Preparation method of hemagglutination inhibition antigen of formaldehyde-inactivated A-type avibacterium paragallinarum

Abstract

The invention relates to the technical field of hemagglutination inhibition antigen preparation, in particular to a preparation method of a formaldehyde-inactivated A-type avibacterium paragallinarum hemagglutination inhibition antigen. According to the technical scheme, the method comprises the following steps: preparing an antigen repair liquid, wherein the antigen repair liquid comprises ethylenediamine tetraacetic acid, sodium citrate dehydrate, tween-20, triethanolamine and purified water; according to the invention, the hemagglutination activity of formaldehyde inactivated A type avibacterium paragallinarum is successfully recovered through an optimized antigen repair liquid and a mild repair process, and the problem of titer loss caused by antigen epitope shielding in a traditional inactivation process is solved; according to the method, the inactivated bacterial liquid is directly utilized, complex culture and crushing steps are omitted, the process is remarkably simplified, the cost is reduced, and meanwhile, the antigen stability and the preservation period are improved; the kit is simple in structure, convenient and safe to operate and high in antigen specificity, can be accurately used for serum antibody detection and vaccine effect evaluation, and provides efficient and reliable technical support for diagnosis, prevention and control of avian infectious rhinitis.

Description

Technical Field

[0001] The invention relates to the technical field of hemagglutination inhibition antigen preparation, in particular to a method for preparing a hemagglutination inhibition antigen of Avibacterium paragallinarum type A inactivated by formaldehyde. Background Art

[0002] Avibacterium paragallinarum type A is the primary pathogen of infectious rhinitis, causing severe respiratory symptoms and decreased egg production in poultry, particularly chickens, resulting in significant economic losses to the global poultry industry. The pathogen's surface hemagglutinin (HA) protein mediates hemagglutination of erythrocytes, and the hemagglutination-inhibiting antibodies (HI antibodies) it induces are a key indicator of immune protection. The development of highly effective and stable HA antigens is crucial for vaccine development and serological diagnosis.

[0003] In the production of vaccine companies, formaldehyde inactivation technology is still one of the core processes in vaccine production, especially in inactivated viral vaccines and toxoid vaccines. The core mechanism of formaldehyde inactivation is to destroy its pathogenicity and retain its immunogenicity through cross-linking reactions with the active groups of antigens (such as viral proteins or bacterial toxins). This cross-linking may cause the antigen epitopes (key areas recognized by the immune system) to be masked or destroyed, thereby affecting the immunogenicity of the vaccine. The degree of cross-linking needs to be precisely controlled to avoid loss of immunogenicity. For example, after formaldehyde inactivation of Avian Bacillus paragallinarum type A, the hemagglutination antigen loses its agglutination effect. The mechanism is as follows: formaldehyde (HCHO) combines with amino (-NH2), sulfhydryl (-SH) and other groups of the pathogen protein through alkylation reaction to form intermolecular cross-links. An appropriate amount of cross-linking can fix the natural conformation of HA protein, but excessive formaldehyde or too long an inactivation time will lead to excessive cross-linking, causing the three-dimensional structure of HA protein to be distorted, shielding the antibody binding epitope (such as the receptor binding domain), and HA to lose the ability to bind to red blood cells, resulting in the inability of the antigen to agglutinate red blood cells in the HI test (false negative), and even if antibodies are present, no inhibitory effect can be shown.

[0004] A Chinese patent (CN 109055253B) discloses a method for preparing hemagglutination antigens from A. paragallinarum. This method involves culturing Staphylococcus suis and using the hyaluronidase in the bacterial solution to treat the capsule of A. paragallinarum to expose antigenic epitopes, thereby increasing the hemagglutination titer. This method is complex and time-consuming.

[0005] The cultivation of A. paragallinarum requires additives such as NAD and chicken serum. The serum demand is high and expensive, which significantly increases the cost of the culture medium. The preparation of hemagglutination inhibition antigens requires KSCN / NaCl treatment of the bacteria and ultrasonic disruption, which is time-consuming and has low titers. In addition, the antigens are unstable and lose hemagglutination activity, resulting in a very short shelf life and uneconomical. Therefore, we proposed a method for preparing hemagglutination inhibition antigens of A. paragallinarum type A that is inactivated by formaldehyde. Summary of the Invention

[0006] The purpose of the present invention is to address the problem that the preparation method of hemagglutination inhibition antigen in the background art is complicated and time-consuming, and to propose a method for preparing hemagglutination inhibition antigen of Avibacterium paragallinarum type A inactivated by formaldehyde.

[0007] The technical solution of the present invention is a method for preparing a hemagglutination inhibition antigen of Avibacterium paragallinarum type A inactivated by formaldehyde, comprising the following steps:

[0008] (1) preparing an antigen repair solution: the antigen repair solution comprises ethylenediaminetetraacetic acid (EDTA), sodium citrate dihydrate, Tween-20, triethanolamine and purified water;

[0009] (2) Initial treatment of bacterial antigen repair: Take formaldehyde-inactivated A. paragallinarum bacterial solution, centrifuge at 3000 rpm and discard the supernatant, collect the bacterial precipitate, mix the antigen repair solution in (1) with the A. paragallinarum bacterial precipitate at a ratio of 40:1 (v / v), incubate at 37°C for 1 hour and 60°C for 30 minutes, then centrifuge at 3000 rpm for 10 minutes, discard the supernatant and retain the precipitate;

[0010] (3) Repeated repair and enhancement: add 10 times the volume of antigen repair solution to the precipitate obtained in step (2), repeat incubation at 37°C for 1 hour and 60°C for 30 minutes, cool, centrifuge at 3000 rpm for 10 minutes, and discard the supernatant;

[0011] (4) Resuspend the precipitate with phosphate buffered saline (PBS) and mix well to obtain the hemagglutination inhibitory antigen of A. paragallinarum type A with hemagglutination activity.

[0012] Optionally, the antigen retrieval solution is composed of: 0.2 g of ethylenediaminetetraacetic acid (EDTA), 1.31 g of sodium citrate dihydrate, 200 μL of Tween-20, and 50 mL of triethanolamine. Water is added to make the volume to 1 L, and the pH is adjusted to 6.0.

[0013] Optionally, the hemagglutination inhibition antigen is used in hemagglutination test (HA) and hemagglutination inhibition test (HI) for detecting antibodies to Avibacterium paragallinarum type A.

[0014] Optionally, the centrifugation conditions in step (2) and step (3) are both 3000 rpm for 10 minutes.

[0015] Optionally, the volume of PBS added in step (4) is equal to the volume of the initial bacterial solution of Avibacterium paragallinarum type A after formaldehyde inactivation.

[0016] Optionally, the hemagglutination titer of the hemagglutination inhibition antigen is detected by a 96-well V-type microplate, and the judgment standard is the maximum dilution multiple of the antigen that causes complete agglutination of red blood cells.

[0017] Optionally, the hemagglutination inhibition test (HI) comprises the following steps: mixing the serum to be tested with 4 hemagglutination units (4HAU) of antigen working solution, adding aldehyde-treated red blood cells, and determining the antibody titer by agglutination inhibition reaction.

[0018] Optionally, the formaldehyde-inactivated Avibacterium paragallinarum type A is derived from a bacterial solution inactivated by formaldehyde during vaccine production.

[0019] Compared with the prior art, the present invention has the following beneficial technical effects:

[0020] Through a specifically formulated antigen repair solution and gradient incubation process, the hemagglutination titer of A. paragallinarum type A was successfully restored after formaldehyde inactivation (HA titer can reach 160-320), solving the technical problem of hemagglutination epitope masking caused by excessive cross-linking in the traditional inactivation process.

[0021] Directly utilizing the formaldehyde-inactivated bacterial solution already available in vaccine production for repair eliminates the complex culture (such as reliance on NAD and chicken serum) and ultrasonic disruption steps in traditional methods, significantly reducing culture medium costs and preparation cycles.

[0022] The repaired antigen showed high stability in the hemagglutination test (HA) and hemagglutination inhibition test (HI), and had a prolonged shelf life, thus avoiding the defect of easy inactivation of traditional antigens.

[0023] The repair process only requires conventional centrifugation and incubation equipment, without the need for complex operations such as high temperature and high pressure, reducing the risk of antigen structure damage and lowering the difficulty of operation for experimenters.

[0024] The prepared antigen only reacts specifically with antibodies of A. paragallinarum type A, and has no cross-reaction with positive sera of A. paragallinarum type B and C and other poultry pathogens (such as avian influenza virus and Newcastle disease virus), ensuring the accuracy of the test results.

[0025] The repaired antigens can be directly used for serological diagnosis (such as HI test to evaluate antibody titer) and vaccine efficacy evaluation, providing an efficient tool for the prevention and control of avian infectious rhinitis.

[0026] The present invention successfully restores the hemagglutination activity of formaldehyde-inactivated A. paragallinarum type A through optimized antigen repair liquid and mild repair process, solving the problem of potency loss caused by antigen epitope shielding in traditional inactivation processes. This method directly utilizes inactivated bacterial liquid, eliminating complex culture and crushing steps, significantly simplifying the process and reducing costs, while improving antigen stability and shelf life. The operation is convenient and safe, and the antigen specificity is strong, which can be accurately used for serum antibody detection and vaccine efficacy evaluation, providing efficient and reliable technical support for the diagnosis and prevention of avian infectious rhinitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A flow chart of a method for preparing a hemagglutination inhibition antigen of Avibacterium paragallinarum type A inactivated by formaldehyde is provided;

[0028] Figure 2 Graph showing the HA test results of formaldehyde-inactivated Avibacterium paragallinarum type A hemagglutination inhibition antigen with and without repair treatment in Example 1;

[0029] Figure 3 This is a graph showing the HA test results of the hemagglutination inhibition antigen of the formaldehyde-inactivated Avibacterium paragallinarum type A provided by the vaccine company in Example 2, with and without repair treatment;

[0030] Figure 4 This is a graph showing the results of a hemagglutination inhibition test using the hemagglutination inhibition antigen prepared by repairing treatment in Example 2 using Avibacterium paragallinarum type A;

[0031] Figure 5 This is a graph showing the results of a HI specificity test using the hemagglutination inhibition antigen of Avibacterium paragallinarum type A prepared using the repair process in Example 2 and positive sera from different backgrounds. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0033] The components of the embodiments of the present invention generally described and shown in the drawings herein may be arranged and designed in a variety of different configurations. Therefore, the following detailed description of the embodiments of the invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the invention.

[0034] Example 1: Hemagglutination (HA) test, such as Figure 1 As shown,

[0035] Step 1: Preparation of antigen retrieval solution: weigh 0.2 g of EDTA (ethylenediaminetetraacetic acid), 1.31 g of sodium citrate dihydrate, 200 μl of Tween-20, and 50 mL of triethanolamine, add water to make up to 1 L, and adjust the pH to 6.0.

[0036] In the second step, take 10 mL of formaldehyde-inactivated A. paragallinarum type A culture, centrifuge, discard the supernatant, and collect the bacterial pellet. Resuspend the bacterial pellet in a 40:1 (v / v) ratio of the antigen retrieval solution prepared in the first step. Vortex thoroughly to form a homogenized suspension. Incubate the suspension at 37°C for 1 hour and 60°C for 30 minutes, then centrifuge at 3000 rpm for 10 minutes. Discard the supernatant and retain the pellet.

[0037] Step 3: Add 10 times the volume of antigen retrieval solution, incubate at 37°C for 1 hour, incubate at 60°C for 30 minutes, and wait for it to cool down naturally.

[0038] Step 4: After returning to room temperature, centrifuge at 3000 rpm for 5 min, remove the supernatant, add 10 mL of PBS to resuspend, and mix thoroughly by pipetting.

[0039] Step 5: Hemagglutination (HA) test

[0040] The operation is best performed at room temperature between 20°C and 25°C. If the room temperature is not within this range, it can be placed in a 37°C incubator. Be sure to cover the container with a lid or sealing film to prevent liquid evaporation.

[0041] 1) HA Assay Procedure: A 96-well V-shaped microplate was used for the assay, with a total reaction volume of 75 μl. The prepared antigen was diluted 5-fold, followed by a 2-fold serial dilution, using bovine serum albumin-PBS (abbreviated as PBSS) according to Table 1. 1% aldehyde-treated red blood cells were then added. After gentle mixing, the cells were allowed to stand at room temperature (15-25°C) for 40-60 minutes, or at 37°C for 40 minutes, until the red blood cells in the aldehyde-treated red blood cell control wells completely precipitated. Detailed procedures are shown in Table 1.

[0042] Table 1 Type A Avibacterium paragallinarum HA test operation procedures

[0043]

[0044] Note: HA titer is determined by the presence of red blood cells flat on the bottom of the well, indicating complete agglutination. Red blood cells clustered in a dot-like pattern in the center of the well indicate complete nonagglutination. Partial agglutination occurs when red blood cells cluster in a dot-like pattern in the center of the well, surrounded by red blood cells flat on the bottom. The maximum dilution of the antigen that causes complete agglutination is the HA titer of that antigen.

[0045] like Figure 2As shown, 1, 2, and 3 are treated A. paragallinarum type A antigens, and according to the judgment rule, the hemagglutination titer is 320. The MOCK group is the hemagglutination antigen titer without treatment after being inactivated by formaldehyde, showing an invalid titer.

[0046] Example 2: HI test detection of Avibacterium paragallinarum type A

[0047] The specific preparation method is as follows:

[0048] Qilu Animal Health Products Co., Ltd. provided 200 mL of Avibacterium paragallinarum type A bacterial solution, which had been inactivated by formaldehyde and had no hemagglutination titer after HA detection.

[0049] Step 1: Weigh 0.2 g of EDTA (ethylenediaminetetraacetic acid), 1.31 g of sodium citrate dihydrate, 200 μl of Tween-20, and 50 mL of triethanolamine, add water to make up to 1 L, and calibrate the pH to 6.0.

[0050] Step 2: Take a formaldehyde-inactivated A. paragallinarum strain A culture and centrifuge it at 3000 rpm for 10 minutes. Discard the supernatant and collect the bacterial pellet. Resuspend the bacterial pellet in the antigen retrieval solution prepared in step 1 at a ratio of 40:1 (v / v). Incubate at 37°C for 1 hour and then at 60°C for 30 minutes. After incubation, centrifuge at 3000 rpm for 10 minutes and discard the supernatant.

[0051] Step 3: Take the bacterial precipitate from the previous step, add 10 times the volume of antigen repair solution, incubate at 37℃ for 1 hour, and then incubate at 60℃ for 30 minutes. After the end, stop heating and cool naturally.

[0052] Step 4: After returning to room temperature, centrifuge at 3000 rpm for 5 minutes, remove the supernatant, and repeat twice. Add 200 mL of PBS and resuspend.

[0053] Step 5: Hemagglutination (HA) test

[0054] The operation steps are carried out according to the HA test operation procedures in Table 2.

[0055] Table 2 HA test operation method for potency determination

[0056]

[0057]

[0058] like Figure 3 As shown, 1, 2, and 3 are treated A. paragallinarum type A antigens, and according to the judgment rule, the hemagglutination titer is 160. The MOCK group is the hemagglutination antigen titer after being inactivated by formaldehyde without treatment, showing an invalid titer.

[0059] Step 6: Prepare a working antigen solution of 4 HA units. Dilute the antigen with bovine serum albumin (BSA) in PBS to a titer of 4 HA units (e.g., if the HA titer is 1:160, dilute the antigen to 1:40). This is a working antigen solution of 4 HA units. To verify the accuracy of the hemagglutination titers of the 4 HA units, add 1.0 mL of the prepared 1:40 dilution to 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, 5.0 mL, and 6.0 mL of PBS, respectively, to achieve final dilutions of 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7. Then, transfer 25 μl of each dilution to a microplate, add 25 μl of PBS, and then add 25 μl of a 1% aldehyde-treated red blood cell suspension and mix thoroughly. Place the microplate at 15-25°C for 40-60 minutes or at 37°C for 40 minutes. If the prepared antigen solution contains 4 HA units, a 1:4 dilution is the endpoint of agglutination. If it is higher than 4 units, a 1:5 or 1:6 dilution may be the endpoint; if it is lower, a 1:2 or 1:3 dilution may be the endpoint. The antigen dilution should be adjusted appropriately based on the test results to ensure that the working solution contains 4 HA units.

[0060] Step 7: HI test

[0061] 1) Serum treatment for HI test: dilute the test serum and negative serum 1:5 with 10% aldehyde-treated red blood cells, incubate at room temperature for 4 hours and then at 4°C overnight, vortexing thoroughly at least 5 times in between. Centrifuge at 1500g for 5 minutes, and collect the supernatant as the 5-fold diluted test serum, positive serum, and negative serum.

[0062] 2) HI Test Procedure: The assay was performed in a 96-well V-shaped microplate. The total reaction volume for the hemagglutination assay was 75 μl. The test serum, positive serum, and negative serum, diluted 5-fold in the above step, were serially diluted in bovine serum albumin-PBS buffer (PBSS). The 4HA working antigen solution was then added and the mixture was allowed to stand at room temperature for 20-30 minutes or at 37°C for 20 minutes. 1% aldehyde-treated red blood cells were then added and thoroughly vortexed to mix. The mixture was then allowed to stand at room temperature (15-25°C) for 40-60 minutes or at 37°C for 40 minutes, until the red blood cells in the red blood cell control well completely sank. The assay includes an aldehyde-treated red blood cell control. Specific procedures are shown in Table 3.

[0063] The results are as follows Figure 4 As shown, 1 and 2 are positive sera for A. paragallinarum type A, which have hemagglutination inhibition effect with A. paragallinarum type A antigen.

[0064] Table 3 HI test operation procedures for Avibacterium paragallinarum type A

[0065]

[0066]

[0067] 3) HI Specificity Test: Specificity was determined by performing a HI test on serum positive for A. paragallinarum (type A), A. paragallinarum (type B), A. paragallinarum (type C), Newcastle disease virus, pullorum, avian influenza virus subtype H5, and Pasteurella multocida, all with clear background. The assay was performed in a 96-well V-shaped microplate. The total hemagglutination reaction volume was 75 μl. After serial dilutions in bovine serum albumin-PBS (PBSS), the 4HA working antigen solution was added and incubated at room temperature for 20-30 minutes or at 37°C for 20 minutes. 1% aldehyde-treated red blood cells (RBCs) were then added and thoroughly vortexed to mix. The RBCs in the RBC control wells were allowed to completely sink. Antigen and aldehyde-treated RBC controls were included in the assay, and the specific procedures were the same as in Table 3.

[0068] The results are as follows Figure 5 As shown, 1-7 are respectively A. paragallinarum (type A)-positive serum, A. paragallinarum (type B)-positive serum, A. paragallinarum (type C)-positive serum, Newcastle disease virus-positive serum, pullorum-positive serum, avian influenza virus H5 subtype-positive serum, and Pasteurella multocida-positive serum. 8 is a PBS control group, in which only the A. paragallinarum type A-positive serum was initially diluted 100-fold, while the others were initially diluted 2-fold. The results showed that the treated A. paragallinarum type A antigen only reacted with A. paragallinarum type A-positive serum to produce an agglutination inhibition reaction, and did not react with the other sera, indicating good specificity.

[0069] The above specific embodiments are merely several optional embodiments of the present invention. Based on the technical solutions of the present invention and the relevant inspirations of the above embodiments, those skilled in the art may make various alternative improvements and combinations to the above specific embodiments.

Claims

1. A method for preparing a hemagglutination inhibition antigen of formaldehyde-inactivated Avibacterium paragallinarum type A, characterized in that: The following steps are involved: (1) preparing an antigen repair solution: the antigen repair solution comprises ethylenediaminetetraacetic acid, sodium citrate dihydrate, Tween-20, triethanolamine and purified water; (2) Initial treatment of bacterial antigen repair: Take formaldehyde-inactivated A. paragallinarum bacterial solution, centrifuge at 3000 rpm, discard the supernatant, collect the bacterial precipitate, mix the above antigen repair solution and A. paragallinarum bacterial precipitate at a ratio of 40:1 (v / v), incubate at 37°C for 1 hour and 60°C for 30 minutes, then centrifuge at 3000 rpm for 10 minutes, discard the supernatant and retain the precipitate; (3) Repeated repair and enhancement: add 10 times the volume of antigen repair solution to the precipitate obtained in step (2), repeat incubation at 37°C for 1 hour and 60°C for 30 minutes, cool and centrifuge at 3000 rpm for 10 minutes, and discard the supernatant; (4) Resuspend the precipitate with phosphate buffered saline (PBS) and mix well to obtain the hemagglutination inhibitory antigen of A. paragallinarum type A with hemagglutination activity.

2. The method for preparing a hemagglutination inhibition antigen of formaldehyde-inactivated Avibacterium paragallinarum type A according to claim 1, characterized in that: The antigen retrieval solution is composed of: 0.2 g of ethylenediaminetetraacetic acid, 1.31 g of sodium citrate dihydrate, 200 μL of Tween-20, and 50 mL of triethanolamine. Water is added to make the volume 1 L, and the pH is adjusted to 6.

0.

3. The method for preparing a hemagglutination inhibition antigen of formaldehyde-inactivated Avibacterium paragallinarum type A according to claim 1, characterized in that: The hemagglutination inhibition antigen is used for hemagglutination test and hemagglutination inhibition test for detecting Avibacterium paragallinarum type A antibodies.

4. The method for preparing a hemagglutination inhibition antigen of formaldehyde-inactivated Avibacterium paragallinarum type A according to claim 1, characterized in that: The centrifugation conditions in step (2) and step (3) are both 3000 rpm for 10 minutes.

5. The method for preparing a hemagglutination inhibition antigen of formaldehyde-inactivated Avibacterium paragallinarum type A according to claim 1, characterized in that: The volume of PBS added in step (4) is equal to the volume of the initial bacterial solution of Avibacterium paragallinarum type A after formaldehyde inactivation.

6. The method for preparing a hemagglutination inhibition antigen of formaldehyde-inactivated Avibacterium paragallinarum type A according to claim 1, characterized in that: The hemagglutination titer of the hemagglutination inhibition antigen is detected by a 96-well V-type microplate, and the judgment standard is the maximum dilution multiple of the antigen that causes complete agglutination of red blood cells.

7. The method for preparing a formaldehyde-inactivated hemagglutination inhibition antigen of Avibacterium paragallinarum type A according to claim 1, characterized in that: The hemagglutination inhibition test comprises the following steps: mixing the serum to be tested with 4 hemagglutination units of antigen working solution, adding aldehyde-treated red blood cells, and determining the antibody titer through agglutination inhibition reaction.

8. The method for preparing a formaldehyde-inactivated Avibacterium paragallinarum type A hemagglutination inhibition antigen according to claim 1, characterized in that: The formaldehyde-inactivated Avibacterium paragallinarum type A is derived from a bacterial solution inactivated by formaldehyde during vaccine production.

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