ELISA antibody detection kit for identifying brucella gene deletion live vaccine (M5-90△26 strain) and wild strain and application

Abstract

The application discloses an ELISA antibody detection kit for identifying Brucella gene deletion live vaccine (M5-90△26 strain) and wild strains and application. The kit comprises enzyme-linked reaction plate 1 and enzyme-linked reaction plate 2. The enzyme-linked reaction plate 1 is coated with LPS extracted from the Brucella gene deletion live vaccine (M5-90△26 strain), and the enzyme-linked reaction plate 2 is coated with a Brucella BP26 antigen epitope polypeptide combination composed of polypeptides with amino acid sequences shown in SEQ ID NO:1-2. The application is suitable for goats and sheep inoculated with the Brucella gene deletion live vaccine (M5-90△26 strain), can effectively distinguish the antibody produced by the Brucella gene deletion live vaccine (M5-90△26 strain) and the antibody produced by wild strain infection, thereby realizing identification and detection, being favorable for prevention and control of brucellosis and optimization of a vaccination strategy, and providing technical support for healthy and sustainable development of animal husbandry.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology. More specifically, this invention relates to an ELISA antibody detection kit and its application, which is suitable for the identification and detection of Brucella gene-deleted live vaccine (M5-90△26 strain) immune antibodies and wild-type strain infection antibodies in sheep serum. Background Technology

[0002] Brucellosis ( Brucellosis Brucellosis, or brucellosis for short, is caused by Brucella bacteria. Brucella Brucellosis, a zoonotic infectious disease caused by brucellosis, is widespread in many countries and regions worldwide. In the newly released "List of Class I, II, and III Animal Diseases" in 2022, brucellosis was classified as a Class II animal disease. Brucellosis is mainly transmitted through contact with infected animals or their secretions. Infected animals often exhibit symptoms such as abortion and infertility, severely impacting livestock production. Human infection can lead to chronic diseases such as fever, joint pain, and fatigue. Currently, global control of the disease is primarily achieved through vaccination and the culling of positive animals.

[0003] Lipopolysaccharide (LPS) is an important component of the Brucella cell wall, consisting of lipid A, a core polysaccharide, and an O-chain. The O-chain is the main immunogenic component of LPS, capable of inducing a strong immune response in the host, especially a humoral immune response. The O-chain plays a crucial role in the diagnosis of Brucella, and most serological detection methods use it as the primary target.

[0004] Currently, commonly used brucellosis vaccines in China (such as S2 strain, A19 strain, M5-90 strain, and Rev.1 strain) share a common deficiency: vaccine strains and wild-type strains may exhibit cross-reactivity in serological testing. Traditional detection methods struggle to distinguish between vaccinated and naturally infected animals, thus affecting precise disease control and the optimization of vaccination strategies. Brucella gene-deleted live vaccine (M5-90△26 strain) is an effective solution to this problem. This vaccine targets sheep, and the BP26 gene-deleted M5-90△26 strain is constructed using homologous recombination technology based on the M5-90 strain. Studies have also shown that naturally infected animals can produce specific antibodies against BP26; therefore, BP26-based detection methods can differentiate between antibodies from the Brucella gene-deleted live vaccine (M5-90△26 strain) and antibodies from wild-type strain infection. Furthermore, ELISA methods using peptides as coating antigens can accurately identify target antibodies and reduce cross-reactivity with non-target antibodies. Peptide antigens are obtained through chemical synthesis, and their sequences and purity are precisely controllable, with high batch-to-batch consistency, ensuring the stability of the detection. Summary of the Invention

[0005] The purpose of this invention is to provide an ELISA antibody detection kit and method of use for differentiating Brucella gene-deleted live vaccine (M5-90△26 strain) from wild-type strains.

[0006] To achieve the above objectives, the present invention provides the following technical approach: This invention extracts LPS from a Brucella gene-deleted live vaccine (M5-90△26 strain) using the phenol-water extraction method, and then employs enzymatic treatment to enhance the purity of the LPS. The purified LPS, as a coating antigen, exhibits high sensitivity and can detect both Brucella vaccine strains and wild-type strains.

[0007] Using chemically synthesized Brucella BP26 epitope peptides as coating antigens, two peptides, SEQ ID NO: 1 and SEQ ID NO: 2, were designed and synthesized. Verification showed that each peptide, used alone as a coating antigen, exhibited excellent detection sensitivity. Furthermore, the peptide combination of SEQ ID NO: 1 and SEQ ID NO: 2, mixed at a mass ratio of 1:1, showed significantly higher detection sensitivity for BP26 antibodies than either peptide alone, indicating a synergistic effect. Since strain M5-90△26 is a BP26 gene-deleted strain and does not produce BP26 antibodies after immunization, while wild-type strains do produce BP26 antibodies in infected animals, this peptide combination possesses differential detection capabilities.

[0008] This invention provides an ELISA antibody detection kit for differentiating Brucella gene-deleted live vaccine (M5-90△26 strain) from wild-type strain, specifically comprising: enzyme-linked reaction plate 1, enzyme-linked reaction plate 2, 20× concentrated washing buffer, sample dilution buffer, positive control serum, negative control serum, enzyme-labeled secondary antibody, substrate solution A, substrate solution B, and stop solution.

[0009] The enzyme-linked reaction plate 1 is coated with LPS extracted from Brucella gene-deleted live vaccine (M5-90△26 strain); the enzyme-linked reaction plate 2 is coated with a chemically synthesized Brucella BP26 antigenic epitope polypeptide combination.

[0010] The ELISA antibody detection kit provided by this invention is suitable for goats and sheep vaccinated with Brucella gene-deleted live vaccine (M5-90△26 strain). The detection results of enzyme-linked reaction plate 1 determine whether the Brucella antibody is positive, and the detection results of enzyme-linked reaction plate 2 determine whether the antibody is an antibody produced by immunization with Brucella gene-deleted live vaccine (M5-90△26 strain) or an antibody produced by infection with wild-type strain.

[0011] The advantages of this invention are as follows: The phenol-water extraction method for LPS effectively removes most proteins and lipids, resulting in high-purity LPS. The extraction scale can be adjusted as needed, making it suitable for both small-scale laboratory extraction and large-scale industrial production. The solid-phase synthesis technique for peptides removes byproducts and unreacted reagents through washing steps at each stage, resulting in relatively pure peptides. Furthermore, the chemically synthesized peptide production process is controllable, and the quality and performance of peptides from different batches are highly consistent. Compared to whole-protein antigens, peptide antigens contain only specific epitopes, avoiding interference from other irrelevant regions in the whole protein, thereby improving detection specificity.

[0012] The kit uses a dual-enzyme-linked reaction plate design with a clear operating procedure, which facilitates laboratory and field testing and provides a supporting detection tool for the promotion and application of Brucella gene-deleted live vaccine (M5-90△26 strain).

[0013] In summary, this invention can be used for differential diagnosis in sheep flocks vaccinated with the Brucella gene-deleted live vaccine (Strain M5-90△26), effectively screening out and culling wild-type infected animals, interrupting the transmission chain of Brucella in the animal population, and accelerating the eradication process. This invention can also be used to evaluate the immunization efficacy of the Brucella gene-deleted live vaccine (Strain M5-90△26), providing a scientific basis for optimizing vaccination strategies. Through the application of this invention, the potential risks of brucellosis to public health can be reduced, promoting the healthy and sustainable development of animal husbandry. Detailed Implementation

[0014] Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0015] Example 1: Extraction of Brucella LPS Brucella LPS was extracted using the phenol-water method, combined with other auxiliary steps to ensure the purity and quality of the LPS. All steps were performed under aseptic conditions to avoid exogenous contamination.

[0016] I. Bacterial Culture Brucella M5-90△26 strain was inoculated into TSB medium containing 5% defibrinated sheep blood and cultured at 37°C in a shaker for 24–48 h until the logarithmic growth phase (OD200). 600nm (Approximately 0.6~0.8). Centrifuge the culture at 10000×g for 15 min and discard the supernatant. Wash the bacterial pellet 2~3 times with PBS to remove any remaining culture medium.

[0017] II. Bacterial lysis 1. Bacterial resuspension Resuspend the bacterial precipitate in an appropriate amount of distilled water (usually 1 g of wet bacteria in 10 mL of distilled water).

[0018] 2. Hot phenol extraction Add an equal volume of 90% phenol solution preheated to 65-68℃, stir rapidly for 30 min, and use it to lyse the bacterial cells and extract LPS.

[0019] III. Phase Separation 1. Cooling and centrifugation The mixture was cooled to 4°C and centrifuged at 10000×g for 30 min. After centrifugation, the mixture separated into two phases: an upper aqueous phase (containing LPS) and a lower phenolic phase (containing proteins and lipids).

[0020] 2. Collect the aqueous phase Use a pipette to aspirate the upper aqueous phase, avoiding contamination with the phenol phase. Repeat the phenol extraction step 1-2 times to improve LPS purity.

[0021] IV. Dialysis and Concentration 1. Dialysis The aqueous phase was placed in a dialysis bag and dialyzed with distilled water for 48 hours. The dialysate was changed every 6 to 8 hours to remove residual phenols and salts.

[0022] 2. Concentrated After dialysis, the LPS solution was concentrated to the required volume using PEG.

[0023] V. Purification The protein and nucleic acid were removed by adding proteinase K (final concentration 100 μg / mL), DNase I and RNase A (final concentration 10 μg / mL) to the LPS solution and incubating at 37°C for 2 h.

[0024] VI. Freeze-drying preservation The purified LPS solution was aliquoted and dried in a Labconco freeze dryer. After packaging, labels were affixed indicating the peptide name, serial number, batch number, concentration, production date, shelf life, and storage conditions. The peptide was stored at -80°C.

[0025] Example 2: Preparation of Brucella BP26 antigenic epitope polypeptide Bioinformatics software was used to analyze the dominant antigenic epitopes of Brucella BP26, and two peptides were designed and prepared, namely the peptide with the amino acid sequence shown in SEQ ID NO: 1 and the peptide with the amino acid sequence shown in SEQ ID NO: 2, as detailed below: SEQ ID NO: 1:LSVLRQAKTAREAMTANNEA SEQ ID NO: 2:ARKRAVANAIAKAKTLADAA The above-mentioned peptides were prepared using the Merrifield solid-phase synthesis method on an Applied Biosystems fully automated peptide synthesizer (model 433A). The production process is as follows: I. Solid-phase synthesis of polypeptides 1. Preparation of synthetic reagents Based on the peptide sequence and synthesis scale, prepare an appropriate amount of Fmoc (9-fluorenylmethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl) modified amino acids (purchased from NOVA) and add them to the corresponding cartridge. Weigh 5g of Rink AmideMBHA resin and add it to the reaction chamber, tighten the top and bottom caps and label them, recording the name of the synthesized peptide, batch number, TARE of the reaction chamber, and resin weight. Place the reaction chamber into the synthesizer, prepare appropriate amounts of synthesis reagents, including 100% NMP, 3% AIM (hexamethylene imidazole), 35% PIP (piperidine), and 100% MeOH (methanol), and add them to the corresponding reagent bottles.

[0026] 2. Synthesizer status detection First, check that all functions of the Applied Biosystem fully automated peptide synthesizer (model 433A) are normal. Power on the instrument and run the Run Self Test program to initiate the instrument's self-test. Simultaneously, manually check that the nitrogen level is sufficient and the system gauge pressure is normal (normal gauge pressure for the 433A is 10.2 psi). Before synthesis, the flow rate of each synthetic reagent needs to be measured. The specific steps are as follows: send the FlowRate1-18 command, select Main Menu > Module Test, locate Module A, Module D, Module I, etc., and press the Start button to measure or observe. If the flow rate does not meet the requirements, adjust the valve pressure until the requirements are met (see Table 1 for testing requirements).

[0027] Table 1. Flow Rate Detection Standards for Peptide Synthesizers reagents Bottle number Module Standard range 35% Piperidine 1 A 1.0~1.2 mL 3% AIM 4 D 1.0~1.2 mL 100% MeOH 9 I 3.5~4.0 mL DIC 8 I 0.45~0.55 g 100% NMP 10 A 2.6~2.8 mL 3. Start the synthesis Edit and save the peptide sequence to be synthesized in the program of the 433A synthesizer, select the Std Fmoc 1.0 SolDIC90 synthesis cycle, and send it to the synthesizer. Click Main Menu>Cycle Monitor>Begin to start the synthesis program.

[0028] 4. Synthesis process The peptide synthesis process proceeds from the C-terminus to the N-terminus, repeating the cyclic steps in Table 2 sequentially. During this process, reagent usage and equipment operation should be observed and recorded.

[0029] Table 2. Peptide synthesis cycle steps Loop steps method Deprotection Deprotect the resin with PIP for 15 min. Washing resin Soak resin in NMP for 20 min condensation The condensation reaction was carried out for 250-350 min. Wearing a hat Capping reaction of the resin with 3.0% AIM for 25-30 min (this step can be omitted if two condensation reactions were performed). Washing resin Soak the resin in NMP for 10 min 5. End synthesis After peptide synthesis is complete, the synthesizer automatically stops. At this point, the peptide is still attached to the resin (called peptide resin). Wash the peptide resin three times with 100% methanol and dry it in a fume hood. Transfer all the peptide resin to a brown polyethylene bottle, seal it, and store it at -20°C.

[0030] II. Peptide Cleavage and Identification 1. Peptide cleavage The peptide lysis buffer was prepared according to a volume ratio of trifluoroacetic acid:triisopropylsilane:phenol:H₂O = 85:8:6:1. An appropriate amount of the synthesized peptide resin was placed in a round-bottom flask, and the prepared lysis buffer and magnetic stir bar were added in a fume hood. The mixture was stirred for 1 h. After the reaction, the product was evaporated using a rotary evaporator with a cold trap for 30–120 min to remove trifluoroacetic acid from the crude product. The peptide was then precipitated with diethyl ether and washed several times with dimethylformamide (DMF). Finally, the product was filtered through a sintered glass funnel to separate the resin and obtain the peptide.

[0031] 2. Identification of polypeptides The obtained peptides were qualitatively and quantitatively analyzed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) and reversed-phase high-performance liquid chromatography (RP-HPLC).

[0032] III. Peptide Purification Ultrafiltration of the peptide solution was performed using a circulating tangential filtration system and corresponding membrane pack at 20–28°C to remove small molecule impurities generated during synthesis and cyclization reactions. Then, sterile filtration was performed using a 0.2 μm pore size filter. The resulting solution was aliquoted into sterile vials, labeled with the peptide name, serial number, production batch number, concentration, production date, shelf life, and storage conditions. After aliquoting, the solutions were temporarily stored at -20°C or -80°C for later use.

[0033] IV. Freeze-drying preservation To facilitate long-term preservation and transportation, the purified peptide solution needs to be freeze-dried. The pre-frozen peptide solution is placed in a Labconco freeze dryer for drying, yielding a solid peptide. After packaging, a label is affixed, indicating the peptide's name, serial number, batch number, concentration, production date, shelf life, and storage conditions. It is then stored at -80°C.

[0034] Example 3: Preparation of positive control serum for the kit Brucellosis live vaccine strain S2 (Chongqing Aolong Biological Products Co., Ltd., approval number veterinary drug production number 230247011, production batch number 2024007) was used as the immunogen. The immunization target was 6-month-old sheep, administered via intramuscular injection at weeks 0, 2, and 4. The immunization dose was twice the dosage specified in the instructions. When the serum antibody titer reached the positive range as identified by a commercially available Brucella cELISA antibody detection kit, aseptic blood was collected from the jugular vein and serum was separated. The obtained serum was appropriately diluted with sample diluent and detected using the ELISA antibody detection kit of this invention. For both enzyme-linked reaction plates 1 and 2, the OD value was set to... 450nm ≥0.8, to prepare positive control serum.

[0035] Example 4: Assembly of the reagent kit Three batches of reagent kits were prepared and assembled according to experimental requirements, with batch numbers ZM-BRU01, ZM-BRU02, and ZM-BRU03. The composition and general preparation method of each batch of reagent kits are as follows, with the main difference being the polypeptide antigen coated on enzyme-linked reaction plate 2. The ELISA antibody detection kit of the present invention specifically includes: (1) Enzyme-linked reaction plate 1: Removable polystyrene enzyme-linked reaction plate coated with Brucella LPS (96 wells / plate, 1 plate).

[0036] (2) Enzyme-linked reaction plate 2: Removable polystyrene enzyme-linked reaction plate coated with Brucella BP26 antigenic epitope polypeptide (96 wells / plate, 1 plate).

[0037] (3) 20× concentrated washing solution: pH 7.4, 0.01 mol / L phosphate buffer containing 1% Tween 20. (50 mL / bottle, 2 bottles).

[0038] (4) Sample dilution: pH 7.4, 0.01 mol / L phosphate buffer containing 5 g / L casein (24 mL / bottle, 1 bottle).

[0039] (5) Positive control serum: Hyperimmune serum prepared by immunizing sheep with Brucella live vaccine as immunogen, and diluted with sample diluent (1.5 mL / tube, 1 tube).

[0040] (6) Negative control serum: serum from healthy sheep that are free from Brucella infection or vaccine immunization (1.5 mL / tube, 1 tube).

[0041] (7) Enzyme-labeled secondary antibody: horseradish peroxidase-labeled rabbit anti-sheep IgG antibody diluted to working concentration (12 mL / bottle, 2 bottles).

[0042] (8) Substrate solution A: Citrate phosphate buffer containing 0.6 mg / mL hydrogen peroxide urea (12 mL / bottle, 1 bottle).

[0043] (9) Substrate solution B: a solution containing 0.2 mg / mL tetramethylbenzidine (12 mL / bottle, 1 bottle).

[0044] (10) Termination solution: 2 mol / L H2SO4 (12 mL / bottle, 1 bottle).

[0045] Depending on the requirements, the kit may also include serum dilution plates (96 wells / plate, 2 plates) for diluting serum samples.

[0046] The specific preparation method of the enzyme-linked reaction plate 1 is as follows: Brucella LPS prepared in Example 1 is dissolved in carbonate buffer at pH 9.6, mixed evenly, and then added to a 96-well polystyrene enzyme-linked reaction plate. 100 μL of LPS at a concentration of 3 μg / mL is added to each well, and the plate is placed in an environment of 2~8℃ for 8~12 h. After completion, 300 μL of PBS buffer at pH 7.4 containing 5 mg / mL bovine serum albumin is added to each well, and the plate is blocked at 37℃ for 2 h. The plate is then removed, shaken dry, and stored in a sealed container at 2~8℃.

[0047] The specific preparation method of the enzyme-linked reaction plate 2 is as follows: The Brucella BP26 antigenic epitope polypeptide prepared in Example 2 is dissolved in carbonate buffer at pH 9.6, mixed thoroughly, and then added to a 96-well polystyrene enzyme-linked reaction plate, 100 μL per well. For batch ZM-BRU01, 2 μg / mL of the polypeptide shown in SEQ ID NO: 1 is added to each well (i.e., 200 ng per well); for batch ZM-BRU02, 2 μg / mL of the polypeptide shown in SEQ ID NO: 2 is added to each well (i.e., 200 ng per well); and for batch ZM-BRU03, a polypeptide combination of SEQ ID NO: 1 and SEQ ID NO: 2 at a mass ratio of 1:1 (i.e., 100 ng of each polypeptide per well, totaling 200 ng) is added to each well. The plate is incubated at 2–8°C for 8–12 h. After completion, 300 mL of PBS buffer at pH 7.4 containing 5 mg / mL bovine serum albumin is added to each well. μL, sealed at 37℃ for 2 h, removed and spun dry, and then stored in a sealed container at 2~8℃ after thorough drying.

[0048] Example 5: How to use the reagent kit (1) Before testing, place each component of the kit at room temperature for 30 min to equilibrate.

[0049] (2) Dilute the 20× concentrated washing solution prepared in the kit with deionized water or distilled water at a ratio of 1:20.

[0050] (3) Take enzyme-linked reaction plate 1 and enzyme-linked reaction plate 2 (which can be used multiple times depending on the number of samples). Set up 2 positive control wells and 2 negative control wells for each test, and the rest are serum wells to be tested.

[0051] (4) Dilute the serum to be tested with sample diluent at a ratio of 1:50.

[0052] (5) Add the diluted serum to be tested, positive control serum and negative control serum to the corresponding wells of enzyme-linked reaction plate 1 and enzyme-linked reaction plate 2 respectively, 100 μL per well.

[0053] (6) After the sample is added, shake to mix and incubate at 37°C for 30 min.

[0054] (7) Remove the reaction plate and shake it dry. Add 300 μL of diluted washing solution to each well, shake gently and discard the washing solution. Wash the plate 3-4 times and pat it dry on absorbent paper.

[0055] (8) Add 100 μL of horseradish peroxidase-labeled rabbit anti-sheep IgG antibody to each well.

[0056] (9) After the sample is added, shake to mix and incubate at 37°C for 60 min.

[0057] (10) Remove the reaction plate and shake it dry. Add 300 μL of diluted washing solution to each well, shake gently and discard the washing solution. Wash the plate 3-4 times and pat it dry on absorbent paper.

[0058] (11) Mix substrate solution A and substrate solution B in equal volumes, add 100 μL of the mixed liquid to each well, and develop color at 37°C in the dark for 15 min.

[0059] (12) Add 50 μL of stop solution to each well.

[0060] (13) Within 10 minutes of adding the stop solution, read the OD of the sample using an ELISA reader. 450nm .

[0061] The method for determining the detection results of the ELISA antibody detection kit of the present invention is as follows: (1) When the OD of each negative control well 450nm ≤0.25, OD of each positive control well 450nm If the result is greater than or equal to 0.8, the result is valid; otherwise, the result is invalid.

[0062] (2) For enzyme-linked reaction plate 1, the OD of the serum to be tested 450nm ≥0.18× OD of positive control well 450nm The average value was used to determine a positive result; the OD value of the serum to be tested was... 450nm <0.18× OD of positive control well 450nmThe average value was determined to be negative.

[0063] (3) For enzyme-linked reaction plate 2, the OD of the serum to be tested 450nm ≥0.35× OD of positive control well 450nm The average value was used to determine a positive result; the OD value of the serum to be tested was... 450nm <0.35× OD of positive control well 450nm The average value was determined to be negative.

[0064] (4) If enzyme-linked reaction plate 1 is negative, it is unrelated to the result of enzyme-linked reaction plate 2. This indicates that Brucella LPS antibody was not detected, suggesting that the Brucella gene-deleted live vaccine (M5-90△26 strain) may not have been successfully administered or that the antibody was negative after immunization and there was no Brucella wild-type virus infection.

[0065] (5) If enzyme-linked reaction plate 1 is positive and enzyme-linked reaction plate 2 is negative, it indicates that the antibody is positive after immunization with Brucella gene deletion live vaccine (M5-90△26 strain).

[0066] (6) If enzyme-linked reaction plate 1 is positive and enzyme-linked reaction plate 2 is positive, it suggests that the tested animal may be infected with Brucella wild-type virus strain, or that it has positive antibodies after being immunized with other Brucella vaccines other than Brucella gene-deleted live vaccine (M5-90△26 strain).

[0067] Example 6: Peptide Sensitivity Detection Using the ELISA antibody detection kits (batch numbers ZM-BRU01~ZM-BRU03) prepared according to the method in Example 4, 80 Brucella-positive sera (derived from farms that had not been immunized with the Brucella gene-deleted live vaccine strain M5-90△26, and verified as positive by a commercially available Brucella cELISA antibody detection kit) were tested using enzyme-linked reaction plates 2 from each batch of kits, following the method described in Example 5. The results are shown in Table 3. The batch number ZM-BRU01 kit (coated with the polypeptide shown in SEQ ID NO: 1) detected 65 positive sera, with a sensitivity of 81.25%; the batch number ZM-BRU02 kit (coated with the polypeptide shown in SEQ ID NO: 2) detected 68 positive sera, with a sensitivity of 85.00%; and the batch number ZM-BRU03 kit (coated with a combination of the polypeptides shown in SEQ ID NO: 1 and SEQ ID NO: 2) detected 78 positive sera, with a sensitivity of 97.50%.

[0068] The above results indicate that the detection sensitivity of the combination of peptides shown in SEQ ID NO: 1 and SEQ ID NO: 2 is significantly higher than that of coating a single peptide. The two peptides, when used together, have a synergistic effect on the detection of BP26 antibody. Therefore, in subsequent performance validation, the highest-performing kit, batch number ZM-BRU03 (coated with the combination of peptides shown in SEQ ID NO: 1 and SEQ ID NO: 2), was selected.

[0069] Table 3 Sensitivity test results reagent kit batch number Number of positive cases Sensitivity ZM-BRU01 (SEQ ID NO: 1) 65 / 80 81.25% ZM-BRU02 (SEQ ID NO: 2) 68 / 80 85.00% ZM-BRU03(SEQ ID NO: 1+SEQ ID NO: 2) 78 / 80 97.50% Example 7, Specificity Detection Using the kit (batch number ZM-BRU03) prepared in Example 4, 30 samples of Brucella antibody-negative sheep serum, 2 samples of Escherichia coli antibody-positive sheep serum, 2 samples of Clostridium perfringens antibody-positive sheep serum, and 2 samples of Salmonella antibody-positive sheep serum were tested according to the method described in Example 5. The results are shown in Table 4. The results showed that both ELISA plate 1 and ELISA plate 2 were negative for all three types of sheep serum: Brucella antibody-negative, Escherichia coli antibody-positive, Clostridium perfringens antibody-positive, and Salmonella antibody-positive.

[0070] Table 4 Specific detection results

[0071] Example 8: Detection of Brucella gene-deleted live vaccine (M5-90△26 strain) in livestock farms Fifty sheep serum samples were collected from farms immunized with Brucella gene-deleted live vaccine (Strain M5-90△26). Verification using a commercially available Brucella cELISA antibody detection kit showed that 48 of the 50 serum samples were positive for Brucella antibodies. Using the kit prepared in Example 4 (batch number ZM-BRU03), the 48 serum samples were tested according to the method described in Example 5. The results are shown in Table 5. The results showed that for ELISA plate 1, the positive detection rate was 97.92% (47 / 48); for ELISA plate 2, the positive detection rate was 0% (0 / 48). According to the kit's determination method, 47 serum samples were determined to be antibody-positive after immunization with Brucella gene-deleted live vaccine (Strain M5-90△26). The kit of this invention has high detection sensitivity for Brucella gene-deleted live vaccine (Strain M5-90△26), and its coated BP26 antigenic epitope polypeptide combination shows no cross-reactivity with the Brucella gene-deleted live vaccine (Strain M5-90△26) immune antibodies.

[0072] Table 5. Results of Brucella gene-deleted live vaccine (M5-90△26 strain) immunization in farms

[0073] Example 9: Brucella positive serum detection Using the kit prepared in Example 4 (batch number ZM-BRU03), 80 Brucella-positive sera samples from Example 6 were tested according to the method described in Example 5. The results are shown in Table 6. The results showed that the positive detection rate was 100% (80 / 80) for ELISA plate 1 and 97.50% (78 / 80) for ELISA plate 2. Based on the kit's diagnostic method, 78 sera samples were determined to be antibody-positive after immunization with any Brucella gene-deleted live vaccine (M5-90△26 strain) or infected with wild-type strains. Since these 80 positive sera samples all originated from farms that had not been immunized with the Brucella gene-deleted live vaccine (M5-90△26 strain), BP26-related antibodies could theoretically be detected. This kit has a universal detection capability for Brucella antibodies and high detection sensitivity for BP26 antibodies, possessing differential diagnostic capabilities.

[0074] Table 6 Results of Brucella positive serum detection

[0075] Example 10: Comparison of detection results with commercially available kits The kit prepared in Example 4 (batch number ZM-BRU03, tested according to the method described in Example 5) and the commercially available Brucella ovis LPS and BP26 protein ELISA antibody detection kit (operated according to the product instructions) were used to simultaneously detect 80 Brucella positive sera (without M5-90△26 strain immunization) described in Example 6 and 48 Brucella gene-deleted live vaccine (M5-90△26 strain) immune positive sera described in Example 8. The experimental results are shown in Table 7. The results showed that for Brucella gene-deleted live vaccine (M5-90△26 strain) immune positive sera, the positive detection rate of the ELISA plate 1 of the kit of the present invention was 97.92% (47 / 48), and the positive detection rate of the LPS plate of the commercially available kit was 93.75% (45 / 48); the detection results of the ELISA plate 2 of the kit of the present invention and the BP26 plate of the commercially available kit were both negative for the above sera. For Brucella positive serum without M5-90△26 immunization, the detection results of the enzyme-linked reaction plate 1 of the kit of the present invention and the LPS plate of the commercial kit were both 100% positive; the positive detection rate of the enzyme-linked reaction plate 2 of the kit of the present invention was 97.50% (78 / 80), and the positive detection rate of the BP26 plate of the commercial kit was 75.00% (60 / 80).

[0076] Table 7. Test results of the reagent kit of this invention and commercially available reagent kits

Claims

1. The application of Brucella LPS as an antigen in the preparation of an ELISA antibody detection kit for differentiating Brucella gene-deleted live vaccine strain M5-90△26 from wild-type strain; wherein the Brucella LPS is LPS extracted from Brucella gene-deleted live vaccine strain M5-90△26.

2. Brucella BP26 antigenic epitope polypeptide, wherein the amino acid sequence of the polypeptide is SEQ ID NO: 1 or SEQ ID NO:

2.

3. A Brucella BP26 antigenic epitope polypeptide combination, which is composed of a polypeptide with the amino acid sequence shown in SEQ ID NO: 1 and a polypeptide with the amino acid sequence shown in SEQ ID NO: 2 in a mass ratio of 1:

1.

4. An ELISA antibody detection kit, comprising an enzyme-linked reaction plate 1, characterized in that, The enzyme-linked reaction plate 1 is coated with Brucella LPS; the Brucella LPS is LPS extracted from Brucella gene-deleted live vaccine strain M5-90△26 by the phenol-water method.

5. The ELISA antibody detection kit according to claim 4, characterized in that, The preparation method of the enzyme-linked reaction plate 1 is as follows: Brucella LPS is dissolved in carbonate buffer at pH 9.6, mixed evenly, and then added to a 96-well polystyrene enzyme-linked reaction plate. 100 μL of Brucella LPS at a concentration of 3 μg / mL is added to each well, and the plate is placed in an environment of 2-8℃ for 8-12 h. After completion, 300 μL of PBS buffer at pH 7.4 containing 5 mg / mL bovine serum albumin is added to each well, and the plate is blocked at 37℃ for 2 h. The plate is then removed, shaken dry, and stored in a sealed container at 2-8℃.

6. The ELISA antibody detection kit according to claim 4, characterized in that, It also includes an enzyme-linked reaction plate 2, which is coated with the Brucella BP26 antigenic epitope polypeptide combination as described in claim 3.

7. The ELISA antibody detection kit according to claim 4, characterized in that, The kit also includes 20× concentrated wash buffer, sample diluent, positive control serum, negative control serum, enzyme-labeled secondary antibody, substrate solution A, substrate solution B, and stop solution; the 20× concentrated wash buffer is pH 7.4 and contains 1% Tween 20 in 0.01 mol / L phosphate buffer; the sample diluent is pH 7.4 and contains 5 g / L casein in 0.01 mol / L phosphate buffer; the positive control serum is hyperimmune serum prepared from sheep immunized with Brucella live vaccine as the immunogen, diluted with the sample diluent; the negative control serum is serum from healthy sheep without Brucella infection or vaccine immunization; the enzyme-labeled secondary antibody is horseradish peroxidase-labeled rabbit anti-sheep IgG antibody; substrate solution A is citrate phosphate buffer containing 0.6 mg / mL hydrogen peroxide urea, and substrate solution B is a 0.2 mg / mL tetramethylbenzidine solution, which should be mixed in equal volumes before use; the stop solution is 2 mol / L H2SO4.

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