A swine erysipelas SpaA protein double antibody sandwich quantitative ELISA kit and a detection method thereof

Abstract

The application relates to a pig erysipelas SpaA protein double antibody sandwich quantitative ELISA kit and a detection method thereof. Mice are immunized by using pig erysipelas SpaA protein, hybridoma cells are screened, and the prepared antibody can specifically recognize SpaA protein. Based on the monoclonal antibody, a sandwich ELISA is established through a series of reaction condition optimizations, can be used for quantitative detection of pig erysipelas SpaA protein, the minimum concentration can reach 0.1 ng / ml, the sensitivity is high, the correlation coefficient R 2 is 0.993, the quantitative accuracy is good, can be applied to quantitative detection of pig erysipelas SpaA protein, and the method overcomes the situation that a traditional quantitative method cannot distinguish between impurities and target proteins at present, so that the accuracy of quantitative data is ensured, and the stability and rigor of a vaccine process are improved.

Description

Technical Field

[0001] This invention relates to a double-antibody sandwich quantitative ELISA kit for SpaA protein in porcine erysipelas, a detection method, and its application, belonging to the field of animal disease diagnosis and biological products. Background Technology

[0002] Swine erysipelas is an acute, febrile infectious disease caused by *Erysipelothrix rhusiopathiae*. Its clinical features include high fever, acute septicemia, skin rashes, chronic verrucous endocarditis, skin necrosis, and polyarthritis (non-suppurative arthritis). This disease is widespread throughout the world, including many regions of my country, and was one of the three major infectious diseases decades ago. It has caused considerable economic losses to the pig farming industry.

[0003] Swine erysipelas is widespread in the natural environment, with infected pigs being the primary source of infection. It spreads through feces, urine, or oral secretions, and can be found in contaminated feed, water, soil, and pens. It can survive and reproduce in the soil and is highly resistant to environmental changes. Pigs of all ages, breeds, and sexes can be infected, but those aged 3-6 months are most susceptible. Factors such as temperature, humidity, nutrition, and fatigue can all trigger the disease. Swine erysipelas is prevalent year-round, but is more common in summer during periods of high temperatures or sudden temperature changes. All pig ages can be infected, but adult pigs are particularly vulnerable, leading to significant losses for pig farms. The incubation period for swine erysipelas can range from a minimum of one day to over seven days. There are also reports of mammals, birds, fish, and amphibians being infected with swine erysipelas. Simultaneously, *Erysipelas erysipelas* is also an important zoonotic bacterium that can cause human infection; human infection with *Erysipelas erysipelas* is called erysipelas-like infection.

[0004] With the increasing prevalence of antibiotic overuse, a large number of drug-resistant bacteria have emerged in clinical practice. Therefore, vaccination is the most effective way to prevent bacterial diseases such as swine erysipelas. Research on swine erysipelas vaccines involves inactivated vaccines, live vaccines, subunit vaccines, and vector vaccines. Currently, traditional vaccines, namely inactivated swine erysipelas vaccines and attenuated live vaccines, are mainly used on the market. The main problem with these vaccines is that attenuated strains can exhibit virulence reversion, and whole-cell inactivated vaccines contain a large number of ineffective components, which can cause serious side effects and excessive, meaningless non-specific immune responses, wasting the body's immune potential. Therefore, developing novel vaccines is the main direction of swine erysipelas vaccine research. Reported novel swine erysipelas vaccine research mainly includes subunit vaccines and lactic acid bacteria vector vaccines.

[0005] Subunit vaccines are genetically engineered vaccines that do not contain nucleic acids, obtained by chemical decomposition, protein synthesis, or expression of immunogenic protein structures from bacteria and viruses. Subunit vaccines contain only a few proteins as antigens, thus avoiding ineffective immune stimulation from excessive irrelevant antigens, conserving the body's immune potential, and reducing vaccine side effects.

[0006] Currently, methods for quantifying antigen proteins in swine erysipelas are limited to BCA and Bradford methods. These methods are limited by their inability to distinguish between contaminating proteins and antigen proteins, leading to significant variations in the final quantitative data and affecting the semi-quantitative results. This ultimately impacts vaccine efficacy. Therefore, there is an urgent need to establish a reliable and reproducible quantitative analysis method for antigen quantification and the quantitative analysis of effective components in vaccine antigens during vaccine production. To ensure analytical reliability, the quantitative method must be thoroughly validated. Key considerations include: specificity (ensuring accurate and specific determination of the analyte even in the presence of interfering components); sensitivity (providing the linear equation and correlation coefficient of the standard curve, including the standard curve and quantitative range); precision (assessing the method's accuracy using intra- and inter-batch coefficients of variation (CV) of quality control samples); and accuracy (the degree to which the measured biological sample concentration closely approximates the true concentration under defined analytical conditions). Summary of the Invention

[0007] The purpose of this invention is to prepare monoclonal antibodies using porcine erysipelas SpaA as an immunogen through multiple immunizations. These monoclonal antibodies can recognize the target protein in Western blotting. In the established double-antibody sandwich ELISA system, this kit exhibits good specificity, high sensitivity, and good stability, enabling rapid, simple, and efficient quantitative detection of porcine erysipelas SpaA protein, with a detection limit of 0.1 ng / mL. This provides a foundation for accurate quantification and quality control of subsequent vaccines.

[0008] This invention first provides a double-antibody sandwich quantitative ELISA kit for porcine erysipelas SpaA. The kit includes an ELISA plate coated with a monoclonal antibody against porcine erysipelas SpaA protein, diluent, blocking buffer, enzyme-labeled antibody, concentrated washing buffer, chromogenic buffer, stop solution, standard, positive control, and negative control. The ELISA plate coated with the monoclonal antibody against porcine erysipelas SpaA protein uses monoclonal antibody 16H7, which is obtained from mouse hybridoma cell (Ery)-16H7 strain. The mouse hybridoma cell (Ery)-16H7 strain was deposited at the Institute of Microbiology, Chinese Academy of Sciences on May 17, 2023, with accession number 45611.

[0009] Furthermore, the enzyme-labeled antibody is a horseradish peroxidase-labeled monoclonal antibody against swine erysipelas SpaA, and the monoclonal antibody against swine erysipelas SpaA is monoclonal antibody 24G12. The monoclonal antibody 24G12 was obtained from mouse hybridoma cell strain 24G12, and the mouse hybridoma cell strain (Ery)-24G12 was deposited at the Institute of Microbiology, Chinese Academy of Sciences on May 17, 2023, with accession number 45612.

[0010] Furthermore, the diluent is a 0.05M carbonate buffer solution with a pH of 9.6, containing 1.59g of Na2CO3 and 2.93g of NaHCO3.

[0011] Furthermore, the blocking solution is a PBS solution containing 5% skim milk.

[0012] The same invention also provides a method for quantitative ELISA detection of SpaA protein in porcine erysipelas using a double-antibody sandwich, characterized by the following steps:

[0013] (1) Dilute the purified monoclonal antibody 16H7 with coating buffer, and coat the microplate at 100 μl / well. Coat at 37°C for 2 h or at 4°C overnight.

[0014] (2) Remove the liquid from the plate, wash the plate 5 times with washing solution for 5 minutes each time, pat dry the plate after the last wash, add blocking solution, 100 μl / well, and incubate at 37°C for 1.5 h.

[0015] (3) Wash the plate 5 times with washing solution, add the antigen sample to be tested and the control sample at a certain dilution, 100 μl / well, and incubate at 37℃. At the same time, set up a control without antigen (using skim milk instead).

[0016] (4) Wash the plate 5 times with washing buffer, add diluted enzyme-labeled antibody 24G12-HRP, 100 μl / well, and incubate at 37℃ for 1 h; (5) Wash the plate 5 times with washing buffer, add TMB, 50 μl / well, and develop color at room temperature for 15 min.

[0017] (6) Add stop solution, 50 μl / well, mix well and measure OD450nm value within 10 min.

[0018] Furthermore, in step (1), the coating solution is diluted with monoclonal antibody 16H7 at a ratio of 1:1000, and in step (4), the enzyme-labeled antibody is diluted at a ratio of 1:2000.

[0019] Compared to existing technologies, the advancement of this invention—a double-antibody sandwich quantitative ELISA kit for porcine erysipelas SpaA protein and its detection method—lies in that it utilizes porcine erysipelas SpaA antigen protein to immunize mice, screens hybridoma cells, and prepares antibodies that specifically recognize porcine erysipelas SpaA protein, which can be used for porcine erysipelas-related detections such as Western blot (WB) and in vitro anaphylaxis (IFA). After antibody pairing screening, a sandwich ELISA was established through a series of optimized reaction conditions. This kit exhibits high specificity, high sensitivity, and good stability, enabling rapid, simple, and efficient quantitative detection of porcine erysipelas SpaA protein, with a lower limit of detection of 0.1 ng / mL, providing a foundation for accurate quantification and quality control of subsequent vaccines. Attached Figure Description

[0020] Figure 1 The image shows the detection results for SpaAWB in swine erysipelas. Markers 1, 2, and 6 are: 1 mg / ml BSA; 2, 1 mg / ml BSA; 4, 0.5 mg / ml BSA; 5, 0.25 mg / ml BSA; 6, 0.125 mg / ml BSA.

[0021] Figure 2 The SpaASDS quantification chart for swine erysipelas is shown. In the chart, 1: maker; 2: BSA-2mg / ml; 3: BSA-1mg / ml; 4: BSA-0.5mg / ml; 5: BSA-0.25mg / ml; 6: BSA-0.125mg / ml.

[0022] Figure 3 The values ​​are OD450 readings obtained from mouse serum by ELISA after three immunizations.

[0023] Figure 4 The OD450 readings of the hybridoma supernatant obtained by ELISA re-examination after cell fusion.

[0024] Figure 5 This is the OD450 reading from the hybridoma supernatant obtained by ELISA after the second subcloning.

[0025] Figure 6 This is the OD450 reading from the ascites fluid ELISA test.

[0026] Figure 7 This is the OD450 reading obtained by antibody ELISA detection after ascites purification.

[0027] Figure 8 This is the OD450 reading from antibody pairing detection.

[0028] Figure 9 This is the result of the paired antibody sensitivity test.

[0029] Figure 10This is the result of Western blot verification of the paired antibody.

[0030] Figure 11 This is the standard curve for a double-antibody sandwich quantitative ELISA of SpaA protein in porcine erysipelas.

[0031] Figure 12 This is a quantitative detection graph of SpaA protein SDS in porcine erysipelas. In the graph, 1: maker; 2: BSA-2mg / ml; 3: BSA-1mg / ml; 4: BSA-0.5mg / ml; 5: BSA-0.25mg / ml; 6: BSA-0.125mg / ml. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0033] The pET28a vector used in this embodiment was purchased from Thermo Fisher Scientific.

[0034] The competent cells BL21 used in this invention were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0035] The TB culture medium used in this invention was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0036] The LB liquid culture medium used in this invention is from Beijing Solarbio Science & Technology Co., Ltd.

[0037] The 96-well microplate used in this invention was purchased from Corning Incorporated.

[0038] The BCA reagent kit used in this invention was purchased from Thermo Fisher Scientific.

[0039] The brand of the ultra-clean workbench is Hujing Purification.

[0040] Thermo CO2 incubator

[0041] The horizontal low-speed centrifuge is from Changzhou Hongke.

[0042] Biological inverted microscope for Shanghai Qibu

[0043] The microplate reader is MDCMaxPlus.

[0044] The electrophoresis apparatus is from Tianneng.

[0045] The vertical electrophoresis tank is 61.

[0046] The wet rotating film tank is 61.

[0047] Horizontal decolorizing shaker for Linbell

[0048] The nucleic acid protein detector is from Hangzhou Aosheng.

[0049] The electrofusion device is NEPAGENE

[0050] Example 1

[0051] I. SpaA-DC1 sequence synthesis and recombinant plasmid construction

[0052] Based on the SpaA sequence of the erysipelas antigen from the LA150627 (GenBank: Kμ214211.1) serological type 1a strain of swine erysipelas reported by NCBI, SpaA with the characteristics of serological type 1a antigen was synthesized; SpaA-DC1 is 1239 bases in size; the sequence was synthesized and ligated between the BamHI and EcoRI restriction sites of the pET28a vector to construct the prokaryotic expression vector pET28a-SpaA-DC1. For specific operation methods, refer to the "Molecular Cloning Experimental Manual".

[0053] II. Construction of expression strains and induction and purification of proteins

[0054] 1. Preparation of SpaA-DC1 protein

[0055] (1) BL21 competent cell conversion and protein-induced expression

[0056] The positive plasmid was transformed into BL21 competent cells. After transformation, single colonies were picked and cultured in LB liquid medium containing kanamycin at 37°C overnight on a shaker. After centrifugation at 8000 r / min for 5 min, the cells were collected, boiled in a water bath for 5 min, centrifuged at 12000 r / min for 3 min, and the supernatant was used for PCR identification. After correct identification, the culture was expanded.

[0057] (2) Cultivation of positive strains and protein induction expression

[0058] PCR-positive strains were inoculated into 150 mL of TB medium and cultured at 37°C in a shaker until OD600 = 0.8. IPTG was added to a final concentration of 1 mmol / L, and induction was performed for 4 h. After centrifugation, the cultures were resuspended in 1 / 10 volume of PBS, sonicated, and the supernatant and precipitate were collected by centrifugation for protein identification. Positive samples were used for subsequent purification. The results are shown in the attached figure. Figure 1 As shown.

[0059] (3) Inclusion body purification

[0060] Add the precipitate to cold washing buffer (20 mM Tris, 2 mM EDTA, 1% Trixon 100, 1% Tween 20, 0.5 mM Murea, pH 8.2) at a ratio of 1:10 (w:v) and stir for 20 minutes. Centrifuge at 12000 rpm for 25 minutes at 2–8 °C, discard the supernatant, and collect the precipitate. Repeat once (washing for 2–4 hours to remove membrane debris and membrane proteins). Wash the resulting precipitate once more with 50 mmol / L Tris-HCl under the same centrifugation conditions, discarding the supernatant. The resulting precipitate is the washed inclusion bodies. Resuspend the washed inclusion body precipitate in inclusion body denaturing and lysing buffer (50 mM Tris, 8 Murea, 5 mM GSH, pH 8.2) at a 1:10 (w:v) ratio. Stir at room temperature for 30–60 minutes to ensure complete dissolution. Centrifuge at 12,000 rpm for 15 minutes at 2–8°C. Discard the precipitate and collect the supernatant. Slowly and evenly add 7 volumes of PBS solution to the supernatant. Incubate overnight at 2–8°C for renaturation. Centrifuge and filter the supernatant through a 0.22 μm filter for sterilization. This is the renatured protein antigen solution.

[0061] III. Inactivation and Quantification of Antigen Proteins

[0062] Inactivation: Add the renatured protein antigen solution to 1 mol / L BEI solution to a final concentration of 0.005 mol / L while stirring continuously until homogeneous. Inactivate at 37°C for 24 hours. After inactivation, add a filtered and sterilized 2 mol / L sodium thiosulfate (Na₂S₂O₃) solution to a final concentration of 0.005 mol / L. Stir thoroughly and react at 37°C for 1 hour to terminate inactivation. Store at 2–8°C.

[0063] Protein content determination: The protein content was determined by SDS method, and the result was 1.32 mg / ml. The results are as follows: Figure 2 As shown.

[0064] IV. Establishment of Monoclonal Cell Lines

[0065] 1. Mouse immunization and antiserum detection

[0066] Five 6-week-old female BalB / C mice were selected, marked and numbered, and 30-50 μl of blood was collected from the tail vein. The pre-immune serum was collected by centrifugation after coagulation.

[0067] First immunization: Calculate the required antigen volume based on an immunization dose of 200 μg per mouse, emulsify the antigen with an equal volume of Freund's complete adjuvant, and administer immunization via multiple subcutaneous injections;

[0068] Second immunization: Two weeks later, a second immunization was performed. The required antigen volume was calculated based on an immunization dose of 100 μg per mouse. An equal volume of Freund's incomplete adjuvant was used to emulsify the antigen, and multiple subcutaneous injections were administered for immunization.

[0069] Third immunization: Two weeks later, the third immunization was performed. The required antigen volume was calculated based on an immunization dose of 100 μg per mouse. An equal volume of Freund's incomplete adjuvant was used to emulsify the antigen, and multiple subcutaneous injections were administered for immunization.

[0070] Serum collection: One week after the third immunization, 30-50 μl of blood was collected from the tail vein, and the serum was collected by centrifugation after coagulation.

[0071] ELISA assay for serum titer: P300396 protein was coated with CBS at a final concentration of 1 μg / ml, 100 μl / well, incubated overnight at 4°C, and then blocked with 0.1% casein-PBS. The plates were washed after blocking at 37°C for 1 h. Mouse serum was initially diluted 1:1000 and serially diluted. PBS served as a negative control. Serum titer was measured by ELISA, and OD450 readings were determined using a microplate reader. Results are shown below. Figure 3 As shown in the figure. The results showed that the serum titers of the five mice after triple spur immunization were similar, all reaching 1:243000. The mouse with the highest value was selected for cell fusion.

[0072] 2. Cell fusion

[0073] (1) Prepare mouse myeloma cells SP2 / 0 at least one week before fusion, adjust the cell state to the logarithmic growth phase, select mice with the highest ELISA titer, and give mice a booster immunization 3 days before fusion, 100μg / mouse, intraperitoneal injection.

[0074] (2) Fusion: Mouse spleens were taken, mechanically broken up and spleen cells were collected, filtered through a 200-mesh sieve, and washed 3 times with PBS; SP2 / 0 cells were collected and washed 3 times with PBS; after cell counting, SP2 / 0:spleen cells were mixed at a ratio of 1:2.5, centrifuged and PBS was discarded, and cell fusion was performed using a cell fusion instrument. After fusion, the cells were centrifuged and added to complete culture medium containing HAT, resuspended and mixed, and then plated in 96-well plates.

[0075] (3) Change the medium: Perform a half change of medium every 5 days after fusion.

[0076] 3. Hybridoma screening

[0077] ELISA assay: The cell culture supernatant was coated with antigen P300396 at a concentration of 1 μg / ml. Seven days after fusion, the supernatant was collected for ELISA testing. Wells with readings greater than 1.0 were recorded, labeled, and the medium was partially replaced. ELISA retest: The next day, the positive wells that had undergone replenishment of medium were retested. The clone numbers with stable titers were recorded. The test results are as follows: Figure 4 As shown.

[0078] 4. Establishment of stable cell lines

[0079] (1) First subcloning: Select positive cells from the previous step, such as 24G12, 12F2, 13H2, 15H6, 10B2, (Ery)-16H7, 13E3, 18A4, and 19E12, for subcloning screening. Use limiting dilution method and complete culture medium to plate cells into 96-well plates with one cell per well.

[0080] (2) After 7 days, observe under a microscope, mark the single clone wells, and perform ELISA testing the next day. Discard the clones that turn negative. For positive clones, select the wells with vigorous growth and perform one fluid replacement.

[0081] (3) Secondary subcloning: Select positive cells from the previous step for subcloning screening, using the limiting dilution method and complete culture medium, and plate them in a 96-well plate with one cell per well.

[0082] (4) After 7 days, observe under a microscope, mark the monoclonal wells, and perform ELISA testing the next day. The test results are as follows: Figure 5 As shown; when the subcloning rate reaches 100% with ELISA detection positivity, select wells with vigorous growth for expanded culture and cryopreservation.

[0083] 5. Preparation of ascites

[0084] Stable hybridoma cells were injected into the peritoneal cavity of mice, 1*10 6 Cells per mouse; mice were sacrificed after 10 days when their peritoneal cavity showed significant distension, and ascites fluid was collected. ELISA was performed to detect the results. Figure 6 As shown.

[0085] 6. Ascites purification and testing

[0086] (1) Equilibration of the purification column: Wash the Protein G purification column with 10 column volumes of deionized water to remove the ethanol protection solution, and then rinse the purification column with 10 column volumes of 0.01MPBS (pH 7.4) to equilibrate the purification column.

[0087] (2) After centrifugation, the ascites fluid was filtered through a 0.45 μm pore size filter membrane and purified by column chromatography.

[0088] (3) Elute with 0.1M glycine (pH 2.5), collect the purified product, and immediately neutralize with 1M pH 9.0 Tris-HCl;

[0089] (4) The purified antibody was dialyzed with 0.01MPBS (pH 7.4), concentrated, and stored at -20℃ for later use;

[0090] (5) Concentration determination: Antibody concentration was determined using a Nano-500.

[0091] (6) Antibody purity detection: SDS-PAGE was used to detect antibody purity;

[0092] (7) Antibody titer detection: P300396 protein was coated with CBS at a final concentration of 1 μg / ml, 100 μl / well, and incubated overnight at 4℃. The plate was then blocked with 0.1% casein-PBS, and washed after 1 hour at room temperature. The initial antibody dilution was 1:2000, with serial dilutions. PBS served as a negative control. Antibody titer was detected by ELISA, and the OD450 reading was measured using a microplate reader. Figure 7 As shown.

[0093] V. Purification of HRP-labeled antibodies

[0094] Antibody labeling: Purified antibodies were labeled with horseradish peroxidase using the classic sodium periodate method.

[0095] 1. Antibody pairing

[0096] (1) Each purified monoclonal antibody was coated with CBS to a final concentration of 1 μg / ml, 100 μl / well, and incubated overnight at 4°C. The plate was then blocked with 0.1% casein-PBS and washed after blocking at 37°C for 1 h.

[0097] (2) Add 50 μl of sandwich antigen, namely P300396 protein (final concentration 100 ng / ml), then add 50 μl of labeled antibody (final concentration 1 / 10000 dilution), and incubate at 37℃ for 1 h.

[0098] (3) Wash the plate 5 times, add 100 μl of TMB working solution to each well, and after 5 min of color development, add 50 μl of stop solution to each well. Measure the absorbance at OD450 using a microplate reader. The results are as follows: Figure 8 As shown.

[0099] 2. Sensitivity Measurement

[0100] CBS-coated capture antibody, with a final concentration of 1 μg / ml, was applied at 100 μl / well. After incubation at 4°C overnight, the plate was blocked with 0.1% casein-PBS and incubated at 37°C for 1 hour, followed by washing. Sandwich antigen P300396 protein was added initially at a final concentration of 3000 ng / ml, serially diluted 3-fold, and then HRP-labeled antibody was added to a final concentration of 0.1 μg / ml. The plate was incubated at 37°C for 1 hour, washed, and the OD450 reading was measured using a microplate reader. The results are as follows: Figure 9 As shown, based on the detection results, a double-sandwich quantitative ELISA method was established using monoclonal antibodies 16H7 and 24G12-HRP. Mouse hybridoma cell lines 16H7 (secreting monoclonal antibody 16H7) and 24G12 (secreting monoclonal antibody 24G12) were then sent to a preservation center for storage.

[0101] 3. Antibody Western Blot Verification

[0102] Gel chromatography: Add 200 ng of antigen Ery-DC1-SpaA and 5 μl of whole Ery-DC1 / DC2 positive control; transfer membrane; block: add PBST + 5% skim milk to the transferred membrane and incubate on a shaker at room temperature for 1 h; incubate with primary antibody: dilute the primary antibody with PBST + 5% skim milk and incubate on a shaker at room temperature for 1 h; wash the membrane 3 times with PBST, 5 min each time; dilute the secondary antibody with PBST + 5% skim milk (dilution ratio 1:10000) and incubate on a shaker at room temperature for 1 h; wash the membrane 3 times with PBST, 5 min each time; add chromogenic buffer, transfer to a chemiluminescence apparatus for imaging, results as shown below. Figure 10 As shown.

[0103] Example 2

[0104] Establishment of a quantitative sandwich ELISA detection method

[0105] 1. Detailed operating steps of sandwich ELISA

[0106] (1) Dilute monoclonal antibody 16H7 1:1000 with 0.05M carbonate buffer (pH 9.6, Na2CO3 1.59g, NaHCO3 2.93g) and add 100μL to each well of a 96-well plate. Coat overnight at 4°C. Discard the liquid in the plate, wash 5 times with 300μl PBST, and pat dry any remaining liquid.

[0107] (2) Add 100 μl of PBS containing % skim milk powder to block for 3 h, discard the liquid in the plate, wash 5 times with 300 μl of PBST, and pat dry the remaining liquid.

[0108] (3) After adding 100 ng / well of porcine erysipelas SpaA antigen, react at 37°C for 60 min, discard the liquid in the plate, wash 5 times with 300 μl PBST, and pat dry the remaining liquid.

[0109] (4) Dilute 24G12-HRP at 1:2000 and add it to the wells respectively. React at 37℃ for 60 min. Discard the liquid in the plate, wash 5 times with 300 μl PBST, and pat dry the remaining liquid.

[0110] (5) Add 50 μl of TMB and develop color at room temperature for 5 min;

[0111] (6) Add 50 μl of 2M H2S O4 Terminate the reaction and take the OD450 reading.

[0112] 2. Establish a standard curve

[0113] Using 90% purified porcine erysipelas SpaA antigen as a standard sample, after BCA quantification, the concentration of porcine erysipelas SpaA was adjusted to 1 μg / mL, followed by a 2-fold serial dilution to obtain different concentrations of porcine erysipelas SpaA standards. The serially diluted standards were added sequentially to 100 μL, yielding concentrations of 100 ng / well, 50 ng / well, 25 ng / well, 12.5 ng / well, 6.25 ng / well, 3.125 ng / well, and 0 ng / well, used to establish a standard curve for porcine erysipelas quantification. The different concentrations and their corresponding OD values ​​were input into Excel for curve fitting, resulting in the corresponding concentration-OD value conversion formula. Figure 11 As shown.

[0114] The results showed that using porcine erysipelas SpaA as a standard sample, a standard curve was established by serially diluting the samples. Statistical analysis was performed on the background-removed OD values ​​and their corresponding concentrations, yielding the corresponding formula. It was found that VLPs exhibited a good linear relationship within the concentration range of 0-1 μg / ml. The calculated formula was y = 1.3558x + 0.0632, with a correlation coefficient R0. 2 It is 0.993.

[0115] 3. Comparison experiment of different quantitative methods

[0116] Different concentrations of purified porcine erysipelas SpaA antigen were tested using a BCA protein concentration assay kit, followed by detection using a sandwich ELISA. Taking three samples with BCA quantification concentrations of 35 μg / mL, 60 μg / mL, and 90 μg / mL as examples, the SpaA-DC1 concentration was calculated based on the standard curve corresponding to the obtained sample OD values ​​and concentrations. The differences between the two concentration quantification methods were analyzed and statistically analyzed. The results showed that the fluctuation range of the sample concentration detected by sandwich ELISA and the protein concentration determined by the BCA method was both less than 5%.

[0117] Example 3

[0118] Evaluation of the SpaA double-antibody sandwich quantitative ELISA method for detecting porcine erysipelas

[0119] (1) Evaluation of detection accuracy: Three proteins from different sources were selected for quantitative detection, including the purified and unpurified porcine erysipelas SpaA-DC1 mentioned above, and a laboratory-prepared bivalent subunit vaccine for porcine erysipelas. The three samples were analyzed and compared using both BCA and SDS methods. The data from ELISA, BCA, and SDS quantitative detection are shown in Table 1 below, and the SDS gel images are shown below. Figure 12 As shown.

[0120] Table 1:

[0121]

[0122] The results showed that for purified samples, the results obtained by the three detection methods were basically consistent and in line with the theory (quantitative data was 1.32 mg / ml); for unpurified samples, the SDS and BCA detection data were compared. The BCA data detected the total protein content but could not distinguish between the target protein and other proteins. Therefore, the BCA detection result was significantly higher than that of SDS, meaning that BCA could not distinguish between the target protein and other proteins. For the results of the porcine erysipelas vaccine product, it was found that because the antigen content of the vaccine product was relatively low (25-30 μg / ml), the SDS grayscale analysis was relatively difficult and could not truly reflect the final result; the BCA detection data contained two antigen components, so it could not truly reflect the accurate content of the porcine erysipelas antigen.

[0123] (2) Sensitivity analysis: Using a zero-concentration standard or sample dilution as the sample, the detection was repeated 20 times, and the absorbance values ​​A of the 20 measurements were obtained. The average value M and standard deviation SD were then calculated. The absorbance value corresponding to M+2SD was substituted into the standard curve equation to obtain the corresponding concentration value, which is the detection sensitivity. This method has a lower limit of detection of 0.1 ng / mL, exhibiting very high sensitivity.

Claims

1. A double-antibody sandwich quantitative ELISA kit for SpaA protein in porcine erysipelas, characterized in that: The kit includes an ELISA plate coated with a monoclonal antibody against SpaA protein of swine erysipelas, diluent, blocking buffer, enzyme-labeled antibody, concentrated washing buffer, chromogenic buffer, stop solution, standards, positive control, and negative control. The ELISA plate coated with the monoclonal antibody against SpaA protein of swine erysipelas uses monoclonal antibody 16H7, which was obtained from mouse hybridoma cell (Ery)-16H7 strain. The mouse hybridoma cell (Ery)-16H7 strain was submitted to the China General Microbiological Culture Collection Center on May 17, 2023, with accession number CGMCC No. 45611.

2. The porcine erysipelas SpaA protein double antibody sandwich quantitative ELISA kit according to claim 1, characterized in that: The enzyme-labeled antibody is a horseradish peroxidase-labeled monoclonal antibody against SpaA in swine erysipelas. The horseradish peroxidase-labeled monoclonal antibody against SpaA in swine erysipelas is monoclonal antibody 24G12. The monoclonal antibody 24G12 was obtained from the mouse hybridoma cell (Ery)-24G12 strain. The mouse hybridoma cell (Ery)-24G12 strain was submitted to the China General Microbiological Culture Collection Center on May 17, 2023, with accession number CGMCC No. 45612.

3. The porcine erysipelas SpaA protein double antibody sandwich quantitative ELISA kit according to claim 1, characterized in that: The blocking solution is a PBS solution containing 5% skim milk.

4. A method for detecting SpaA protein double-antibody sandwich quantitative ELISA kit for porcine erysipelas as described in claim 2, characterized in that, Includes the following steps: (1) Dilute the purified monoclonal antibody 16H7 with coating buffer, and coat the microplate with 100 μl / well. Coat at 37°C for 2 h or at 4°C overnight. (2) Remove the liquid from the plate, wash the plate 5 times with washing solution for 5 minutes each time, pat dry the plate on the last wash, add blocking solution, 100 μl / well, and incubate at 37°C for 1.5 h; (3) Wash the plate 5 times with washing solution, add the diluted antigen sample to be tested and the control sample, 100 μl / well, and incubate at 37°C. At the same time, set up a control without antigen by replacing it with skim milk. (4) Wash the plate 5 times with washing buffer, add diluted enzyme-labeled antibody 24G12, 100 μl / well, and incubate at 37°C for 1 h; (5) Wash the plate 5 times with washing solution, add color development solution, 50 μl / well, and develop color at room temperature for 15 min; (6) Add stop solution, 50 μl / well, mix well and measure OD450nm value within 10 min.

5. The detection method of the porcine erysipelas SpaA protein double antibody sandwich quantitative ELISA kit according to claim 4, characterized in that: In step (1), the coating solution is diluted with monoclonal antibody 16H7 at a ratio of 1:1000, and in step (4), the enzyme-labeled antibody is diluted at a ratio of 1:2000.

6. The application of the porcine erysipelas SpaA protein double antibody sandwich quantitative ELISA kit as described in claim 1 in the antigen detection of porcine erysipelas SpaA antigen.

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