Brucella BP26 protein ELISA antibody detection kit and application thereof

Abstract

The invention discloses a Brucella BP26 protein ELISA (enzyme-linked immuno sorbent assay) antibody detection kit and an application of the Brucella BP26 protein ELISA antibody detection kit. The kit comprises an enzyme-linked reaction plate and an enzyme-labeled secondary antibody. The enzyme-linked reaction plate is coated with a Brucella BP26 protein antigen epitope polypeptide composition. The epitope polypeptide composition is any one or two or more than two of polypeptide shown as a sequence 1, polypeptide shown as a sequence 2 and polypeptide shown as a sequence 3 in a sequence table. According to the kit, the chemically synthesized peptide is used as the coating antigen, so that the interference of impure protein on the detection result is effectively reduced. The polypeptide composition disclosed by the invention is excellent in performance, high in sensitivity and strong in specificity, and can be used for accurately detecting a Brucella BP26 protein antibody in a sheep serum sample. The method is suitable for sheep brucellosis screening and antibody monitoring, and is beneficial to stable promotion of livestock brucellosis prevention and control and purification work in China.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and more specifically, the invention relates to a Brucella BP26 protein ELISA antibody detection kit, which is suitable for detecting Brucella antibodies in sheep serum samples. Background Art

[0002] Brucellosis, also known as brucellosis, is a zoonotic infectious disease caused by intracellular parasites of Brucella. It is prevalent in more than 170 countries and regions, posing a huge threat to the development of animal husbandry and human health. my country has classified it as a Class II animal epidemic disease. Infection in livestock often leads to inflammation of the reproductive organs, causing miscarriage, infertility and local lesions of various tissues.

[0003] Brucella is a Gram-negative bacterium belonging to the Brucella genus of the Brucella family of the Rhizobiales order of the phylum Alphaproteobacteria. So far, a total of 12 species of Brucella have been discovered, and different species have the ability to cross-infect each other. Four species can infect humans, namely, Brucella of sheep, pig, cattle and dog, among which Brucella of sheep is the most pathogenic to humans. Brucella can be infected through skin and mucous membrane contact or through the digestive tract, or it can form aerosols in a contaminated environment and infect through the respiratory tract. Brucella has a strong ability to survive in the natural environment and can survive for several months in the secretions, excrement, organs, fur and milk of sick animals. It has strong resistance to dry and cold environments, but is sensitive to heat. It can be killed in an environment of 60°C for 30 minutes and will die immediately when boiled.

[0004] At present, there are three main types of vaccines used for animal brucellosis prevention and control in China: Brucellosis live vaccine (S2 strain), Brucellosis live vaccine (A19 strain), and Brucellosis live vaccine (M5 strain) series. Vaccination is the most effective measure for the prevention and control of brucellosis at present, and different types of vaccines have their own advantages and disadvantages. S2 is a naturally weakened strain of swine Brucella type 1, which can produce good immunity in cattle, sheep, and pigs. The vaccine can be used for oral immunization or injection immunization. It is the weakest brucellosis vaccine strain and also the brucellosis vaccine strain with the broadest immune spectrum. The A19 strain is an international brucellosis reference strain. It was isolated from milk in 1923 and weakened by room temperature storage in the laboratory. The vaccine is a special vaccine for bovine brucellosis, which can produce good and lasting immunity for cattle. The longest immunity period can reach 6 years. It is the best choice for brucellosis prevention in cattle farms. M5 is a weakened strain isolated from Brucella melitensis and cultivated by my country. It can produce good immunity for both cattle and sheep. Because the strain is highly virulent and there is a risk of reversion to virulence, a series of modifications were carried out later to make it safer.

[0005] At present, the most authoritative "gold standard" for brucellosis diagnosis is still bacterial isolation and identification, but the in vitro culture of Brucella is difficult and time-consuming, and needs to be carried out in a biosafety level 3 laboratory, which cannot meet the needs of rapid on-site diagnosis. Serological diagnosis methods are convenient to operate and more suitable for large-scale screening and diagnosis. Traditional serological diagnosis methods have their own advantages and disadvantages. Red tiger plate agglutination (RBT) is a designated method for the diagnosis of brucellosis in many animals in international trade. This method has high sensitivity and is easy to operate. It is suitable for population surveys of brucellosis, but its high sensitivity often leads to false positive results. Compared with RBT, the test tube agglutination test (SAT) has higher accuracy and can be used for early diagnosis of brucellosis. However, as an agglutination test, the principle of SAT is also to identify the O-chain antigen of Brucella LPS, and the O-chain antigen of LPS exists in many Gram-negative bacteria, so SAT also has a certain probability of false positives. The complement fixation test (CFT) mainly observes whether the sheep red blood cells are hemolyzed to determine whether there are antibodies in the tested serum. It has strong specificity, but its sensitivity is lower than that of RBT and ELISA. In contrast, the enzyme-linked immunosorbent assay (ELISA) has good specificity and sensitivity. The time from sampling to result determination can be controlled within two hours. The operating procedures are clearly defined and easy to use, thus avoiding human errors to the greatest extent.

[0006] The Brucella genome is about 3.29Mb in size, containing two chromosomes, chromosome I is about 2.11Mb, chromosome II is about 1.18Mb, and there are about 3200 open reading frames. The cell membrane has three layers, namely the outer membrane, the periplasmic membrane and the cytoplasmic membrane. The outer membrane and peptidoglycan form the cell wall. The outer membrane contains lipopolysaccharide (LPS), outer membrane proteins (OMPs) and phospholipid layer. Outer membrane proteins are important virulence factors of Brucella, which can interfere with the immune presentation activity of macrophages and are also important antigens that mediate immunity. The antibodies produced by the body's immune response to them are one of the main antibodies in brucellosis detection.

[0007] BP26 protein is the dominant outer membrane protein of Brucella, with an open reading frame of 753 bp, encoding 250 amino acids, and high expression abundance. It has good immune activity, and naturally infected animals can produce specific antibodies against the protein, which has the potential to be used as a target protein for serological diagnosis. Summary of the invention

[0008] The invention analyzes the dominant epitopes of Brucella BP26 protein, prepares chemically synthesized peptides targeting the dominant epitopes, and selects a polypeptide composition for coating, thereby effectively eliminating the interference of foreign proteins and improving the accuracy of the detection result.

[0009] The present invention provides an indirect ELISA kit for detecting Brucella antibodies and a use method thereof. The kit uses Brucella BP26 protein antigen epitope polypeptide as a coating antigen, can quickly detect Brucella antibodies in sheep serum samples, and has the advantages of high sensitivity, strong specificity and convenient operation.

[0010] Generally, if a single polypeptide is used as a coating antigen, there is generally a problem of low sensitivity, and a more sensitive polypeptide is difficult to meet the detection requirements in terms of specificity. To solve this problem, the present invention screens out Brucella BP26 protein antigen epitope polypeptides and polypeptide compositions with excellent performance through research, and the specific Brucella BP26 protein antigen epitope polypeptides are polypeptides shown in sequence 1, polypeptides shown in sequence 2, or polypeptides shown in sequence 3 in the sequence table, and the composition is any combination of two or more polypeptides shown in sequence 1, polypeptides shown in sequence 2, and polypeptides shown in sequence 3 in the sequence table. When the polypeptide combination is two of the polypeptide shown in sequence 1, the polypeptide shown in sequence 2, and the polypeptide shown in sequence 3, the mass ratio of the two polypeptides is (0.5-1.5):(0.5-1.5), and preferably, the mass ratio is 1:1; when the polypeptide combination is three of the polypeptide shown in sequence 1, the polypeptide shown in sequence 2, and the polypeptide shown in sequence 3, the mass ratio of the three polypeptides is (0.5-1.5):(0.5-1.5):(0.5-1.5), and preferably, the mass ratio is 1:1:1.

[0011] The Brucella BP26 protein ELISA antibody detection kit of the present invention specifically comprises: an enzyme-linked reaction plate, a 20× concentrated washing solution, a sample diluent, a positive control serum, a negative control serum, an enzyme-labeled secondary antibody, a substrate solution A, a substrate solution B, and a stop solution.

[0012] The enzyme-linked reaction plate is a detachable 96-well enzyme-labeled plate, coated with a Brucella BP26 protein antigen epitope polypeptide composition artificially synthesized by a chemical method.

[0013] The preparation method of the enzyme-linked reaction plate is to dissolve the Brucella BP26 protein antigen epitope polypeptide composition in a carbonate buffer solution of pH 9.6, mix well and add to a 96-well polystyrene enzyme-linked reaction plate, add 100 μL of a polypeptide or polypeptide composition with a concentration of 2 μg / mL to each well, place in a 2-8°C environment for 8-12 hours, and allow the coated material to be fully adsorbed on the enzyme-linked reaction plate. After the coating is completed, 300 μL of bovine serum albumin (BSA) PBS buffer solution (pH 7.4) with a concentration of 5 mg / mL is added to each well, sealed at 37°C for 2 hours, taken out and dried, and sealed and stored at 2-8°C after being fully dried.

[0014] The 20× concentrated washing solution is a 0.01 mol / L phosphate buffer (pH 7.4) containing 1% Tween 20.

[0015] The sample diluent is a 0.01 mol / L phosphate buffer (pH 7.4) containing 5 g / L casein.

[0016] The positive control serum is prepared by extracting high-immune serum from sheep after immunizing them three times with Brucella live vaccine as the immunogen, and then diluting the positive control serum to a certain extent with the sample diluent.

[0017] The negative control serum is the serum of healthy sheep without Brucella infection or vaccine immunization.

[0018] The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled rabbit anti-sheep IgG antibody.

[0019] The substrate solution A is a citric acid phosphate buffer solution containing 0.6 mg / mL hydrogen peroxide urea, and the substrate solution B is a 0.2 mg / mL tetramethylbenzidine solution. The two need to be mixed at a ratio of 1:1 before use.

[0020] The stop solution is 2 mol / L H2SO4.

[0021] The kit of the present invention is suitable for detecting Brucella BP26 protein antibodies (IgG) in sheep serum samples. Generally speaking, the presence of the antibodies indicates that the tested animal is infected with Brucella or produces immune antibodies after being vaccinated with Brucella vaccine.

[0022] The specific method of using the Brucella BP26 protein ELISA antibody detection kit of the present invention is:

[0023] (1) Before testing, equilibrate all components of the kit to room temperature;

[0024] (2) Dilute the 20× concentrated washing solution provided in the kit with deionized water or distilled water at a ratio of 1:20;

[0025] (3) Take an ELISA plate (which can be used multiple times according to the number of samples). For each test, two positive control wells, two negative control wells, and an appropriate number of blank control wells should be set up, and the rest should be wells for the serum to be tested;

[0026] (4) Dilute the test serum, positive control serum, and negative control serum at a ratio of 1:50 using sample diluent;

[0027] (5) Add 100 μL of the diluted corresponding serum to each well of the serum to be tested, the positive control well, and the negative control well;

[0028] (6) After adding the sample, shake to mix and incubate at 37°C for 30 min;

[0029] (7) Remove the reaction plate and spin dry. Add 300 μL of diluted washing solution to each well. After slight shaking, discard the washing solution. Wash the plate 3 to 4 times and pat dry on absorbent paper.

[0030] (8) Add 100 μL of horseradish peroxidase-labeled rabbit anti-goat IgG antibody to each well;

[0031] (9) After adding the sample, shake to mix and incubate at 37°C for 60 min;

[0032] (10) Remove the reaction plate and spin dry. Add 300 μL of diluted washing solution to each well. After gentle shaking, discard the washing solution. Wash the plate 3 to 4 times and pat dry on absorbent paper.

[0033] (11) Mix substrate solution A and substrate solution B at a ratio of 1:1, add 100 μL of the mixed substrate solution to each well, and incubate at 37°C in the dark for 15 min;

[0034] (12) Add 50 μL of stop solution to each well;

[0035] (13) Add the stop solution and read the sample OD with an ELISA reader within 10 minutes. 450nm value.

[0036] The method for determining the detection result of the Brucella BP26 protein ELISA antibody detection kit of the present invention is as follows:

[0037] (1) When the OD of the negative control well is 450nm The average value is ≤0.25. The result is valid when the value of each positive control well is between 1.0 and 2.5, otherwise the result is invalid.

[0038] (2) Calculate the critical value: critical value = 0.33 × positive control well OD 450nm average value;

[0039] (3) Serum OD to be tested 450nm ≥ critical value, it is judged as BP26 antibody positive; serum OD to be tested 450nm < critical value, it was judged as BP26 antibody negative.

[0040] The present invention adopts bioinformatics method to analyze the dominant antigenic epitopes of Brucella BP26 protein, screens out a polypeptide composition with excellent performance, can quickly detect Brucella BP26 protein antibodies in sheep serum samples, and has the advantages of high sensitivity, strong specificity and good repeatability.

[0041] The present invention adopts solid phase synthesis technology to synthesize polypeptides, and the synthesized peptides have high purity and stable properties; chemically synthesized polypeptides are used as coating antigens to avoid contact with bacteria, effectively remove the interference of foreign proteins, improve the specificity of detection, and thus reduce the probability of false positives.

[0042] The Brucella BP26 protein ELISA antibody detection kit of the present invention can accurately detect Brucella antibodies in sheep, is suitable for brucellosis screening and antibody monitoring, and is conducive to the steady advancement of brucellosis prevention, control and purification work among livestock in my country. DETAILED DESCRIPTION

[0043] The methods in the following embodiments are conventional methods unless otherwise specified.

[0044] Example 1 Preparation of Brucella BP26 Protein ELISA Antibody Detection Kit Coated Antigen

[0045] Bioinformatics software was used to analyze the dominant antigenic epitopes of Brucella melitensis biovar Abortusstrain S19BP26 (bp26) gene (AY166766.1) in the GenBank library. After comprehensive evaluation, three polypeptides were screened out. The sequences are shown in the sequence table as sequence 1, sequence 2, and sequence 3. A polypeptide with a purity of about 80% was synthesized as a coating antigen.

[0046] The above-mentioned peptides were prepared using an Applied Biosystem fully automatic peptide synthesizer (model 433A). The Merrifield solid phase synthesis method was used, using Fmoc (9-fluorenylmethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl) modified amino acids and Rink Amide MBHA resin as a solid phase carrier. The production process includes four parts: peptide solid phase synthesis, peptide cleavage and identification, peptide purification, and freeze-drying storage:

[0047] 1. Solid Phase Synthesis of Peptides

[0048] 1. Preparation of Synthetic Reagents

[0049] The amino acid sequences of the synthetic polypeptides are shown in Sequence 1, Sequence 2, and Sequence 3 in the sequence listing.

[0050] Sequence 1: NEAMTKVLDAMKKAGIEDRD

[0051] Sequence 2: IQPIYVYPDDKNHLKEPTLT

[0052] Sequence 3: ANVGKILDESVTRGVNQGGD

[0053] According to the peptide sequence and the amount of synthesis, prepare an appropriate amount of Fmoc-modified amino acids (purchased from NOVA) and add them to the corresponding cartridge. According to the synthesis scale, weigh 5g of resin, add it to the reaction chamber, tighten the upper and lower lids, label it, record the name of the synthesized peptide, the batch number, the TARE of the reaction chamber and the weight of the weighed resin, load the reaction chamber into the synthesizer, prepare an appropriate amount of synthesis reagents, including 100% NMP, 3% AIM (acetyl imidazole), 35% PIP (piperidine), 100% MeOH (methanol), and add them to the corresponding reagent bottles.

[0054] 2. Detection of synthesizer status

[0055] First, check whether the functions of the Applied Biosystem fully automatic peptide synthesizer (model 433A) are normal, turn on the machine, run the Run Self Test program, and start the instrument self-test process. In addition, manually check whether the nitrogen is sufficient and whether the system gauge pressure is normal (the normal gauge pressure of 433A is 10.2psi). Before synthesis, measure the flow rate of each synthesis reagent. The specific steps are: send Flow Rate1-18 to the synthesizer, select Main Menu—Module Test—press Prer or next to find ModuleA, ModuleD, ModuleI, ModuleI, Module A—press Start—press More to measure or observe. If the flow rate is not appropriate, adjust the valve pressure until it meets the requirements (see Table 1 for specific test requirements).

[0056] Table 1 Standard table for flow rate detection of peptide synthesizer

[0057] Reagents Bottle No. Module Standard range 35% Piperidine 1 A 1.0~1.2mL 3% AIM 4 D 1.0~1.2mL 100% MeOH 9 I 3.5~4.0mL DIC 8 I 0.45～0.55g 100% NMP 10 A 2.6–2.8 mL

[0058] 3. Start synthesis

[0059] In the 433A synthesizer program, send the amino acid sequence to be synthesized to the synthesizer as Std Fmoc 1.0Sol DIC90, edit the peptide sequence and save it, then edit the Cycles and save it, send it to the synthesizer, click MainMenu—Cycle Monitor—begin to start running.

[0060] 4. Synthesis process

[0061] Removal of Fmoc group: The electron-withdrawing effect of the fluorene ring system of the Fmoc group makes 9-H acidic and easily removed by weaker bases. During the reaction, piperidine (PIP) attacks 9-H, β-elimination forms dibenzofluorene, and after the Fmoc group is removed, the "-NH2" group is exposed for the synthesis reaction. Then, the next Fmoc group-protected amino acid and 1-hydroxybenzotriazole (HOBT) are added to the reactor.

[0062] If the peptide synthesis is from C-terminus to N-terminus, the synthesis steps are repeated in the cycle sequence in Table 2, and the reagent dosage and equipment operation status are observed and recorded during the process.

[0063] Table 2 Peptide synthesis cycle steps

[0064]

[0065] 5. End the synthesis

[0066] After the peptide synthesis is completed, the synthesizer automatically stops, and the peptide resin (peptide is still connected to the resin) is basically washed. Remove the reactor from the peptide synthesizer, wash the peptide resin 3 times with 100% methanol, blow dry in a fume hood, and then transfer all the peptide resin to a brown polyethylene bottle, seal it, and store it at -20°C.

[0067] Dipeptide cleavage and identification

[0068] 1. Peptide cleavage

[0069] Prepare the peptide lysis solution according to the volume ratio of (trifluoroacetic acid: triisopropylsilane: phenol: H2O = 85:8:6:1), take out the synthesized peptide resin, put it into a round-bottom flask, add the prepared lysis solution and magnetic stirring bar to the flask in a fume hood, stir for 1 hour, and then use a rotary evaporator with a cold trap to evaporate for 30 to 120 minutes to remove TFA in the crude product, then collect the precipitated peptide with ether, wash the crude peptide antigen with dimethylformamide (DMF) several times, and finally filter the mixed resins with a sand core funnel to obtain the peptide.

[0070] 2. Identification of Peptides

[0071] Matrix-assisted laser desorption time-of-flight mass spectrometry (MODAL-TOF) and reversed-phase high pressure liquid chromatography (RP-HPLC) were used for qualitative and quantitative analysis.

[0072] Purification of three peptides

[0073] Use a circulating tangential filtration membrane package to ultrafilter the polypeptide at 20-28°C (TangentialFlowDevice circulating tangential filtration membrane package and its matching peristaltic pump). This process can filter out small molecular impurities formed in the early synthesis and late cyclization reactions, and then sterilize with a 0.2μm pore size filter. The resulting solution is packaged into sterile bottles and labeled with the name, number, production batch number, concentration, production date, shelf life and storage conditions of the polypeptide. After packaging, store at -20°C or -80°C for future use.

[0074] 4. Freeze-dried storage

[0075] In order to facilitate long-term storage and transportation, the peptide needs to be freeze-dried. The pre-frozen peptide is placed on a Labconco freeze dryer for drying to obtain a solid peptide. After packaging, a label is affixed, indicating the name, number, production batch number, concentration, production date, shelf life and storage conditions of the peptide.

[0076] Example 2 Preparation of positive control serum for Brucella BP26 protein ELISA antibody detection kit

[0077] Brucellosis live vaccine (Chongqing Aolong Biological Products Co., Ltd., approval number veterinary medicine 230247011, production batch number 2024007) was used as the immunogen. The immunized subjects were 6-month-old sheep. The injection method was subcutaneous injection in the neck. Three immunizations were performed at week 0, week 2, and week 4, respectively. The immunization doses were 2, 2, and 4, respectively. Blood was collected 7 to 10 days after the three immunizations to determine the serum antibody titer. When the serum antibody titer reached the positive range after identification by the commercial Brucella cELISA antibody detection kit, a large amount of blood was collected and the serum was separated. The obtained serum was appropriately diluted with sample diluent to make OD 450nm The value was between 1.0 and 2.5, and the positive control serum was prepared.

[0078] Example 3 Assembly of Brucella BP26 Protein ELISA Antibody Detection Kit

[0079] The Brucella BP26 protein ELISA antibody detection kit of the present invention specifically comprises:

[0080] (1) A detachable polystyrene enzyme-linked reaction plate (96 wells, 2 plates) coated with a synthetic peptide antigen of Brucella BP26 protein.

[0081] (2) 20× concentrated washing solution: 0.01 mol / L phosphate buffer containing 1% Tween 20 at pH 7.4 (50 mL / bottle, 2 bottles).

[0082] (3) Sample diluent: 0.01 mol / L phosphate buffer containing 5 g / L casein at pH 7.4 (24 mL / bottle, 1 bottle).

[0083] (4) Positive control serum: Brucella live vaccine was used as the immunogen. Highly immune serum was extracted and prepared after immunizing sheep three times. The positive control serum (1.5 mL / tube, 1 tube) was prepared by diluting the sample diluent to a certain extent.

[0084] (5) Negative control serum: serum from healthy sheep without Brucella infection or vaccine immunization (1.5 mL / tube, 1 tube).

[0085] (6) Enzyme-labeled secondary antibody: horseradish peroxidase-labeled rabbit anti-goat IgG antibody (Sigma) diluted 1:30,000 (12 mL / bottle, 2 bottles).

[0086] (7) Substrate solution A: citrate phosphate buffer containing 0.6 mg / mL urea hydrogen peroxide (12 mL / bottle, 1 bottle).

[0087] (8) Substrate solution B: 0.2 mg / mL tetramethylbenzidine solution (12 mL / bottle, 1 bottle).

[0088] (9) Stop solution: 2 mol / L H2SO4 solution (12 mL / bottle, 1 bottle).

[0089] According to the requirements, the kit can also be equipped with a serum dilution plate (96 wells, 2 pieces) for dilution of serum samples.

[0090] The method for preparing an enzyme-linked reaction plate coated with a synthetic peptide antigen of Brucella BP26 protein is as follows: the polypeptide antigen prepared in Example 1 is dissolved in a carbonate solution of pH 9.6, and then added to a 96-well polystyrene enzyme-linked reaction plate, with a total of 200 ng of polypeptide in each well (ZM202401 has 200 ng of polypeptide shown in sequence 1 per well, ZM202402 has 200 ng of polypeptide shown in sequence 2 per well, ZM202403 has 200 ng of polypeptide shown in sequence 3 per well, ZM202404 has 100 ng of polypeptide shown in sequence 1 and 100 ng of polypeptide shown in sequence 2 per well, and ZM202405 has 100 ng of polypeptide shown in sequence 1 and 100 ng of polypeptide shown in sequence 3 per well) , ZM202406 100ng of the polypeptide shown in sequence 2 and 100ng of the polypeptide shown in sequence 3 per well, ZM202407 66.7ng of the polypeptide shown in sequence 1, 66.7ng of the polypeptide shown in sequence 2, and 66.7ng of the polypeptide shown in sequence 3 per well, coated at 2-8°C for 8-12h. After coating, 300μL of 5mg / mL bovine serum albumin (BSA) PBS buffer (pH7.4) was added to each well, blocked at 37°C for 2h, taken out and dried, and sealed and stored at 2-8°C after sufficient drying.

[0091] Example 4: Method for using the Brucella BP26 protein ELISA antibody detection kit

[0092] (1) Before testing, equilibrate all components of the kit to room temperature;

[0093] (2) Dilute the 20× concentrated washing solution provided in the kit with deionized water or distilled water at a ratio of 1:20;

[0094] (3) Take an ELISA plate (which can be used multiple times according to the number of samples). For each test, two positive control wells, two negative control wells, and an appropriate number of blank control wells should be set up, and the rest should be wells for the serum to be tested;

[0095] (4) Dilute the test serum, positive control serum, and negative control serum at a ratio of 1:50 using sample diluent;

[0096] (5) Add 100 μL of the diluted corresponding serum to each well of the serum to be tested, the positive control well, and the negative control well;

[0097] (6) After adding the sample, shake to mix and incubate at 37°C for 30 min;

[0098] (7) Remove the reaction plate and spin dry. Add 300 μL of diluted washing solution to each well. After slight shaking, discard the washing solution. Wash the plate 3 to 4 times and pat dry on absorbent paper.

[0099] (8) Add 100 μL of horseradish peroxidase-labeled rabbit anti-goat IgG antibody to each well;

[0100] (9) After adding the sample, shake to mix and incubate at 37°C for 60 min;

[0101] (10) Remove the reaction plate and spin dry. Add 300 μL of diluted washing solution to each well. After gentle shaking, discard the washing solution. Wash the plate 3 to 4 times and pat dry on absorbent paper.

[0102] (11) Mix substrate solution A and substrate solution B at a ratio of 1:1, add 100 μL of the mixed substrate solution to each well, and incubate at 37°C in the dark for 15 min;

[0103] (12) Add 50 μL of stop solution to each well;

[0104] (13) Add the stop solution and read the sample OD with an ELISA reader within 10 minutes. 450nm value.

[0105] Method for determining the test results of Brucella BP26 protein ELISA antibody detection kit:

[0106] (1) When the OD of the negative control well is 450nmThe average value is ≤0.25. The result is valid when the value of each positive control well is between 1.0 and 2.5, otherwise the result is invalid.

[0107] (2) Calculate the critical value: critical value = 0.33 × positive control well OD 450nm average value;

[0108] (3) Serum OD to be tested 450nm ≥ critical value, it is judged as BP26 antibody positive; serum OD to be tested 450nm < critical value, it was judged as BP26 antibody negative.

[0109] Example 5 Sensitivity Test

[0110] Using 7 batches of Brucella BP26 protein ELISA antibody detection kits (batches ZM202401-ZM202407) prepared according to the method of Example 3, 120 antibody-positive sheep sera collected from the sheep farm (all identified as antibody-positive by commercial Brucella cELISA antibody detection kits) were detected according to the method of using the Brucella BP26 protein ELISA antibody detection kit described in Example 4. The test results are shown in Table 3. The Brucella BP26 protein ELISA antibody detection kit of batch number ZM202401 of the present invention detected a total of 79 copies, and the sensitivity of the kit to the 120 copies of sera to be tested was 65.8%; the Brucella BP26 protein ELISA antibody detection kit of batch number ZM202402 of the present invention detected a total of 74 copies, and the sensitivity of the kit to the 120 copies of sera to be tested was 61.7%; the Brucella BP26 protein ELISA antibody detection kit of batch number ZM202403 ...4 of the present invention detected a total of 74 copies, and the sensitivity of the kit to the 120 copies of sera to be The ISA antibody detection kit detected 66 samples in total, and the sensitivity of the kit to 120 samples of serum to be tested was 55.0%; the Brucella BP26 protein ELISA antibody detection kit of batch number ZM202404 of the present invention detected 105 samples in total, and the sensitivity of the kit to 120 samples of serum to be tested was 87.5%; the Brucella BP26 protein ELISA antibody detection kit of batch number ZM202405 of the present invention detected 98 samples in total, and the sensitivity of the kit to 120 samples of serum to be tested was 81.7%; the Brucella BP26 protein ELISA antibody detection kit of batch number ZM202406 of the present invention detected 90 samples in total, and the sensitivity of the kit to 120 samples of serum to be tested was 75.0%; the Brucella BP26 protein ELISA antibody detection kit of batch number ZM202407 of the present invention detected 115 samples in total, and the sensitivity of the kit to 120 samples of serum to be tested was 95.8%.

[0111] The results of the sensitivity test showed that the combination of two or three of the polypeptides shown in sequence 1, sequence 2 and sequence 3 was better than a single polypeptide, indicating that these polypeptides have a synergistic effect in detection.

[0112] Table 3 Sensitivity test results

[0113] Kit batch number Detection rate Sensitivity ZM202401(sequence 1) 79 / 120 65.8％ ZM202402 (sequence 2) 74 / 120 61.7％ ZM202403 (sequence 3) 66 / 120 55.0％ ZM202404(sequence 1+sequence 2) 105 / 120 87.5％ ZM202405 (sequence 1 + sequence 3) 98 / 120 81.7％ ZM202406 (sequence 2 + sequence 3) 90 / 120 75.0％ ZM202407(sequence 1+sequence 2+sequence 3) 115 / 120 95.8％

[0114] Example 6 Specificity Test

[0115] 7 batches of Brucella BP26 protein ELISA antibody detection kits (batches ZM202401-ZM202407) prepared according to the method of Example 3 were used, and 100 negative sheep sera, 2 sheep Escherichia coli antibody positive sera, 2 sheep Clostridium antibody positive sera, and 2 sheep Streptococcus antibody positive sera (the above sera are all provided by China Animal Husbandry Industry Co., Ltd.) were tested respectively according to the method of using the Brucella BP26 protein ELISA antibody detection kit described in Example 4. The specificity test results of the kit are shown in Table 4, and the test results of 100 negative sheep sera show that the specificity of all batches of kits is 100.0%. The test results of 2 sheep Escherichia coli antibody positive sera, 2 sheep Clostridium antibody positive sera, and 2 sheep Streptococcus antibody positive sera are all negative, so the specificity of the 7 batches of kits for the above-mentioned related positive serum detection is 100%.

[0116] Table 4 Specificity test results

[0117]

[0118]

Claims

1. Brucella BP26 protein antigen epitope polypeptide, which is the polypeptide shown in sequence 1 in the sequence list, the polypeptide shown in sequence 2 in the sequence list, or the polypeptide shown in sequence 3 in the sequence list.

2. The Brucella BP26 protein antigen epitope polypeptide composition is any combination of two or more of the polypeptides shown in Sequence 1 in the sequence list, the polypeptides shown in Sequence 2 in the sequence list, and the polypeptides shown in Sequence 3 in the sequence list.

3. The Brucella BP26 protein antigen epitope polypeptide composition according to claim 2, characterized in that: When the polypeptide combination is two of the polypeptide shown in sequence 1, the polypeptide shown in sequence 2, and the polypeptide shown in sequence 3, the mass ratio of the two polypeptides is (0.5-1.5):(0.5-1.5), and preferably, the mass ratio is 1:1; when the polypeptide combination is three of the polypeptide shown in sequence 1, the polypeptide shown in sequence 2, and the polypeptide shown in sequence 3, the mass ratio of the three polypeptides is (0.5-1.5):(0.5-1.5):(0.5-1.5), and preferably, the mass ratio is 1:1:

1.

4. A Brucella BP26 protein ELISA antibody detection kit, comprising an enzyme-linked reaction plate and an enzyme-labeled secondary antibody; wherein: The enzyme-linked reaction plate is coated with the Brucella BP26 protein antigen epitope polypeptide according to claim 1 or the Brucella BP26 protein antigen epitope polypeptide composition according to claim 2 or 3.

5. Brucella BP26 protein ELISA antibody detection kit according to claim 4, characterized in that: The enzyme-linked reaction plate is a detachable 96-well enzyme-labeled plate; the Brucella BP26 protein antigen epitope polypeptide is a chemically synthesized peptide.

6. Brucella BP26 protein ELISA antibody detection kit according to claim 4, characterized in that: The preparation method of the enzyme-linked reaction plate is to dissolve the Brucella BP26 protein antigen epitope polypeptide composition in a carbonate buffer solution with a pH of 9.6, mix well and add to a 96-well polystyrene enzyme-linked reaction plate, add 100 μL of Brucella BP26 protein antigen epitope polypeptide or Brucella BP26 protein antigen epitope polypeptide composition with a concentration of 1 μg / mL to each well, place in a 2-8°C environment for 8-12 hours, so that the coated material is fully adsorbed on the enzyme-linked reaction plate, and after the coating is completed, add 300 μL of PBS buffer solution with a concentration of pH 7.4 and containing 5 mg / mL bovine serum albumin to each well, seal at 37°C for 2 hours, take out and spin dry, and seal and store at 2-8°C after being fully dried.

7. Brucella BP26 protein ELISA antibody detection kit according to claim 4, characterized in that: The kit also includes 20× concentrated washing solution, sample diluent, positive control serum and negative control serum; the 20× concentrated washing solution is a 0.01 mol / L phosphate buffer with a pH of 7.4 and containing 1% Tween20; the sample diluent is a 0.01 mol / L phosphate buffer with a pH of 7.4 and containing 5 g / L casein; the positive control serum is prepared by extracting high-immune serum from sheep after immunizing sheep three times with Brucella live vaccine as an immunogen, and then diluting the positive control serum to a certain extent with the sample diluent; the negative control serum is the serum of healthy sheep without Brucella infection or vaccine immunization; and the enzyme-labeled secondary antibody is a rabbit anti-sheep IgG antibody labeled with horseradish peroxidase.

8. Brucella BP26 protein ELISA antibody detection kit according to claim 4, characterized in that: The kit also includes substrate solution A, substrate solution B and stop solution; the substrate solution A is a citric acid phosphate buffer solution containing 0.6 mg / mL hydrogen peroxide urea, and the substrate solution B is a 0.2 mg / mL tetramethylbenzidine solution, and the two need to be mixed in a 1:1 ratio before use; the stop solution is 2 mol / L H2SO4.

9. Use of the Brucella BP26 protein antigen epitope polypeptide according to claim 1 or the Brucella BP26 protein antigen epitope polypeptide composition according to claim 2 or 3 in preparing a kit for detecting Brucella antibodies.

10. The use according to claim 9, characterized in that: The detection of Brucella antibodies refers to detecting whether Brucella BP26 protein antibodies exist in sheep serum samples. If the antibodies exist, it indicates that the sheep being tested is infected with Brucella or produces immune antibodies after being vaccinated with Brucella vaccines.

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