μl. The twelfth well is the negative control well.

[0135] (3) Add 25 μl of red blood cells sensitized with different buffer solutions to each row. Shake the blood coagulation plate to mix well, and let it stand at room temperature for 1.5 to 2 hours. Determine the titer when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0136] (4) The pH value of the buffer solution corresponding to the sensitized red blood cells with the highest titer in the tested sample is the optimal sensitization pH value.

[0137] 2.3 Results The experimental results are shown in Table 2-1. As can be seen from Table 2-1, the sensitization effect of the buffer solution with pH 4.6 to pH 5.4 is better, and the sensitized red blood cells can detect rabies virus antigen sensitively and specifically.

[0138] Table 2-1 Reverse indirect hemagglutination titers of the tested samples (log2)

[0139] Buffer pH pH 3.8 pH 4.2 pH 4.6 pH 5.0 pH 5.4 pH 5.8 valence / / 10 9 8 6

[0140] Note: " / " indicates a negative control agglutination, and the result is invalid.

[0141] Example 3: Determination of the optimal sensitization concentration of rabies virus monoclonal antibody

[0142] 3.1 Materials: 1% aldehyde-tannic acid-treated sheep red blood cells prepared by conventional methods.

[0143] The rabies monoclonal antibody, designated C11 (3 mg / ml), was purified by the ammonium octanoate sulfate method and protein G chromatography.

[0144] 0.1 mol / L pH 5.0 acetate buffer (abbreviated as pH 5.0 buffer).

[0145] Diluent: 0.1 mol / L phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0146] Rabies virus inactivated antigen (hereinafter referred to as the test sample).

[0147] 3.2 Methods: Dilute C11 rabies monoclonal antibody with pH 5.0 buffer to achieve dilution ratios of 200, 400, 800, 1600, 3200, and 6400 times.

[0148] 1% aldehyde-tannic acidified sheep red blood cells were mixed with antibodies of various dilutions at a 1:1 ratio, incubated at 37°C for 30 min, centrifuged to deposit the red blood cells, and resuspended in diluent to prepare a 1% sensitized red blood cell suspension.

[0149] (1) Add 25 μl of diluent to each well of a 96-well V-bottom blood coagulation plate;

[0150] (2) Add 25 μl of rabies antigen to the first well of each row, and perform serial dilution from the first well to the eleventh well. After mixing, discard the 25 μl. The twelfth well is the negative control well.

[0151] (3) Add 25 μl of antibody-sensitized red blood cells of different dilutions to each row. Shake the blood coagulation plate to mix well, and let it stand at room temperature for 1.5 to 2 hours. Determine the titer when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0152] (4) The highest dilution of the antibody corresponding to the sensitized red blood cells with the highest titer in the tested sample is the optimal antibody dilution.

[0153] 3.3 Results: The aggregation results for each pore are shown in the figure. Figure 2 The experimental results are shown in Table 3-1. Table 3-1 shows that sensitizing red blood cells with 3 mg / ml C11 rabies monoclonal antibody diluted 200–1600 times yielded the highest titer in the tested samples. This means that C11 rabies monoclonal antibody at concentrations of 2–15 μg / ml can sensitize red blood cells, and a concentration of 2 μg / ml of C11 rabies monoclonal antibody constitutes one sensitizing unit.

[0154] Table 3-1 Reverse indirect hemagglutination titers of the tested samples (log2)

[0155] antibody dilution factor 200 times 400 times 800 times 1600 times 3200 times 6400 times valence 10 10 10 9 7 ＜1

[0156] Example 4: Study on the repeatability (intra-batch, inter-batch), specificity (comparison of canine distemper, canine adenovirus, and canine parvovirus), and sensitivity (live virus detection amount, comparison of titers before and after inactivation) of a reverse indirect hemagglutination method for detecting rabies antigen.

[0157] 4.1 Materials

[0158] 1% aldehyde-tannic acid-treated sheep red blood cells, 3 batches, batches 01 / 02 / 03 respectively;

[0159] Three batches of rabies monoclonal antibody, designated C11, were purified by the ammonium octanoate sulfate method and protein G chromatography. The contents of batches 01, 02, and 03 were 3.0 mg / ml, 2.2 mg / ml, and 1.8 mg / ml, respectively.

[0160] Three batches of 0.1 mol / L pH 5.0 acetate buffer (abbreviated as pH 5.0 buffer) were designated as batches 01, 02, and 03.

[0161] Diluent: Phosphate buffer at pH 7.2 containing 1% healthy rabbit serum, 3 batches, batches 01 / 02 / 03;

[0162] The tested samples and Sample 1 were live viruses; Sample 2 was inactivated Sample 1; Sample 3 was Sample 2 concentrated 5 times; Sample 4 was Sample 3 purified by chromatography; Sample 5 was a canine distemper sample; Sample 6 was a canine adenovirus sample; Sample 7 was a canine parvovirus sample; Sample 8 had a content of 10 8.0 TCID 50 / ml of rabies virus inactivated sample.

[0163] 4.2 Methods

[0164] 4.2.1 Preparation of sensitized red blood cells: Dilute C11 rabies monoclonal antibody with pH 5.0 buffer to a final concentration of 4 μg / ml. Mix 1% aldehyde-tannic acid-modified sheep red blood cells with antibodies of various dilutions at a 1:1 ratio, incubate at 37°C for 30 min, centrifuge to deposit the red blood cells, and resuspend them in diluent to prepare a 1% sensitized red blood cell suspension.

[0165] 4.2.2 Detection Method

[0166] (1) Add 25 μl of diluent to each well of a 96-well V-bottom blood coagulation plate;

[0167] (2) Add 25 μl of each of the samples 1 to 8 to the first well of each row, and perform serial dilution from the first well to the 11th well. After mixing, discard 25 μl.

[0168] (3) Add 25 μl of antibody-sensitized red blood cells of different dilutions to each row. Shake the blood coagulation plate to mix well, and let it stand at room temperature for 1.5 to 2.5 hours. The result is determined when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0169] (4) The highest dilution factor that produces 50% hemoglobin agglutination is taken as the potency of the tested sample.

[0170] 4.2.3 Experimental Implementation Method

[0171] Three researchers prepared five batches of sensitized red blood cells using three batches of materials (Table 4-1); the three researchers then used the five batches of sensitized red blood cells to test samples 1 through 8.

[0172] Table 4-1 Information on operators and raw materials for 5 batches of sensitized red blood cells

[0173]

[0174] 4.3 Results

[0175] The test results are shown in Table 4-2.

[0176] Table 4-2 Results of reverse indirect hemagglutination assay of sensitized red blood cells by 3 individuals (log2)

[0177]

[0178]

[0179] Table 4-2 shows that 3 operators tested 8 samples from 5 batches of sensitized red blood cells and obtained 15 titer results, including 2 at 6log2, 7 at 7log2, and 6 at 8log2, indicating that 10 8.0 TCID 50 A sample of 8 ml, diluted approximately 100-fold (7 log2), can induce agglutination of sensitized red blood cells, meaning the detection sensitivity is 10. 6.0 TCID 50 / ml.

[0180] Table 4-2 shows that this method can detect live rabies virus, inactivated virus, and chromatographically purified virus (sensitized red blood cells specifically agglutinate with live rabies virus, inactivated virus, and chromatographically purified virus). However, it cannot detect canine distemper, canine adenovirus, or canine parvovirus samples (sensitized red blood cells do not specifically agglutinate with these samples). This indicates good method specificity. When different researchers use the same batch of red blood cells to test the same sample, most results are consistent, with a few inconsistencies. The highest to lowest difference between inconsistent results is 1 log2, which is attributed to random factors and thus shows intra-batch stability. Different batches of sensitized red blood cells test the same sample differently, with a highest to lowest difference of 2 log2. This indicates inter-batch variability among sensitized red blood cells, which is the main factor contributing to significant differences in test results. This characteristic is consistent with the characteristics of indirect hemagglutination inhibition assays. Since the difference is no greater than 2 log2, inter-batch stability is within an acceptable range.

[0181] Example 5: Comparison of this method with a dilution reference sample method and a method without a reference sample.

[0182] 5.1 Materials: Rabies monoclonal antibody-sensitized red blood cells: batches X01, X02, and X03

[0183] Diluent: Phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0184] Sample tested: Rabies virus inactivated sample

[0185] Reference sample: 10 samples of inactivated rabies virus with known content. 8.0 TCID 50 / ml

[0186] 1.5ml centrifuge tube, or simply centrifuge tube

[0187] 5.2 Methods

[0188] 5.2.1 This method is used to determine the potency of the tested sample.

[0189] Add 50 μl of diluent to centrifuge tubes 2, 3, and 4 respectively. Add 200 μl of the test sample to centrifuge tube 2, mix well, then add 200 μl to centrifuge tube 3, mix well, and then add 200 μl to centrifuge tube 4. Centrifuge tubes 2, 3, and 4 contain 80%, 64%, and 51% of the test sample, respectively.

[0190] Using the same method, reference samples were obtained in samples 6, 7, and 8, yielding 80%, 64%, and 51% respectively.

[0191] (1) One 96-well V-bottom blood coagulation plate, with 8 rows in total, add 25 μl of diluent to each well;

[0192] (2) Add 25 μl of each of the test sample, 80% of the test sample, 64% of the test sample, 51% of the test sample, reference sample, 80% of the reference sample, 64% of the reference sample, and 51% of the reference sample to each well in column 1. Perform serial dilution from well 1 to well 11. After mixing, discard 25 μl.

[0193] (3) Add 25 μl of sensitized red blood cells to each well. Shake the blood coagulation plate to mix the contents, and let it stand at room temperature for 1.5 to 2.5 hours. The result is determined when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0194] (4) Determine the agglutination results of each well and fill in the result record table. Record 4, 3, 2, 1 and 0 points for 100%, 75%, 50%, 25% and 0% red blood cell agglutination, respectively.

[0195] (5) Calculate the potency: The total score of each well in columns 1-11 of the tested sample, 80% of the tested samples, 64% of the tested samples, and 51% of the tested samples is A; the total score of each well in columns 1-11 of the reference sample, 80% of the reference sample, 64% of the reference sample, and 51% of the reference sample is B; C = (AB) ÷ 16; Test sample potency = Reference sample potency × 2 C .

[0196] 5.2.2 Determining the potency of the test sample using a dilution reference sample method

[0197] (1) One 96-well V-bottom blood coagulation plate, add 25 μl of diluent to each well in the first and fourth rows;

[0198] (2) Add 25 μl of the test sample and the reference sample to each well in column 1. Perform serial dilution from well 1 to well 11. After mixing, discard 25 μl.

[0199] (3) Add 25 μl of sensitized red blood cells to each well. Shake the blood coagulation plate to mix the contents, and let it stand at room temperature for 1.5 to 2.5 hours. The result is determined when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0200] (4) Determine the reverse indirect hemagglutination titer of the test sample and the reference sample respectively: The highest dilution that causes 50% agglutination of sensitized red blood cells is the reverse indirect hemagglutination titer of the test sample. If there is no 50% agglutination, the highest dilution is 75%. If there is no 75% agglutination, the highest dilution is 100% agglutination.

[0201] (5) Calculate the titer of the tested sample: Titer of tested sample = (Reverse indirect hemagglutination titer of tested sample ÷ Reverse indirect hemagglutination titer of reference sample) × Titer of reference sample

[0202] 5.2.3 Determination of potency of test samples without reference samples

[0203] (1) One 96-well V-bottom blood coagulation plate, add 25 μl of diluent to each well in the first row;

[0204] (2) Add the test sample to each well in column 1 and dilute it by 2 times from well 1 to well 11. After mixing, discard 25 μl.

[0205] (3) Add 25 μl of sensitized red blood cells to each well. Shake the blood coagulation plate to mix the contents, and let it stand at room temperature for 1.5 to 2.5 hours. The result is determined when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0206] (4) Determining the reverse indirect hemagglutination titer of the test sample: The highest dilution that causes 50% agglutination of sensitized red blood cells is taken as the reverse indirect hemagglutination titer of the test sample. If no 50% agglutination occurs, 75% is used; if no 75% agglutination occurs, 100% agglutination is used as the highest dilution that causes agglutination. Test sample titer = Reverse indirect hemagglutination titer of the test sample.

[0207] 5.2.4 Multiple people repeatedly tested the potency of the tested samples using methods 5.2.1, 5.2.2, and 5.2.3, and compared the stability and accuracy of the test results from the three methods.

[0208] (Note: The first and fourth rows of steps (1), (2), and (3) in 5.2.1 are the same as the first and fourth rows of steps (1), (2), and (3) in 5.2.2. The first row of steps (1), (2), and (3) in 5.2.1 is the same as the first row of steps (1), (2), and (3) in 5.2.3. Therefore, there is no need to repeat the operations in 5.2.2 and 5.2.3. The results of 5.2.1 can be used to perform the calculations in steps (4) and (5).)

[0209] 5.3 Results

[0210] 5.3.1 Operators 1, 2, and 3 performed two determinations of the potency of the tested samples according to this method. The agglomeration results of each well were determined and scored. The specific scores are recorded in Tables 5-1, 5-2, 5-3, 5-4, 5-5, and 5-6. The calculated potency results of the tested samples are shown in Table 5-7.

[0211] Table 5-1 Operator 1-1 times - Batch X01 Sensitized Red Blood Cells

[0212] 1 2 3 4 5 6 7 8 9 10 11 12 Tested sample 4 4 4 4 4 3 2 0 0 0 0 / 80% of the tested samples 4 4 4 4 4 4 2 1 0 0 0 / 64% of the tested samples 4 4 4 4 4 3 2 0 0 0 0 / 51% of the tested samples 4 4 4 4 4 3 1 0 0 0 0 / Reference Sample 4 4 4 4 4 3 1 0 0 0 0 / 80% of the reference samples 4 4 4 4 3 2 1 0 0 0 0 / 64% of the reference samples 4 4 4 4 3 1 0 0 0 0 0 / 51% of the reference samples 4 4 4 4 2 1 0 0 0 0 0 /

[0213] Table 5-2 Operator 1-2 times - Batch X01 Sensitized Red Blood Cells

[0214] 1 2 3 4 5 6 7 8 9 10 11 12 Tested sample 4 4 4 4 4 4 3 1 0 0 0 / 80% of the tested samples 4 4 4 4 4 4 3 1 0 0 0 / 64% of the tested samples 4 4 4 4 4 3 2 1 0 0 0 / 51% of the tested samples 4 4 4 4 4 3 1 0 0 0 0 / Reference Sample 4 4 4 4 4 3 2 0 0 0 0 / 80% of the reference samples 4 4 4 4 3 2 1 0 0 0 0 / 64% of the reference samples 4 4 4 4 3 1 0 0 0 0 0 / 51% of the reference samples 4 4 4 4 2 2 0 0 0 0 0 /

[0215] Table 5-3 Operator 2-1 times - Batch X02 Sensitized Red Blood Cells

[0216]

[0217]

[0218] Table 5-4 Operator 2-2 times - X02 batch of sensitized red blood cells

[0219] 1 2 3 4 5 6 7 8 9 10 11 12 Tested sample 4 4 4 4 4 4 4 3 1 0 0 / 80% of the tested samples 4 4 4 4 4 4 4 2 1 0 0 / 64% of the tested samples 4 4 4 4 4 4 3 2 1 0 0 / 51% of the tested samples 4 4 4 4 4 3 3 1 0 0 0 / Reference Sample 4 4 4 4 4 4 3 2 1 0 0 / 80% of the reference samples 4 4 4 4 4 4 3 1 0 0 0 / 64% of the reference samples 4 4 4 4 4 3 2 0 0 0 0 / 51% of the reference samples 4 4 4 4 4 2 1 0 0 0 0 /

[0220] Table 5-5 Operator 3-1 times - Batch X03 Sensitized Red Blood Cells

[0221] 1 2 3 4 5 6 7 8 9 10 11 12 Tested sample 4 4 4 4 4 4 4 4 2 0 0 / 80% of the tested samples 4 4 4 4 4 4 4 3 1 0 0 / 64% of the tested samples 4 4 4 4 4 4 3 2 0 0 0 / 51% of the tested samples 4 4 4 4 4 4 3 2 0 0 0 / Reference Sample 4 4 4 4 4 4 3 1 0 0 0 / 80% of the reference samples 4 4 4 4 4 3 2 0 0 0 0 / 64% of the reference samples 4 4 4 4 4 3 2 0 0 0 0 / 51% of the reference samples 4 4 4 4 4 2 0 0 0 0 0 /

[0222] Table 5-6 Operator 3-2 times - Batch X03 Sensitized Red Blood Cells

[0223] 1 2 3 4 5 6 7 8 9 10 11 12 Tested sample 4 4 4 4 4 4 4 3 2 1 0 / 80% of the tested samples 4 4 4 4 4 4 4 3 2 0 0 / 64% of the tested samples 4 4 4 4 4 4 3 2 1 0 0 / 51% of the tested samples 4 4 4 4 4 4 4 1 0 0 0 / Reference Sample 4 4 4 4 4 4 4 3 1 0 0 / 80% of the reference samples 4 4 4 4 4 4 3 1 0 0 0 / 64% of the reference samples 4 4 4 4 4 4 2 0 0 0 0 / 51% of the reference samples 4 4 4 4 4 3 2 0 0 0 0 /

[0224] Table 5-7 Potency of the tested samples obtained by this method

[0225]

[0226] 5.3.2 The potency of the test sample was determined using the method with a one-dilution reference sample. Operators 1, 2, and 3 performed two determinations each on the test sample and the reference sample. The agglutination results for each well are shown in Table 5-8 (derived from Tables 5-1 to 5-6). The calculated potency of the test sample is shown in Table 5-9.

[0227] Table 5-8 Results of Aggregation Judgment for Each Well

[0228]

[0229] Table 5-9 Potency of the tested sample obtained by the reference sample method at a dilution level.

[0230]

[0231] 5.3.3 Determination of the potency of the test sample without a reference sample: Operators 1, 2, and 3 each measured the test sample twice. The agglutination results for each well are shown in Table 5-10 (derived from the results of Tables 5-1 to 5-6). The calculated potency results of the test sample are shown in Table 5-11.

[0232] Table 5-10 Results of Aggregation Judgment for Each Well

[0233]

[0234] Table 5-11 Reverse indirect hemagglutination titers of tested samples obtained without reference samples.

[0235]

[0236] 5.3.4 Summary and Analysis of Experimental Results The deviations of the results obtained by the three detection methods were summarized and calculated. The results are shown in Table 5-12.

[0237] Table 5-12 Potency of tested samples obtained by different methods

[0238]

[0239] As shown in Table 5-12, the results of various measurements by different operators are the most stable using this method. That is, the method of calculating the potency of the test sample by using the agglutination results of test samples and reference samples of different concentrations has the smallest deviation in the test results. The method with a reference sample of one dilution is not very stable. That is, the method of calculating the potency of the test sample by using the agglutination potency of the test sample and reference sample has a large deviation in the test results. The method without a reference sample is not very stable. That is, the method of directly measuring the agglutination potency of the test sample has a large deviation in the test results.

[0240] Example 6: Determination of Rabies Virus Antigen Content in 5 Samples

[0241] 6.1 Materials: Rabies monoclonal antibody-sensitized red blood cells: Batch X01

[0242] Diluent: Phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0243] Samples tested: Rabies virus antigen, numbered 01, 02, 03, 04, 05.

[0244] Reference sample: 10 samples of inactivated rabies virus with known content. 8.0 TCID 50 / ml

[0245] 6.2 Method: Add 50 μl of diluent to centrifuge tubes 1-2, 1-3, and 1-4 respectively. Add 200 μl of sample 01 to centrifuge tube 1-2, mix well, and then add 200 μl of the mixture to centrifuge tube 1-3. Repeat this process, adding 200 μl of the mixture to centrifuge tube 1-4. Centrifuge tubes 1-2, 1-3, and 1-4 contain 80%, 64%, and 51% of the sample, respectively.

[0246] The same procedure was used on samples 02, 03, 04, and 05, as well as the reference sample, to obtain 80%, 64%, and 51% of the samples 02, 03, 04, and 05, respectively.

[0247] (1) Three 96-well V-bottom blood coagulation plates were prepared in 24 rows. 25 μl of diluent was added to each well.

[0248] (2) In column 1, add the following to each well: sample 01, 80% of sample 01, 64% of sample 01, and 51% of sample 01; sample 02, 80% of sample 02, 64% of sample 02, and 51% of sample 02; sample 03, 80% of sample 03, 64% of sample 03, and 51% of sample 03; sample 04... 80% of test sample 04, 64% of test sample 04, and 51% of test sample 04; 80% of test sample 05, 64% of test sample 05, and 51% of test sample 05; and 25 μl each of reference sample, 80% of reference sample, 64% of reference sample, and 51% of reference sample. Serially dilute each sample 2-fold from well 1 to well 11, mixing thoroughly and discarding the 25 μl.

[0249] (3) Add 25 μl of sensitized red blood cells to each well. Shake the blood coagulation plate to mix the contents, and let it stand at room temperature for 1.5 to 2.5 hours. The result is determined when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0250] (4) Determine the agglutination results of each well and fill in the result record table. Record 4, 3, 2, 1 and 0 points for 100%, 75%, 50%, 25% and 0% red blood cell agglutination, respectively.

[0251] (5) Calculate the potency of the tested sample, 80% of the tested samples, 64% of the tested samples, and 51% of the tested samples. Let A be the total score of each well in columns 1-11 of the reference sample, 80% of the reference sample, 64% of the reference sample, and 51% of the reference sample. Let B be the total score of each well in columns 1-11 of the reference sample. C = (AB) ÷ 16. Potency of the tested sample = Reference sample × 2 C .

[0252] 6.3 Test Results: As shown in Table 6-1, the calculated potency of the tested samples is shown in Table 6-2. The test results can be used to determine whether the antigen is qualified, or to determine the antigen concentration factor, or to determine the antigen dilution factor when preparing vaccines, etc. The potency of the tested samples was obtained within 3 hours using this method, which has strong timeliness; since samples with 4 dilutions (100%, 80%, 64%, and 51%) were tested and calculated comprehensively, the accuracy of the results is high; since a reference sample was used for calculation, the results data are comparable to the potency of other samples measured at other times, and data comparison analysis can be performed; the antigen content of the inactivated tested antigen was obtained, which solved the technical problem that inactivated antigens could not be quantitatively detected, making the quality of the subsequently prepared vaccines more reliable.

[0253] Table 6-1 Results of Aggregation Judgment for Each Well

[0254]

[0255]

[0256] Table 6-2 Potency of the tested samples

[0257] Sample number <![CDATA[Potency (×10 8.0 TCID 50 / ml)]]> 01 3.0 02 4.6 03 6.4 04 8.0 05 0.9

[0258] Example 7: Determination of the optimal sensitization concentration of PCV2-Cap monoclonal antibody

[0259] 7.1 Materials: 1% aldehyde-tannic acid-treated sheep red blood cells prepared by conventional methods.

[0260] PCV2-Cap monoclonal antibody (3 mg / ml), designated 3F2, was purified by the ammonium octanoate sulfate method and protein G chromatography.

[0261] 0.1 mol / L pH 5.0 acetate buffer (abbreviated as pH 5.0 buffer).

[0262] Diluent: 0.1 mol / L phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0263] The sample to be tested was PCV2-Cap antigen diluted 100 times.

[0264] 7.2 Methods: Dilute PCV2-Cap monoclonal antibody with pH 5.0 buffer to achieve dilutions of 500, 1000, 2000, 4000, 8000, and 16000 times.

[0265] 1% aldehyde-tannic acidified sheep red blood cells were mixed with antibodies of various dilutions at a 1:1 ratio, incubated at 37°C for 30 min, centrifuged to deposit the red blood cells, and resuspended in diluent to prepare a 1% sensitized red blood cell suspension.

[0266] (1) Add 25 μl of diluent to each well of a 96-well V-bottom blood coagulation plate;

[0267] (2) Add 25 μl of the test sample to the first well of each row, and perform serial dilution from the first well to the eleventh well. After mixing, discard the 25 μl. The twelfth well is the negative control well.

[0268] (3) Add 25 μl of antibody-sensitized red blood cells of different dilutions to each row. Shake the blood coagulation plate to mix well, and let it stand at room temperature for 1.5 to 2 hours. Determine the titer when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0269] (4) The highest dilution of the antibody corresponding to the sensitized red blood cells with the highest titer in the tested sample is the optimal antibody dilution.

[0270] 7.3 Results: The aggregation results for each well are shown in the figure. Figure 3The experimental results are shown in Table 7-1. Table 7-1 shows that the highest titer in the tested samples was achieved when PCV2-Cap monoclonal antibody was diluted 2000 times to sensitize red blood cells at a concentration of 3 mg / ml. Therefore, 2000-fold dilution is the optimal antibody dilution. This means that PCV2-Cap monoclonal antibody at a concentration of 1.5 μg / ml can sensitize red blood cells, and a concentration of 1.5–2 μg / ml is considered one sensitizing unit.

[0271] Table 7-1 Reverse indirect hemagglutination titers of the tested samples (log2)

[0272] antibody dilution factor 500 times 1000 times 2000 times 4000 times 8000 times 16000 times valence 7 7 7 6 3 1

[0273] Example 8: Preparation of sensitized red blood cells using PCV2-Cap monoclonal antibody

[0274] 8.1 Materials: 1% aldehyde-tannic acid-treated sheep red blood cells prepared by conventional methods

[0275] Diluent: Phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0276] 0.1 mol / L pH 5.0 acetate buffer (abbreviated as pH 5.0 buffer)

[0277] PCV2-Cap monoclonal antibody (3 mg / ml), serial number 3F2, was purified by the ammonium octanoate sulfate method and protein G chromatography.

[0278] 8.2 Methods: Preparation of monoclonal antibody solution: Add 50 μl of PCV2-Cap monoclonal antibody to 100 ml of pH 5.0 buffer to dilute to a factor of 2000.

[0279] Mix 100 ml of 1% aldehyde-tannic acid-treated sheep red blood cells with 100 ml of PCV2-Cap monoclonal antibody solution, incubate at 37°C for 30 min, centrifuge the deposited red blood cells, suspend them in 100 ml of diluent, add 1 ml of 1% thimerosal and mix well to obtain 100 ml of 1% sensitized red blood cell suspension.

[0280] Example 9: This method was used to detect 6 PCV2-Cap antigen samples.

[0281] 9.1 Materials: 1% sensitized red blood cells prepared in Example 8

[0282] Diluent: Phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0283] Samples 1 to 6 were: PCV2-Cap expressed by insect baculovirus (Samples 1 to 3) and PCV2-Cap expressed by insect baculovirus after chromatographic purification (Samples 4 to 6), respectively. The PCV2-Cap antigen content detected by competitive ELISA was 47, 146, 169, 500, 470, and 633 μg / ml, respectively.

[0284] Reference sample: PCV2-Cap expressed by insect baculovirus was purified by chromatography, density gradient centrifugation, and diluted to 25.1 μg / ml.

[0285] 1.5ml centrifuge tube, or simply centrifuge tube.

[0286] 9.2 Methods: Samples 1 to 6 were diluted 100 times with diluent before subsequent testing.

[0287] Add 50 μl of diluent to centrifuge tubes 2, 3, and 4 respectively. Add 200 μl of the test sample to centrifuge tube 2, mix well, then add 200 μl to centrifuge tube 3, mix well, and then add 200 μl to centrifuge tube 4. Centrifuge tubes 2, 3, and 4 contain 80%, 64%, and 51% of the test sample, respectively.

[0288] The same method was used to test 6 samples and 1 reference sample.

[0289] (1) Add 25 μl of diluent to each well of a 96-well V-bottom blood coagulation plate;

[0290] (2) Add 25 μl of each of the test sample, 80% of the test sample, 64% of the test sample, 51% of the test sample, reference sample, 80% of the reference sample, 64% of the reference sample, and 51% of the reference sample to each well in column 1. Perform serial dilution from well 1 to well 11. After mixing, discard 25 μl.

[0291] (3) Add 25 μl of sensitized red blood cells to each well. Shake the blood coagulation plate to mix the contents, and let it stand at room temperature for 1.5 to 2.5 hours. The result is determined when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0292] (4) Determine the agglutination results of each well and fill in the result record table. Record 4, 3, 2, 1 and 0 points for 100%, 75%, 50%, 25% and 0% red blood cell agglutination, respectively.

[0293] (5) Calculate the potency of the tested sample, 80% of the tested samples, 64% of the tested samples, and 51% of the tested samples. Let A be the total score of each well in columns 1-11 of the reference sample, 80% of the reference sample, 64% of the reference sample, and 51% of the reference sample. Let B be the total score of each well in columns 1-11 of the reference sample. C = (AB) ÷ 16. Potency of the tested sample = Reference sample × 2C .

[0294] 9.3 Results The test results are shown in Table 9-1. The calculated titers of the tested samples are shown in Table 9-2. The results show that the titers measured by this method correspond to those measured by the ELISA method. This indicates that this method can replace the ELISA method as a quantitative detection method for PCV2-Cap antigen, shortening the detection time and reducing the detection cost while ensuring accurate test results.

[0295] Table 9-1 Test Results

[0296]

[0297]

[0298] Table 9-2 Calculation results of sample titers

[0299]

[0300] Example 10: Specificity of the PCV2-Cap reverse indirect coagulation method

[0301] 10.1 Materials: 1% sensitized red blood cells prepared in Example 8.

[0302] Diluent: Phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0303] The samples tested: Samples 1 to 6 are cell culture media of the following viruses: baculovirus, classical swine fever virus, porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus, transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV).

[0304] 1.5ml centrifuge tube, or simply centrifuge tube.

[0305] 10.2 Methods: (1) 96-well V-bottom blood coagulation plate, add 25 μl of diluent to each well;

[0306] (2) Add 25 μl of the test sample to the first well of each row, and perform serial dilution from the first well to the eleventh well. After mixing, discard the 25 μl. The twelfth well is the negative control well.

[0307] (3) Add 25 μl of antibody-sensitized red blood cells of different dilutions to each row. Shake the blood coagulation plate to mix well, and let it stand at room temperature for 1.5 to 2 hours. Determine the titer when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0308] 10.3 Results: Culture media of baculovirus, classical swine fever virus, porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus, transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV) did not agglutinate 1% of sensitized red blood cells sensitized with PCV2-Cap monoclonal antibody, as detailed in Table 10-1. This indicates that the method of the present invention has good specificity.

[0309] Table 10-1 Detection Results (log2)

[0310]

[0311] Note: <1: 1% of sensitized red blood cells sensitized with PCV2-Cap monoclonal antibody were added to the virus sample at the highest concentration (2-fold dilution), and no 50%, 75%, or 100% agglutination was observed.

[0312] Example 11: Determination of the optimal sensitization concentration of PRRSV monoclonal antibody

[0313] 11.1 Materials: 1% aldehyde-tannic acid-treated sheep red blood cells prepared by conventional methods.

[0314] PRRSV monoclonal antibody (3 mg / ml) with serial number 3B9 was purified by the ammonium octanoate sulfate method and protein G chromatography.

[0315] 0.1 mol / L pH 5.0 acetate buffer (abbreviated as pH 5.0 buffer).

[0316] Diluent: 0.1 mol / L phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0317] Sample tested: PRRSV antigen.

[0318] 11.2 Methods: PRRSV monoclonal antibody was diluted with pH 5.0 buffer to make dilutions of 40, 80, 160, 320, 640, and 1280 times; 1% aldehyde-tannic acid-treated sheep red blood cells were mixed with each dilution of antibody at a 1:1 ratio, incubated at 37°C for 30 min, centrifuged to deposit the red blood cells, and resuspended in diluent to prepare a 1% sensitized red blood cell suspension.

[0319] (1) Add 25 μl of diluent to each well of a 96-well V-bottom blood coagulation plate;

[0320] (2) Add 25 μl of the test sample to the first well of each row, and perform serial dilution from the first well to the eleventh well. After mixing, discard the 25 μl. The twelfth well is the negative control well.

[0321] (3) Add 25 μl of antibody-sensitized red blood cells of different dilutions to each row. Shake the blood coagulation plate to mix well, and let it stand at room temperature for 1.5 to 2 hours. Determine the titer when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0322] (4) The highest dilution of the antibody corresponding to the sensitized red blood cells with the highest titer in the tested sample is the optimal antibody dilution.

[0323] 11.3 Results The experimental results of the study on the optimal antibody dilution are shown in Table 11-1. Table 11-1 shows that a 3 mg / ml PRRSV monoclonal antibody diluted 160-fold to sensitize red blood cells resulted in the highest titer in the tested samples; therefore, a 160-fold dilution is the optimal antibody dilution. That is, a PRRSV monoclonal antibody concentration of 20 μg / ml can sensitize red blood cells, meaning that a PRRSV monoclonal antibody concentration of 2 μg / ml constitutes one sensitization unit.

[0324] Table 11-1 Reverse indirect hemagglutination titers of the tested samples (log2)

[0325] antibody dilution factor 40 times 80 times 160 times 320 times 640 times 1280 times valence 6 6 6 5 3 0

[0326] Example 12 PRRSV monoclonal antibody sensitized red blood cells

[0327] 12.1 Materials: Same as materials in 8.1 of Example 8, except that the monoclonal antibody was replaced with PRRSV monoclonal antibody number 3F2 (3 mg / ml) purified by ammonium caprylate sulfate method and protein G chromatography column.

[0328] 12.2 Method: 700 μl of PRRSV monoclonal antibody was added to 100 ml of pH 5.0 buffer to dilute to approximately 150 times. Then, 100 ml of 1% aldehyde-tannic acid-treated sheep red blood cells were mixed with 100 ml of PRRSV monoclonal antibody solution and incubated at 37°C for 30 min. The deposited red blood cells were centrifuged and resuspended in 100 ml of diluent. 1 ml of 1% thimerosal was added and mixed well to obtain 100 ml of 1% sensitized red blood cell suspension.

[0329] Example 13: This method detects PRRSV antigen.

[0330] 13.1 Materials: 1% sensitized red blood cells prepared in Example 12.

[0331] Diluent: Phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0332] Sample tested: PRRSV antigen after inactivation, concentration and purification.

[0333] Reference sample: PRRSV antigen, whose geometric mean calculated after multiple measurements of its reverse indirect hemagglutination titer is 24 (or 4.6 log2 or 2). 4.6 ).

[0334] 1.5ml centrifuge tube, or simply centrifuge tube.

[0335] 13.2 Method: Add 50 μl of diluent to centrifuge tubes 2, 3, and 4 respectively. Add 200 μl of the test sample to centrifuge tube 2, mix well, then add 200 μl to centrifuge tube 3, mix well, and then add 200 μl to centrifuge tube 4. Centrifuge tubes 2, 3, and 4 contain 80%, 64%, and 51% of the test sample, respectively.

[0336] The same method was used for the same operation as the reference sample.

[0337] (1) Add 25 μl of diluent to each well of a 96-well V-bottom blood coagulation plate;

[0338] (2) Add 25 μl of each of the test sample, 80% of the test sample, 64% of the test sample, 51% of the test sample, reference sample, 80% of the reference sample, 64% of the reference sample, and 51% of the reference sample to each well in column 1. Perform serial dilution from well 1 to well 11. After mixing, discard 25 μl.

[0339] (3) Add 25 μl of sensitized red blood cells to each well. Shake the blood coagulation plate to mix the contents, and let it stand at room temperature for 1.5 to 2.5 hours. The result is determined when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0340] (4) Determine the agglutination results of each well and fill in the result record table. Record 4, 3, 2, 1 and 0 points for 100%, 75%, 50%, 25% and 0% red blood cell agglutination, respectively.

[0341] (5) Calculate the potency of the tested sample, 80% of the tested samples, 64% of the tested samples, and 51% of the tested samples. Let A be the total score of each well in columns 1-11 of the reference sample, 80% of the reference sample, 64% of the reference sample, and 51% of the reference sample. Let B be the total score of each well in columns 1-11 of the reference sample. C = (AB) ÷ 16. Potency of the tested sample = Reference sample × 2 C .

[0342] 13.3 Results The agglomeration results are shown in Figure 4 The test results are recorded in Table 13-1. The calculated potency of the tested sample was 92. This can be understood as the tested sample diluted 92 times being able to cause 50% agglutination of sensitized red blood cells. Using the same method, the potency of more batches of PRRSV antigen was determined, and the "mean value - 2 × standard deviation" was calculated. The "mean value - 2 × standard deviation" was used as the quality standard for PRRSV antigen. Antigens below this standard were considered unqualified antigens. In other words, the quality of the finished vaccine was controlled by controlling the quality of PRRSV antigen.

[0343] Table 13-1 Test Results

[0344]

[0345] Example 14 Specificity

[0346] 14.1 Materials: 1% sensitized red blood cells prepared in Example 12.

[0347] Diluent: Phosphate buffer at pH 7.2 containing 1% healthy rabbit serum.

[0348] The samples tested: Samples 1 to 6 are cell culture media of the following viruses: baculovirus, classical swine fever virus, porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus, transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV).

[0349] 14.2 Methods

[0350] (1) Add 25 μl of diluent to each well of a 96-well V-bottom blood coagulation plate;

[0351] (2) Add 25 μl of the test sample to the first well of each row, and perform serial dilution from the first well to the eleventh well. After mixing, discard the 25 μl. The twelfth well is the negative control well.

[0352] (3) Add 25 μl of antibody-sensitized red blood cells of different dilutions to each row. Shake the blood coagulation plate to mix well, and let it stand at room temperature for 1.5 to 2 hours. Determine the titer when button-shaped deposits of red blood cells appear at the bottom of the 12th well.

[0353] 14.3 Results: Culture media of baculovirus, classical swine fever virus, porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus, transmissible gastroenteritis virus (TGEV), and porcine epidemic diarrhea virus (PEDV) did not agglutinate 1% sensitized red blood cells sensitized with PRRSV monoclonal antibody, as detailed in Table 14-1. This indicates that the method of the present invention has good specificity.

[0354] Table 14-1 Detection Results (log2)

[0355]

[0356]

[0357] Note: <1: No 50%, 75%, or 100% agglutination was observed when 1% of sensitized red blood cells were added to the virus sample at the highest concentration (2-fold dilution).

[0358] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for detecting antigen content for non-diagnostic purposes, characterized in that, The detection method includes the following steps: S10. Prepare 4 test samples and 4 reference samples with different concentrations. Use diluent to serially dilute the 4 test samples and 4 reference samples with different concentrations on a 96-well blood coagulation plate to obtain test samples and reference samples with different dilutions. S20. Sensitized red blood cell suspension is added to the wells of test samples and reference samples at different dilutions, mixed, and allowed to stand. The agglutination results of each well are then determined. Scores are assigned and recorded based on the agglutination results of each well. The scores of all test samples are summed and recorded as A, and the total scores of all reference samples are summed and recorded as B. The step of scoring and recording the agglutination results of the sensitized red blood cell suspension in the agglutination mixture of multiple diluted test samples as A includes: the sensitized red blood cell suspension... The cell agglutination results include any one of 100% sensitized red blood cell agglutination, 75% sensitized red blood cell agglutination, 50% sensitized red blood cell agglutination, 25% sensitized red blood cell agglutination, and 0% sensitized red blood cell agglutination. The agglutination results of 100% sensitized red blood cell agglutination, 75% sensitized red blood cell agglutination, 50% sensitized red blood cell agglutination, 25% sensitized red blood cell agglutination, and 0% sensitized red blood cell agglutination are recorded as 4, 3, 2, 1, and 0 points, respectively. S30. According to the formula, the antigen content of the tested sample = antigen content of the reference sample × 2 （A-B） / 16 .

2. The method for detecting antigen content for non-diagnostic purposes as described in claim 1, characterized in that, Before step S10, the preparation of a sensitized red blood cell suspension is also included, and the steps for preparing the sensitized red blood cell suspension are as follows: S101. After washing, aldehyde-modifying, and tanning sheep red blood cells, the cells are resuspended in buffer solution to obtain a resuspended solution. S102. A monoclonal antibody is added to the resuspended solution to sensitize it, and then a preservative is added to obtain a sensitized red blood cell suspension.

3. The method for detecting antigen content for non-diagnostic purposes as described in claim 2, characterized in that, In step S101, the buffer solution is an acetate buffer solution; Wherein, the pH value of the acetate buffer solution is 4.6~5.4; and / or, The concentration of the acetate buffer solution is 0.01~0.1 mol / L.

4. The method for detecting antigen content for non-diagnostic purposes as described in claim 2, characterized in that, In step S102, the monoclonal antibody includes any one of the following monoclonal antibodies: Monoclonal antibodies against rabies N protein or monoclonal antibodies against rabies G protein; Monoclonal antibodies targeting the PCV2-Cap protein; Monoclonal antibodies against PRRSV antigen.

5. The method for detecting antigen content for non-diagnostic purposes as described in claim 1, characterized in that, In step S20, the four concentrations of the tested samples include: samples containing 100% of the tested sample, samples containing 80% of the tested sample, samples containing 64% of the tested sample, and samples containing 51% of the tested sample; and / or, The four reference samples include: 100% reference sample, 80% reference sample, 64% reference sample, and 51% reference sample.

6. The method for detecting antigen content for non-diagnostic purposes as described in claim 1, characterized in that, In step S20, the reference sample includes any one of the following antigens: a solution or lyophilized product containing inactivated rabies virus, a solution containing PCV2-Cap antigen, and a solution containing PRRSV antigen.

7. The method for detecting antigen content for non-diagnostic purposes as described in claim 1, characterized in that, In step S10, the diluent is a phosphate buffer containing 1% healthy rabbit serum; Wherein, the pH value of the phosphate buffer solution is 7-7.4; and / or, The concentration of the phosphate buffer solution is 0.01~0.1 mol / L.

8. The method for detecting antigen content for non-diagnostic purposes as described in claim 1, characterized in that, The antigens include any one of rabies virus antigen, PCV2-Cap antigen, and PRRSV antigen.

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