A method, kit, and application for detecting PCV2 Cap protein using a double-antibody sandwich quantitative ELISA.

Abstract

This invention relates to the field of animal biological product detection technology, specifically to a detection method, kit, and application of a PCV2 Cap protein double-antibody sandwich quantitative ELISA. Based on a combination of PCV2 Cap protein monoclonal antibody, standard antigen, PCV2 rabbit polyclonal antibody, and anti-rabbit HRP enzyme-labeled antibody, this invention establishes a PCV2 Cap protein double-antibody sandwich quantitative ELISA detection method by adjusting and optimizing the concentrations of the monoclonal antibody coating, detection antibody, and enzyme-labeled antibody, the concentration range of the standard antigen, and the reaction temperature, time, and ELISA reaction system solution formulation for each step. The raw materials used in this detection method are readily available, the preparation is simple, the polyclonal antibody can bind to more antigen sites, resulting in good blocking effect, a lower limit of detection, high sensitivity, and more accurate detection results. The PCV2 Cap protein quantification results are accurate and stable, providing effective assurance for the differential diagnosis of PCV2 and the quality control of PCV2 vaccines.

Description

Technical Field

[0001] This invention relates to the field of animal biological product detection technology, specifically to a detection method, kit, and application of a PCV2 Cap protein double antibody sandwich quantitative ELISA. Background Technology

[0002] The Cap protein (capsid protein) of porcine circovirus type 2 (PCV2) is the most important and characteristic structural protein of the virus. It plays a central role in the virus's life cycle and pathogenicity, and is also a major target for diagnostics and vaccine development.

[0003] Currently, the main methods for quantifying PCV2 Cap protein are SDS-PAGE and HPLC. SDS-PAGE quantifies the denatured Cap protein of PCV2, while HPLC quantifies the VLP structure of PCV2. Since each method quantifies different protein forms and structures, the quantitative results vary considerably. ELISA, on the other hand, quantifies immunologically active antigen proteins, allowing for the relative quantification of antigens that exert an immune effect. This is of great significance in providing data support for the effective antigen content of vaccines and the immunization efficacy of vaccines.

[0004] Therefore, for the quantitative detection of PCV2 Cap antigen, the classic double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) method is still used. For example, Chinese patent CN103033622A discloses a porcine circovirus type 2 ELISA antigen detection kit, which uses a traditional sandwich ELISA preparation method for PCV2-Cap protein. However, the traditional sandwich ELISA method is limited by the labeling density of the enzyme-labeled detection antibody itself, which is prone to false negatives and has poor sensitivity. In addition, the detection antibodies need to be individually labeled with HRP, which is cumbersome and costly to prepare.

[0005] Therefore, developing a PCV2Cap protein double antibody sandwich quantitative ELISA detection method with high sensitivity, stronger specificity, lower detection limit, and more stable detection results is of great practical significance. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the shortcomings of the existing technology, such as unstable quantitative results of PCV2 Cap protein, low sensitivity and insufficient specificity of detection methods, which cannot provide effective guarantee for the differential diagnosis of PCV2 and the quality control of PCV2 vaccines. The present invention provides a PCV2 Cap protein double antibody sandwich quantitative ELISA detection method.

[0007] Another technical problem to be solved by the present invention is to provide a PCV2 Cap protein double antibody sandwich quantitative ELISA kit.

[0008] Another technical problem to be solved by the present invention is to provide the application of the PCV2 Cap protein double antibody sandwich quantitative ELISA kit in the preparation of PCV2 antigen or PCV2 vaccine products.

[0009] The final technical problem to be solved by the present invention is to provide a reagent for stabilizing the PV2 Cap protein standard antigen.

[0010] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides a method for detecting PCV2 Cap protein using a double-antibody sandwich quantitative ELISA, comprising the following steps:

[0012] (1) Coat the ELISA plate with PCV2 Cap monoclonal antibody and block the coated ELISA plate with blocking solution;

[0013] (2) Dilute the PV2 Cap protein standard antigen and the sample to be tested with sample diluent, add them to the enzyme-labeled plate for incubation, wash with PBST buffer, add the detection antibody for incubation, wash with PBST buffer, and then add the enzyme-labeled antibody for incubation.

[0014] (3) Finally, after TMB light-shielded color development and termination of the reaction with a stop solution, the OD value was read. 450nm The value is used to determine the result;

[0015] The blocking solution and sample diluent are the same, containing 0.1–1 M PBS buffer, 1% (w / v) BSA, 5% (w / v) sucrose, 0.2% (v / v) Proclin 300, 160,000 units of gentamicin, and 0.01% (w / v) auxiliary component; wherein the auxiliary component is MgCl2 or ZnCl2. Specifically, 1 g bovine serum albumin, 0.2 mL Proclin 300, 16 mg gentamicin, 5 g sucrose, and 0.01 g MgCl2 or ZnCl2 are dissolved in 0.1–1 M PBS buffer and the volume is adjusted to 100 mL.

[0016] Preferably, the blocking solution and the sample diluent are the same, containing 0.1~1 M PBS buffer, 1% (w / v) BSA, 5% (w / v) sucrose, 0.2% (v / v) Proclin 300, 160,000 units of gentamicin and 0.01% (w / v) auxiliary component; wherein the auxiliary component is MgCl2.

[0017] In step (1), the PCV2 Cap monoclonal antibody is a monoclonal antibody secreted by hybridoma cell line 1A6 with accession number CCTCC NO: C2022151.

[0018] Specifically, the hybridoma cell line 1A6 with accession number CCTCC NO: C2022151 and the monoclonal antibody it secretes have been disclosed in Chinese patent CN114958776B.

[0019] In step (1), the PCV2 Cap monoclonal antibody is coated on the enzyme-labeled plate at a concentration of 0.1~1 μg / mL.

[0020] Preferably, the PCV2 Cap monoclonal antibody is coated onto the ELISA plate at a concentration of 0.1–0.2 μg / mL.

[0021] More preferably, the PCV2 Cap monoclonal antibody is coated on the ELISA plate at a concentration of 0.1 μg / mL.

[0022] In step (2), the PV2 Cap protein standard antigen and the test sample are diluted with sample diluent, specifically, the standard antigen and the test sample are diluted with sample diluent to 1 μg / mL, and then serially diluted 2 times to the 7th gradient.

[0023] The PV2 Cap protein standard antigen has been published in the article "Optimization of porcine circovirus type 2 Cap protein expressed by recombinant baculovirus and study on protein immunogenicity".

[0024] In step (2), the detection antibody is diluted 2000 to 3000 times, preferably 3000 times, and is diluted with sample diluent.

[0025] In step (2), the enzyme-labeled antibody is diluted 5,000 to 10,000 times, preferably 10,000 times, and is diluted with enzyme-labeled antibody diluent, wherein the enzyme-labeled antibody diluent is a sample diluent containing 0.05% complex enzyme stabilizer.

[0026] Specifically, the compound enzyme stabilizer includes, but is not limited to, AES, purchased from Xibao Biotechnology, catalog number ACE0070A.

[0027] In step (2), the incubation conditions are: incubation at 37℃ for 0.5~2 h; preferably, the incubation conditions are: incubation at 37℃ for 0.5~1 h.

[0028] In some embodiments of the present invention, the incubation conditions in the ELISA plate are: incubation at 37°C for 1 h; the incubation conditions for the detection antibody are: incubation at 37°C for 1 h; and the incubation conditions for the ELISA-labeled antibody are: incubation at 37°C for 0.5 h.

[0029] The detection antibody is a PCV2 rabbit polyclonal antibody, which is prepared in-house. The preparation method is as follows: healthy rabbits are immunized with a PCV2 monoclonal vaccine containing PCV2-rCap protein, and positive serum is collected 63 days after immunization; Lot: 20220615 (2-2).

[0030] The enzyme-labeled antibody was anti-rabbit HRP, purchased from Abcam; REF: ab205718, Lot: GR3411617-4.

[0031] In step (2), the PBST washing solution is formulated as follows: 0.05% Tween-20 PBS solution, that is, 0.5 mL Tween-20 is added to 1 L phosphate buffer.

[0032] In step (3), the TMB color development is performed at 37°C in the dark for 5-20 minutes. Preferably, the TMB color development is performed at 37°C in the dark for 15 minutes.

[0033] The TMB colorimetric solution was purchased from Thermo Fisher Scientific, REF: 34029, Lot: YA3822901.

[0034] In step (3), the termination solution is prepared by mixing hydrochloric acid and water at a volume ratio of 1:11.

[0035] The detection method described herein is used for the specific quantification of PCV2 Cap protein, with a lower limit of detection of 0.0625 μg / mL. The CV value of the detection results for the same sample at different times is <10%, indicating that the present invention provides an accurate, stable and effective detection method for the quantification of PCV2 Cap protein.

[0036] In step (3), the result determination specifically refers to: the reaction OD of the positive sample well. 450nm Value > 1.0; OD of reaction in negative sample wells 450nm Value < 0.3; Use Relpot software to calculate the sample RP value. When the RP value of the sample to be tested is > 1.0, the sample to be tested is deemed qualified.

[0037] Secondly, the present invention provides a PCV2 Cap protein double antibody sandwich quantitative ELISA kit.

[0038] The PCV2 Cap protein double antibody sandwich quantitative ELISA kit includes an enzyme-labeled plate coated with PCV2 Cap monoclonal antibody, blocking buffer, standard antigen, sample diluent, PCV2 rabbit polyclonal antibody, anti-rabbit HRP, enzyme-labeled antibody diluent, PBST washing buffer, chromogenic solution, and stop solution.

[0039] The blocking solution and the sample diluent are the same, containing 0.1-1 M PBS buffer, 1% BSA, 5% sucrose, 0.2% Proclin 300, 160,000 units of gentamicin, and 0.01% auxiliary components; wherein the auxiliary components are MgCl2 or ZnCl2; preferably, the auxiliary components are MgCl2.

[0040] Thirdly, the present invention provides the application of the PCV2 Cap protein double antibody sandwich quantitative ELISA kit in the preparation of PCV2 antigen or PCV2 vaccine products.

[0041] Fourthly, the present invention provides a reagent for stabilizing the PV2 Cap protein standard antigen.

[0042] The reagent is a sample diluent, formulated as follows: 0.1–1 M PBS buffer, 1% BSA, 5% sucrose, 0.2% Proclin 300, 160,000 units of gentamicin, and 0.01% auxiliary component; wherein the auxiliary component is MgCl2 or ZnCl2. Specifically, 1 g bovine serum albumin, 0.2 mL Proclin 300, 16 mg gentamicin, 5 g sucrose, and 0.01 g MgCl2 or ZnCl2 are dissolved in 0.1–1 M PBS buffer and the volume is adjusted to 100 mL.

[0043] Preferably, the sample diluent comprises 0.1-1 M PBS buffer, 1% (w / v) BSA, 5% (w / v) sucrose, 0.2% (v / v) Proclin 300, 160,000 units of gentamicin, and 0.01% (w / v) auxiliary component; wherein the auxiliary component is MgCl2.

[0044] In some embodiments of the present invention, the PV2 Cap protein standard antigen is stored in the sample diluent for more than 8 months and remains stable, with the CV value of the antigen detection OD value being only 5.9%.

[0045] Beneficial effects:

[0046] (1) The double-antibody sandwich quantitative ELISA detection method for PCV2 Cap protein established in this invention has excellent blocking effect, can significantly reduce non-specific background, and obtain detection results with high signal-to-noise ratio; it has high detection sensitivity and good detection effect, and the lowest detection limit can reach 0.0625 μg / mL, which can be efficiently used for the differential diagnosis of PCV2 and the quality control of PCV2 vaccine.

[0047] (2) The detection method of PCV2 Cap protein by double antibody sandwich quantitative ELISA established in this invention has a CV value of <10%. The system is less affected by environmental factors and the quantitative detection results are stable and accurate.

[0048] (3) The detection method of this invention uses a sample diluent and blocking solution with consistent formulations for the first time. While ensuring high sensitivity and accuracy of detection, it can also be used for long-term preservation of PCV2 Cap protein standard antigen. After being stored in this solution for 8 months, the PCV2 Cap protein standard antigen still maintains highly stable biological activity and OD value, without significant degradation or inactivation. This characteristic greatly extends the shelf life of the kit and solves the problem of activity decay of biological reagents during transportation and storage, and has extremely high application value. Attached Figure Description

[0049] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0050] Figure 1 Figure 1 shows the results of the specificity verification experiment for the PCV2 Cap protein double antibody sandwich ELISA quantitative detection method.

[0051] Figure 2 The result of establishing a linear relationship for the standard curve is shown in the graph.

[0052] Figure 3 This is a graph showing the results of verifying the shelf life of the standard antigen in the sample dilution solution. Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0054] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0055] The specific experimental reagents and materials used in this invention are as follows:

[0056] (1) PCV2 Cap monoclonal antibody coated plate (Lot: F403 P4875): original concentration 1087 mg / mL (quantitative detection at A280 nm);

[0057] (2) PCV2 Cap protein standard antigen: concentration 1425 mg / mL. The PCV2 Cap protein standard antigen is the PCV2 Cap protein, which has been published in the article “Optimization of recombinant baculovirus expression of porcine circovirus type 2 Cap protein and study on protein immunogenicity” (BCA quantitative detection).

[0058] (3) PCV2 rabbit polyclonal antibody: prepared internally by the company. The preparation method is as follows: healthy rabbits are immunized with PCV2 monoclonal antibody containing PCV2-rCap protein, and positive serum is collected 63 days after immunization; Lot:20220615(2-2).

[0059] (4) Anti-rabbit HRP: purchased from Abcam, REF: ab205718, Lot: GR3411617-4;

[0060] (5) Coating solution: Prepare 0.05 M carbonate buffer solution with the following formula: 2.93 g NaHCO3 and 1.59 g Na2CO3 dissolved in distilled water, and diluted to 1 L;

[0061] (6) PBST washing solution: PBS solution containing 0.05% Tween-20, i.e., 0.5 mL Tween-20 added to 1 L phosphate buffer.

[0062] (7) TMB developer: Thermo Fisher Scientific REF: 34029, Lot: YA3822901;

[0063] (8) Termination solution: Hydrochloric acid and water are mixed in a volume ratio of 1:11 to prepare the termination solution.

[0064] Example 1: Verification of the binding of monoclonal antibody to PCV2 Cap protein

[0065] The binding of serially diluted PCV2 Cap monoclonal antibody to PCV2 Cap protein standard antigen was verified. The specific procedure is as follows:

[0066] 1. PCV2 Cap monoclonal antibody gradient dilution coating

[0067] 1.1 The PCV2 Cap monoclonal antibody (i.e., the monoclonal antibody secreted by hybridoma cell line 1A6 with accession number CCTCC NO: C2022151, which has been disclosed in Chinese patent CN114958776B) was serially diluted with carbonate buffer at pH 9.6 to prepare monoclonal antibody solutions of the following concentrations: 0.8 μg / mL, 0.4 μg / mL, 0.2 μg / mL, 0.1 μg / mL, 0.05 μg / mL, 0.025 μg / mL, 0.0125 μg / mL, and 0.00625 μg / mL, 100 μL / well. The solutions were added to an ELISA plate and incubated overnight at 2–8°C for coating.

[0068] 1.2 Remove the coated monoclonal antibody plate, discard the liquid in the wells, add PBST washing buffer, 260~300 μL / well, wash 4 times, and on the last wash, invert the monoclonal antibody plate onto absorbent paper and pat dry.

[0069] 1.3 Add blocking solution (1% BSA), 150 μL / well, and incubate at 37℃ for 2 h;

[0070] 1.4 Discard the liquid in the wells, add PBST washing buffer, 260~300 μL / well, wash 4 times, and on the last wash, invert the monoclonal antibody plate onto absorbent paper and pat dry.

[0071] 1.5 After the coated monoclonal antibody plates have dried until there are no watermarks on the plates and no obvious water droplets on the surface, seal them with sealing film and store them in a refrigerator at 2~8℃ for later use.

[0072] 2. Add PCV2 Cap protein standard antigen

[0073] 2.1 Dilute the PCV2 Cap protein standard antigen with sample diluent (1% BSA) to a concentration of 1 μg / mL (upper limit of detection for the standard curve).

[0074] 2.2 Add the diluted PCV2 Cap protein standard antigen to the corresponding wells of the monoclonal antibody plate coated in step 1, 100 μL / well. After sealing the plate with sealing film, incubate at 37°C for 1 h.

[0075] 2.3 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0076] 3. Add detection antibodies

[0077] 3.1 The PCV2 rabbit polyclonal antibody was diluted 3000 times with the sample dilution buffer, 100 μL / well, sealed with sealing film, and incubated at 37℃ for 0.5 h;

[0078] 3.2 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0079] 4. Add enzyme-labeled antibody

[0080] 4.1 Dilute anti-rabbit HRP 10,000 times with enzyme-labeled antibody dilution buffer (PBS buffer), 100 μL / well, seal with sealing film, and incubate at 37℃ for 0.5 h;

[0081] 4.2 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0082] 5. Color development, termination, and reading

[0083] 5.1 Add substrate solution TMB colorimetric reagent to develop color, 100 μL / well, seal the plate with sealing film, and develop color at 37℃ in the dark for 10 minutes;

[0084] 5.2 Add stop solution, 50 μL / well, gently mix the liquid in the well, and read the OD on a microplate reader within 10 minutes after stopping the reaction. 450nm value.

[0085] The results are shown in Table 1. The PCV2 Cap protein standard antigen bound well to the serially diluted monoclonal antibodies at concentrations of 0.1–0.2 μg / mL. The OD value tended to stabilize in the higher concentration range, and the monoclonal antibody coating concentration was basically saturated.

[0086] Table 1. Binding of serially diluted monoclonal antibodies to PCV2 Cap protein

[0087]

[0088] Example 2: Verification of the binding of serially diluted PCV2 Cap protein to a fixed coating concentration of monoclonal antibody

[0089] The binding efficiency of two different coating concentrations (0.1 μg / mL and 1 μg / mL) of PCV2 Cap monoclonal antibody with serially diluted PCV2 Cap protein standard antigen (starting from a concentration of 1 μg / mL and serially diluted 2-fold, i.e., 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, and 0.0156 μg / mL) was further evaluated. The specific procedure is as follows:

[0090] 1. PCV2 Cap monoclonal antibody dilution and coating

[0091] 1.1 The PCV2 Cap monoclonal antibody (i.e., the monoclonal antibody secreted by hybridoma cell line 1A6 with accession number CCTCC NO: C2022151, which has been disclosed in Chinese patent CN114958776B) was diluted with carbonate buffer at pH 9.6 to 1 μg / mL and 0.1 μg / mL, respectively, with a coating volume of 100 μL / well, and placed in a refrigerator at 2~8℃ overnight.

[0092] 1.2 Remove the coated monoclonal antibody plate, discard the liquid in the wells, add PBST washing buffer, 260~300 μL / well, wash 4 times, and on the last wash, invert the monoclonal antibody plate onto absorbent paper and pat dry.

[0093] 1.3 Add blocking solution (1% BSA), 150 μL / well, and incubate at 37℃ for 2 h;

[0094] 1.4 Discard the liquid in the wells, add PBST washing buffer, 260~300 μL / well, wash 4 times, and on the last wash, invert the monoclonal antibody plate onto absorbent paper and pat dry.

[0095] 1.5 After the coated monoclonal antibody reaction plate has dried until there are no watermarks on the plate and no obvious water droplets on the surface, seal it with sealing film and store it in a refrigerator at 2~8℃ for later use.

[0096] 2. Add PCV2 Cap protein standard antigen

[0097] 2.1 Dilute the PCV2 Cap protein standard antigen to a concentration of 1 μg / mL using sample dilution buffer (1% BSA), and then perform serial dilutions of 2-fold to the 7th concentration gradient;

[0098] 2.2 Add the diluted PCV2 Cap protein standard antigen to the corresponding wells of the monoclonal antibody plate coated in step 1, 100 μL / well. After sealing the plate with sealing film, incubate at 37°C for 1 h.

[0099] 2.3 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0100] 3. Add detection antibodies

[0101] 3.1 The PCV2 rabbit polyclonal antibody was diluted 3000 times with the sample dilution buffer, 100 μL / well, sealed with sealing film, and incubated at 37℃ for 0.5 h;

[0102] 3.2 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0103] 4. Add enzyme-labeled antibody

[0104] 4.1 Dilute anti-rabbit HRP 10,000 times with enzyme-labeled antibody dilution buffer (PBS buffer), 100 μL / well, seal with sealing film, and incubate at 37℃ for 0.5 h;

[0105] 4.2 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0106] 5. Color development, termination, and reading

[0107] 5.1 Add substrate solution TMB colorimetric reagent to develop color, 100 μL / well, seal the plate with sealing film, and develop color at 37℃ in the dark for 10 minutes;

[0108] 5.2 Add stop solution, 50 μL / well, gently mix the liquid in the well, and read the OD on a microplate reader within 10 minutes after stopping the reaction. 450nm value.

[0109] The results are shown in Table 2. At an antigen concentration of 1 μg / mL, the OD values ​​of the 0.1 μg / mL monoclonal antibody-coated wells (OD≈1.804) and the 1 μg / mL monoclonal antibody-coated wells (OD≈2.094) showed a relatively small difference (approximately 16%). Considering the need to save antibody costs, and given that the data in Table 1 showed similar OD values ​​for antigen detection at low and high monoclonal antibody concentrations, 0.1 μg / mL was chosen as the concentration for subsequent experiments.

[0110] Table 2. Mean OD values ​​of the antigen reaction between monoclonal antibodies and serially diluted standards.

[0111]

[0112] Example 3: Screening and determination of components of blocking solution and sample dilution solution

[0113] 1. Screening of protein components in blocking liquid

[0114] Examples 1 and 2 determined the effective specific binding of the antigen and antibody, verifying the feasibility of establishing a PCV2 Cap protein double-antibody sandwich ELISA quantitative detection method. To obtain lower non-specific binding background and improve the sensitivity of the method, the protein components of the blocking solution in the ELISA reaction system were further screened and optimized.

[0115] Specifically, 1% BSA, or 1% casein, or 1% protamine, or 1% ovalbumin (OVA) were prepared using 0.1 M PBS buffer as blocking solutions. The ELISA method was used to detect 1 μg / mL standard antigen and negative control. The results are shown in Table 3 below.

[0116] Table 3. Mean OD values ​​of the protein components in the blocking liquid screening experiment.

[0117]

[0118] As shown in Table 3, when using 1% BSA as the blocking solution, the test results have lower negative control values ​​and higher positive control values, with the highest P / N ratio and the best blocking effect. This is followed by 1% OVA. Therefore, 1% BSA is selected as the optimal blocking solution.

[0119] 2. Investigation into the influence of sample diluent and blocking solution compatibility on detection results

[0120] To ensure the stability of the ELISA detection system and reduce nonspecific background, the impact of different combinations of sample diluent and blocking solution on detection performance was further evaluated.

[0121] Using 1% BSA as the preferred blocking solution, two sets of sample dilution solutions, 1% BSA and 1% OVA, were set up for comparative verification experiments. The test results are shown in Table 4.

[0122] Table 4. Results of mean OD values ​​in experiments involving combinations of blocking and diluent solutions.

[0123]

[0124] Experimental data showed that when both the sample diluent and blocking buffer were 1% BSA, the P / N value reached 14.51. This value was not significantly different from the optimal P / N value of 15.53 obtained in Table 3, and was significantly higher than the control group using 1% OVA as the sample diluent. This indicates that using 1% BSA as the blocking buffer, and simultaneously using it as the sample diluent, can effectively suppress non-specific adsorption, minimize background signal interference, and improve detection stability. Therefore, 1% BSA was determined to be the optimal blocking buffer and sample diluent for this ELISA system.

[0125] 3. Screening for the optimal combination of other additives in the sample diluent / blocking solution

[0126] To extend the shelf life of the PCV2 Cap protein standard antigen and maintain its bioactivity and detection stability, other additives were introduced into the sample dilution / blocking buffer. The optimized ELISA method was used to detect the 1 μg / mL standard antigen and negative control to determine the optimal combination of additives. The results are shown in Table 5.

[0127] Table 5. Screening Results of Combinations of Added Components to Sample Diluent / Blocking Solution

[0128]

[0129] The results showed that the P / N values ​​of each group differed significantly after introducing different additives. Combination 10 exhibited the best detection performance, with the highest P / N value of 13.48, followed by Combination 12, which also achieved a P / N value of 12.01. Although these values ​​were lower than those of the pure BSA system (P / N = 14.51), both remained within the high signal-to-noise ratio range. This result indicates that Combinations 10 and 12, with different additives, are expected to effectively maintain the spatial conformation and long-term stability of the PCV2 Cap protein antigen while minimizing non-specific background interference, ensuring a high signal-to-noise ratio and method stability, and potentially achieving a balance between detection sensitivity and reagent storage stability. Subsequently, Combination 10 was selected as the optimal formulation for sample diluent / blocking solution, and its shelf life was validated in subsequent experiments on the standard antigen.

[0130] Example 4: Determination of the dilution factor between detection antibody and enzyme-labeled antibody

[0131] Using the optimal formulation of sample dilution / blocking buffer and a preferred concentration of 0.1 μg / mL to coat the monoclonal antibody, the PCV2Cap protein standard antigen was diluted to a concentration of 1 μg / mL. This was then reacted with detection antibodies diluted 2000-fold and 3000-fold, and enzyme-labeled antibodies diluted 5000-fold and 10000-fold, respectively. Based on the reaction OD values, the P / N ratio was calculated to determine the optimal combination of dilution factors for the detection antibody and enzyme-labeled antibody, thereby obtaining a high signal-to-noise ratio and a suitable detection range. The specific process is as follows:

[0132] 1. PCV2 Cap monoclonal antibody dilution and coating

[0133] 1.1 The PCV2 Cap monoclonal antibody (i.e., the monoclonal antibody secreted by hybridoma cell line 1A6 with accession number CCTCC NO: C2022151, which has been disclosed in Chinese patent CN114958776B) was diluted to 0.1 μg / mL with carbonate buffer at pH 9.6, and the coating volume was 100 μL / well. The wells were then placed in a refrigerator at 2~8℃ overnight.

[0134] 1.2 Remove the coated monoclonal antibody plate, discard the liquid in the wells, add PBST washing buffer, 260~300 μL / well, wash 4 times, and on the last wash, invert the monoclonal antibody plate onto absorbent paper and pat dry.

[0135] 1.3 Add blocking solution, 150 μL / well, and incubate at 37℃ for 2 h;

[0136] 1.4 Discard the liquid in the wells, add PBST washing buffer, 260~300 μL / well, wash 4 times, and on the last wash, invert the microplate onto absorbent paper and pat dry.

[0137] 1.5 After the coated monoclonal antibody reaction plate has dried until there are no watermarks on the plate and no obvious water droplets on the surface, seal it with sealing film and store it in a refrigerator at 2-8℃ for later use.

[0138] 2. Add PCV2 Cap protein standard antigen

[0139] 2.1 Dilute the PCV2 Cap protein standard antigen to a concentration of 1 μg / mL using sample dilution buffer;

[0140] 2.2 Add the diluted PCV2 Cap protein standard antigen to the corresponding wells of the monoclonal antibody plate coated in step 1, 100 μL / well. After sealing the plate with sealing film, incubate at 37°C for 1 h.

[0141] 2.3 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0142] 4. Add detection antibodies

[0143] 3.1 The PCV2 rabbit polyclonal antibody was diluted 2000 times and 3000 times with the sample dilution buffer, 100 μL / well, sealed with sealing film, and incubated at 37℃ for 0.5 h;

[0144] 3.2 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0145] 5. Add enzyme-labeled antibody

[0146] 4.1 Dilute anti-rabbit HRP 5000-fold and 10000-fold with enzyme-labeled antibody dilution buffer (sample dilution buffer containing 0.05% enzyme stabilizer), 100 μL / well, seal with sealing film, and incubate at 37℃ for 0.5 h;

[0147] 4.2 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0148] 5. Color development, termination, and reading

[0149] 5.1 Add substrate solution TMB colorimetric reagent to develop color, 100 μL / well, seal the plate with sealing film, and develop color at 37℃ in the dark for 10 minutes;

[0150] 5.2 Add stop solution, 50 μL / well, gently mix the liquid in the well, and read the OD on a microplate reader within 10 minutes after stopping the reaction. 450nm value.

[0151] The results are shown in Table 6. The P / N ratios for positive and negative results were highest when the rabbit polyclonal antibody was diluted 3000 times and the anti-rabbit HRP was diluted 10000 times. Therefore, this condition was selected as the optimal dilution factor for the detection antibody and the enzyme-labeled antibody.

[0152] Table 6. Mean OD values ​​of the experiment determining the dilution factor of the detection antibody and the enzyme-labeled antibody.

[0153]

[0154] Example 5: Determination of incubation conditions for standard antigen / analyte antigen and detection antibody

[0155] To avoid the influence of room temperature variations on the experimental results, a constant temperature incubator at 37℃ was used to complete all incubation steps. Based on the determined optimal formulations of sample diluent / blocking buffer, optimal monoclonal antibody coating concentration (0.1 μg / mL), optimal detection antibody (rabbit polyclonal antibody) dilution factor (3000-fold), and optimal enzyme-labeled antibody (anti-rabbit HRP) dilution factor (10000-fold), the PCV2 Cap protein standard antigen was diluted to 1 μg / mL as a positive control, and the sample diluent served as a negative control. The incubation times for the standard antigen / analyte antigen and the detection antibody rabbit polyclonal antibody were set to 2 h, 1 h, and 0.5 h, respectively. Cross-condition screening was conducted to determine the optimal combination of incubation times in the 37℃ constant temperature incubator. Based on the reaction OD value results, the P / N value was calculated to obtain a high signal-to-noise ratio and a stable detection signal.

[0156] For the specific experimental procedure, please refer to the experimental procedure in Example 5.

[0157] As can be seen from the experimental results in Table 7, the P / N value is the largest when the antigen is incubated for 1 h and the rabbit polyclonal antibody is incubated for 1 h, indicating that the difference between the mean negative OD value and the mean positive OD value is the largest at this time, which is the optimal incubation condition for antigen and detection antibody.

[0158] Table 7. Mean OD values ​​of the experiments determining the incubation conditions for standard antigens / analyte antigens and detection antibodies.

[0159]

[0160] Example 6: Determination of color development conditions

[0161] To avoid the influence of room temperature variations on experimental results at different times, colorimetric development was still performed in a 37°C incubator. Based on all previously optimized conditions (optimal formulation of sample diluent / blocking buffer, monoclonal antibody coating concentration of 0.1 μg / mL, 3000-fold dilution of rabbit polyclonal antibody, 10000-fold dilution of anti-rabbit HRP, 1 h of antigen incubation, and 1 h of rabbit polyclonal antibody incubation), different colorimetric development times (5 min, 10 min, 15 min, and 20 min) were set to complete the colorimetric development and then the readings were stopped. The optimal colorimetric development time for the TMB substrate under constant temperature and light-protected conditions at 37°C was selected to obtain a high signal-to-noise ratio and a stable endpoint signal.

[0162] For the specific experimental procedure, please refer to the experimental procedure in Example 5.

[0163] The experimental results are shown in Table 8. When the color development time was increased from 5 min to 15 min, the P / N value reached its maximum at 15 min. Therefore, 37℃ and light-protected color development for 15 minutes was selected as the optimal color development condition.

[0164] Table 8. Determination of color development time conditions and mean OD values.

[0165]

[0166] Example 7: Specificity, Sensitivity and Standard Curve Establishment

[0167] 1. Specificity test

[0168] Using the established PCV2 Cap protein double-antibody sandwich ELISA detection method, common swine diseases such as classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine parvovirus (PPV), porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (RV), and porcine circovirus type 2 (PCV2) were detected. The results are shown in Table 9. Figure 1 As shown, only a P / N value > 2.1 for PCV2 is considered positive, while the detection results for other non-PCV2 pathogens are all negative. This indicates that the established PCV2 double-antibody sandwich quantitative ELISA detection method has no cross-reactivity with other swine viruses and has good specificity.

[0169] Table 9. Results of the specificity validation experiment for the PCV2 Cap protein double antibody sandwich ELISA quantitative detection method.

[0170]

[0171] 2. Sensitivity test

[0172] The PCV2 antigen protein was diluted to 1 μg / mL, and then serially diluted 2-fold to the 7th concentration gradient. The last gradient was treated with sample diluent as a negative control. Two replicates were performed for each concentration gradient. The ELISA detection method was used for detection, and the results are shown in the table below. The lowest concentration value corresponding to a P / N value > 2.1 was selected as the sensitivity of this method. According to the results in Table 10, the sensitivity of this method is 0.0625 μg / mL.

[0173] Table 10. Results of the sensitivity verification experiment for the PCV2 Cap protein double antibody sandwich ELISA quantitative detection method.

[0174]

[0175] 3. Experiment on establishing the standard curve

[0176] PCV2 standard protein was serially diluted seven times from a concentration of 1 μg / mL, and OD was measured using the PCV2 Cap protein double-antibody sandwich ELISA method described in this invention. 450nm Value detection, using R 2 The concentration range of 0.016 μg / mL to 1 μg / mL corresponding to ≥0.99 was used as the detection range for the standard curve, and the established standard curve is as follows. Figure 2 As shown, i.e., y = 1.9571x + 0.2405, R 2 =0.9928, where y = standard protein concentration, x = OD 450nm The value, and the protein concentration of the standard sample is related to the OD value. 450nm The values ​​show good linearity and can serve as a basic guarantee for calculating sample RP values ​​using software.

[0177] Example 8: Establishment of a quantitative ELISA method for PCV2 Cap protein double antibody sandwich assay

[0178] Based on all the aforementioned optimization conditions, a quantitative ELISA method for PCV2 Cap protein detection using a double-antibody sandwich assay was established. The specific process is as follows:

[0179] 1. PCV2 Cap monoclonal antibody gradient dilution coating

[0180] 1.1 The PCV2 Cap monoclonal antibody (i.e., the monoclonal antibody secreted by hybridoma cell line 1A6 with accession number CCTCC NO: C2022151, which has been disclosed in Chinese patent CN114958776B) was diluted to 0.1 μg / mL with carbonate buffer at pH 9.6, and 100 μL / well was added to the microplate and coated overnight in a refrigerator at 2~8℃.

[0181] 1.2 Remove the coated monoclonal antibody plate, discard the liquid in the wells, add PBST washing buffer, 260~300 μL / well, wash 4 times, and on the last wash, invert the monoclonal antibody plate onto absorbent paper and pat dry.

[0182] 1.3 Add blocking buffer (0.1 M PBS buffer containing 1% BSA, 0.2% Proclin 300 preservative, 160,000 units of gentamicin antibiotic, 5% sucrose and 0.01% MgCl2), 150 μL / well, and incubate at 37℃ for 2 h;

[0183] 1.4 Discard the liquid in the wells, add PBST washing buffer, 260~300 μL / well, wash 4 times, and on the last wash, invert the monoclonal antibody plate onto absorbent paper and pat dry.

[0184] 1.5 After the coated monoclonal antibody plates have dried until there are no watermarks on the plates and no obvious water droplets on the surface, seal them with sealing film and store them in a refrigerator at 2-8℃ for later use.

[0185] 2. Add PCV2 Cap protein standard antigen and the sample to be tested.

[0186] 2.1 Dilute the PCV2 Cap protein standard antigen to a concentration of 1 μg / mL using sample diluent (same as blocking buffer), then perform 2-fold serial dilutions to the 7th gradient in a dilution plate. Add diluent to the last gradient as a negative control well. Dilute the vaccine sample to be tested 100-fold using sample diluent, then perform 2-fold serial dilutions to the 8th gradient in a dilution plate. Perform 3 replicates for both the standard and the sample. The well distribution for the standard and sample dilutions is shown in Table 11 below.

[0187] Table 11. Well distribution of samples for PCV2 Cap protein double antibody sandwich quantitative ELISA detection.

[0188]

[0189] 2.2 Add the diluted standard and sample to the corresponding wells of the monoclonal antibody plate coated in step 1, 100 μL / well. After sealing the plate with sealing film, incubate at 37℃ for 1 h.

[0190] 2.3 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0191] 3. Add detection antibodies

[0192] 3.1 The PCV2 rabbit polyclonal antibody was diluted 3000 times with the sample diluent, 100 μL / well, sealed with sealing film, and incubated at 37℃ for 1 h;

[0193] 3.2 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0194] 4. Add enzyme-labeled antibody

[0195] 4.1 Dilute anti-rabbit HRP 10,000 times with enzyme-labeled antibody dilution buffer, 100 μL / well, seal with sealing film, and incubate at 37°C for 30 minutes;

[0196] 4.2 Discard the liquid in the wells, add PBST washing buffer to the reaction plate, 260~300 μL / well, wash 4 times, shake for 30 s each time, and finally invert the reaction plate onto absorbent paper to dry.

[0197] 5. Color development, termination, and reading

[0198] 5.1 Add the substrate solution TMB colorimetric reagent to develop the color, 100 μL / well, seal the plate with sealing film, and develop the color at 37℃ in the dark for 15 minutes;

[0199] 5.2 Add stop solution, 50 μL / well, gently mix the liquid in the well, and read the OD on a microplate reader within 10 minutes after stopping the reaction. 450nm value.

[0200] 6. Result Determination

[0201] 6.1 Determination of Experimental Validity

[0202] The reaction OD of the standard well 450nm The value should be in the range of 1.0~2.2 for the reaction OD of the positive sample well. 450nm The value should be >1.0; the OD of the reaction in the negative sample well. 450nm The value should be <0.3; if the above conditions are not met, the initial dilution ratio of the sample to be tested should be adjusted and the test should be repeated.

[0203] 6.2 Calculation of Experimental Results

[0204] 6.2.1 After inputting the test data using Relpot 4.0 software, the RP value for each sample is automatically calculated;

[0205] 6.2.2 If the RP value of a vaccine sample is greater than 1.0, the vaccine product is deemed qualified;

[0206] 6.2.3 Protein content of the sample to be tested = relative content of the sample (RP value) × protein content of the standard (1 μg / mL) × initial dilution factor of the sample.

[0207] Example 9: Stability study of standard antigens during shelf life

[0208] The PCV2 Cap protein standard antigen was placed in sample dilution buffer (same as the blocking buffer, i.e., 0.1 M PBS buffer determined by the combination screening, containing 1% BSA, 0.2% Proclin 300 preservative, 160,000 units of gentamicin antibiotic, 5% sucrose, and 0.01% MgCl2) and PBS buffer, and stored at 2–8°C. Samples were taken at different time points (0–240 days) for ELISA detection, and the OD at each time point was recorded. 450nm The stability of the PCV2 Cap protein standard antigen in the sample dilution solution was examined.

[0209] The results are shown in Table 12 and Figure 3 As shown, the PCV2 Cap protein standard antigen placed in the sample dilution buffer has an average OD value of [missing information]. 450nm With a standard deviation of 2.068 and a CV value of only 5.9%, the extremely low standard deviation and CV value demonstrate that this sample diluent provides an extremely stable microenvironment for the PCV2 Cap protein standard. In this sample diluent, 0.2% Proclin 300 preservative and 160,000 units of gentamicin antibiotic ensured a sterile and stable state in a sample diluent containing a large amount of other non-specific proteins and high concentrations of sugars. The high concentration of sugars, through hydration, encapsulates the protein molecular structure, forming a protective hydration layer that resists damage to the protein structure from external physical factors. Simultaneously, 0.01% MgCl2 effectively maintained the conformational stability of the protein, regulating ionic strength and optimizing the binding efficiency of antigen and antibody. These components work synergistically, enabling the PCV2 Cap protein standard antigen to maintain high activity and high stability during long-term storage.

[0210] Table 12 Results of Standard Antigen Shelf Life Verification Experiment

[0211]

[0212] Example 11: Stability Verification of the Method

[0213] The same vaccine aqueous antigen PCV2 Cap sample after demulsification was tested at different times, and the test data are shown in Table 13 below. It can be seen that, through the adjustments and optimizations of this invention, the PCV2 Cap protein double-antibody sandwich quantitative ELISA detection method provides stable quantitative detection results for PCV2 Cap protein. The CV values ​​of the detection results for the same sample at different times are all <10%, providing an accurate, stable, and effective method for the quantification of PCV2 Cap protein.

[0214] Table 13. Detection results of the same sample at different times.

[0215]

[0216] This invention provides a detection method, kit, and application concept and method for PCV2 Cap protein double antibody sandwich quantitative ELISA. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for detecting PCV2 Cap protein using a double-antibody sandwich quantitative ELISA, characterized in that, Includes the following steps: (1) Coat the ELISA plate with PCV2 Cap monoclonal antibody and block the coated ELISA plate with blocking solution; (2) Dilute the PV2 Cap protein standard antigen and the sample to be tested with sample diluent, add them to the enzyme-labeled plate for incubation, wash with PBST buffer, add the detection antibody for incubation, wash with PBST buffer, and then add the enzyme-labeled antibody for incubation. (3) After the TMB color development and the termination of the reaction by the termination solution, the OD value is read and the results are determined. 450nm value is read and the results are determined. The blocking solution and the sample diluent are the same, containing 0.1-1 M PBS buffer, 1% BSA, 5% sucrose, 0.2% Proclin 300, 160,000 units of gentamicin, and 0.01% auxiliary components; wherein the auxiliary components are MgCl2 or ZnCl2.

2. The detection method according to claim 1, characterized in that, The blocking solution and the sample diluent are the same, containing 0.1-1 M PBS buffer, 1% BSA, 5% sucrose, 0.2% Proclin 300, 160,000 units of gentamicin, and 0.01% MgCl2.

3. The detection method according to claim 1, characterized in that, In step (1), the PCV2 Cap monoclonal antibody is coated on the ELISA plate at a concentration of 0.1~1 μg / mL; preferably, the PCV2 Cap monoclonal antibody is coated on the ELISA plate at a concentration of 0.1 μg / mL.

4. The detection method according to claim 1, characterized in that, In step (2), the dilution step involves diluting the standard antigen and the test sample to 1 μg / mL with sample diluent, and then performing a 2-fold serial dilution to the 7th gradient.

5. The detection method according to claim 1, characterized in that, In step (2), the detection antibody is diluted 2000-3000 times with sample diluent; the enzyme-labeled antibody is diluted 5000-10000 times with enzyme-labeled antibody diluent; preferably, the enzyme-labeled antibody diluent is a sample diluent containing 0.05% enzyme stabilizer; more preferably, the detection antibody is diluted 3000 times with sample diluent; and the enzyme-labeled antibody is diluted 10000 times with enzyme-labeled antibody diluent.

6. The detection method according to claim 1, characterized in that, In step (2), the incubation conditions are: incubation at 37°C for 0.5~2 h; preferably, the incubation conditions are: incubation at 37°C for 0.5~1 h.

7. The detection method according to claim 1, characterized in that, In step (3), the reaction conditions for TMB color development are: 37°C, protected from light, for 5-20 min; preferably, the reaction conditions for TMB color development are: 37°C, protected from light, for 15 min.

8. A PCV2 Cap protein double antibody sandwich quantitative ELISA kit, characterized in that, The kit includes an ELISA plate coated with PCV2 Cap monoclonal antibody, blocking buffer, standard antigen, sample dilution buffer, PCV2 rabbit polyclonal antibody, anti-rabbit HRP, ELISA antibody dilution buffer, PBST washing buffer, chromogenic solution, and stop solution. The blocking solution and the sample diluent are the same, containing 0.1-1 M PBS buffer, 1% BSA, 5% sucrose, 0.2% Proclin 300, 160,000 units of gentamicin, and 0.01% auxiliary components; wherein the auxiliary components are MgCl2 or ZnCl2; preferably, the auxiliary components are MgCl2.

9. The use of the PCV2 Cap protein double antibody sandwich quantitative ELISA kit according to claim 8 in the preparation of PCV2 antigen or PCV2 vaccine products.

10. A reagent for stabilizing the PV2 Cap protein standard antigen of claim 1, characterized in that, The reagent is a sample diluent, and its formulation is as follows: 0.1~1 M PBS buffer, 1% BSA, 5% sucrose, 0.2% Proclin 300, 160,000 units of gentamicin, and 0.01% auxiliary components; wherein the auxiliary components are MgCl2 or ZnCl2.

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