Kit for detecting livestock brucella antibody and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a kit for detecting a livestock brucella antibody and application of the kit. The magnetic particle chemiluminescence method comprises the following steps: coating magnetic beads, a signal tracer agent, a reaction diluent and the like; the coated magnetic beads are magnetic beads coated with Brucella specific lipopolysaccharide antigens, and the dilution concentration of the coated magnetic beads is 0.05-0.15 mg / mL; the signal tracer agent is recombinant streptococcus protein G marked by acridinium ester, and the dilution ratio is 1: (8000-12000); the reaction diluent is a phosphate buffer, the concentration of the phosphate buffer is 0.01-0.03 M, and the pH value of the phosphate buffer is 7.3-7.5. The detection method provided by the invention has high specificity, sensitivity, repeatability and reliability, and can be used for the detection purposes of brucellosis epidemiological investigation, immune antibody evaluation and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a kit for detecting Brucella antibodies in livestock and its application. Background Technology

[0002] Brucellosis is a globally distributed zoonotic infectious disease caused by bacteria of the genus Brucella. In animals, brucellosis typically leads to abortion and infertility, causing significant economic losses to livestock production. Typical clinical symptoms of brucellosis in humans include undulant fever, hyperhidrosis, and arthritis, seriously endangering human health. Timely and accurate diagnosis is crucial for effective brucellosis control.

[0003] Currently, the main methods for detecting brucellosis include bacterial isolation and identification, etiological detection, and serological detection. To date, various etiological detection methods have been developed and applied, including conventional polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), droplet digital PCR (ddPCR), and recombinase-assisted amplification (RAA) technology. Compared to etiological detection, serological detection is more widely used in the diagnosis of suspected infections, epidemic surveillance, and epidemiological assessment. Currently, commonly used serological detection methods include the Rose Bengal Plate Test (RBT), Stem Agglutination Test (SAT), Complement Fixation Test (CFT), Indirect Enzyme-Linked Immunosorbent Assay (iELISA), and Competitive Enzyme-Linked Immunosorbent Assay (cELISA). The main advantages and disadvantages of different methods include: RBT is simple to operate, time-saving, and low-cost, but prone to cross-reactivity; SAT can quantify antibody titers, but has low sensitivity; CFT is sensitive and specific, but the operation is complex; ELISA has excellent diagnostic performance, high throughput, and relatively simple operation, but is not suitable for automated equipment. This study aims to develop a new, sensitive, specific, high-throughput, and automated method for detecting Brucella antibodies.

[0004] Chemiluminescence immunoassay (CLIA) is a labeled immunoassay that combines a chemiluminescence system with an immune reaction. Due to its high sensitivity and specificity, CLIA has been widely used in life science research, clinical medical research, food analysis and testing, and environmental monitoring. Compared to several commonly used serological detection methods for brucellosis, CLIA offers advantages such as high sensitivity, a wide linear range, ease of automation, and high throughput. In veterinary medicine, CLIA has been reported for the diagnosis of various diseases, including African swine fever, foot-and-mouth disease, and mycoplasma pneumoniae; however, research on its application in brucellosis detection is limited. This study established a novel magnetic particle chemiluminescence method (Bru-CLIA) for detecting Brucella antibodies in livestock serum, using extracted lipopolysaccharide (LPS)-coated magnetic microparticles (LPS-MPs) as the diagnostic antigen and acridine ester (AE)-labeled recombinant streptococcal protein G (AE-SPG) as the signal tracer. This method boasts advantages such as high sensitivity, a wide linear range, and high automation, providing technical support for brucellosis prevention and control.

[0005] This invention provides a magnetic particle chemiluminescence method for detecting Brucella antibodies in bovine and ovine serum. This method offers advantages such as short detection time, high sensitivity, specificity, and high throughput. It is particularly suitable for large-scale animal population screening and immune antibody assessment, and is of great significance for shortening the diagnostic window period for diseases and blocking disease transmission. It also provides an innovative solution for the prevention and control of zoonotic diseases in my country. Summary of the Invention

[0006] The purpose of this invention is to provide a kit for detecting Brucella antibodies in livestock and its application.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a kit for detecting Brucella antibodies in livestock, comprising: coated magnetic beads, a signal tracer, and a reaction diluent; the coated magnetic beads are magnetic beads coated with Brucella-specific lipopolysaccharide antigen, with a dilution concentration of 0.05–0.15 mg / mL; the signal tracer is acridinium-labeled recombinant streptococcal protein G, with a dilution ratio of 1:8000–12000; the reaction diluent is phosphate buffer, with a concentration of 0.01–0.03 M and a pH of 7.3–7.5.

[0009] Preferably, the kit further includes: a pre-activation solution and an activation solution.

[0010] Preferably, the lipopolysaccharide is extracted by inactivation after culturing Brucella bovis strain A19.

[0011] Preferably, the method for preparing the coated magnetic beads is as follows: after washing the magnetic beads, resuspend them in binding buffer, add Brucella-specific lipopolysaccharide and catalytic reagent solution, incubate, block, incubate again, and wash.

[0012] Preferably, the method for preparing the signal tracer is as follows: recombinant streptococcal protein G is dissolved in acridinium ester labeling buffer 4, and after ultrafiltration, a recombinant streptococcal protein G solution is obtained; then, acridinium ester solution is added for reaction in the dark, blocked, incubated, and acridinium ester labeling buffer 5, glycerol and acridinium ester labeling protectant are added to the reaction solution.

[0013] The present invention also provides the application of the kit for detecting Brucella antibodies in the serum of livestock.

[0014] Preferably, the method of application is as follows:

[0015] (1) Mix and incubate the coated magnetic beads, serum sample and dilution buffer;

[0016] (2) After washing the incubated solution system, add the signal tracer, pre-activation solution and activation solution, mix and incubate again, detect and judge the results.

[0017] Preferably, the volume ratio of the coated magnetic beads, serum sample, and dilution buffer is 50–100:5:50–100.

[0018] Preferably, the incubation temperature in steps (1) and (2) is 36-38°C and the incubation time is 8-12 min.

[0019] Preferably, the volume ratio of the signal tracer, pre-excitation solution and excitation solution in step (2) is 8-12:8-20:8-20.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This study developed a chemiluminescent immunoassay (Bru-CLIA) for brucellosis antibodies. The method uses lipopolysaccharide (LPS)-coated magnetic microparticles (LPS-MPs) extracted from Brucella a19 strain as the serum diagnostic antigen, and acridine ester (AE)-labeled recombinant streptococcal protein G (AE-SPG) as the signal tracer. After optimizing the Bru-CLIA operating parameters, the detection sensitivity of this method for Brucella antibodies in sheep and bovine serum was approximately 1 IU / mL and 2 IU / mL, respectively. This method showed no cross-reactivity with positive sera from Escherichia coli O157:H7, Mycobacterium tuberculosis, Vibrio cholerae, Legionella, Salmonella, foot-and-mouth disease virus types O and A, bovine viral diarrhea virus, Mycoplasma sheep, goatpox virus, and peste des petits ruminants virus, exhibiting good specificity. After testing 81 sheep serum samples and 96 bovine serum samples, the concordance rates between Bru-CLIA and the commercial ID-VET kit in sheep and bovine samples were 87.65% and 93.75%, respectively. The results indicate that Bru-CLIA possesses excellent specificity, sensitivity, repeatability, and reliability, and has good application potential in livestock brucellosis epidemiological surveys, immune antibody assessment, and disease-free eradication. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 Establishment of Brucella chemiluminescent immunoassay (Bru-CLIA): (A) Optimization of LPS-MPs concentration: Three concentrations of 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL were evaluated, with each concentration repeated three times. (B) Optimization of AE-SPG dilution: Four dilution ratios of 1:5000, 1:10000, 1:15000, and 1:20000 were evaluated, with each dilution ratio repeated three times. (C) Optimization of serum dilution: Four dilution ratios of 1:5, 1:10, 1:20, and 1:40 were evaluated, with each dilution ratio repeated three times. (D) Optimization of reaction diluents: Chemiluminescent reactions were performed using seven different reaction diluents under the same conditions, with each dilution component repeated three times. (E) Reaction procedures: One-step and two-step incubation reactions of antigen and antibody were evaluated, including four procedures.

[0024] Figure 2Determination of ROC curves and interactive scatter plots for Bru-CLIA; (A) and (B) are ROC curves and interactive scatter plots of Bru-CLIA used to detect sheep serum samples. (C) and (D) are ROC curves and interactive scatter plots of Bru-CLIA used to detect bovine serum samples. Detailed Implementation

[0025] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1

[0027] 1. Source of materials

[0028] Bovine standard positive serum (titer 4000 IU / mL) was purchased from the China Institute of Veterinary Drug Control. Sheep standard positive serum (titer 400 IU / mL) was identified and provided by the National Animal Brucellosis Specialty Laboratory of the China Animal Health and Epidemiology Center. Thirty-three sheep serum samples (13 brucellosis positive sera and 20 brucellosis negative sera) and twenty-three bovine serum samples (11 brucellosis positive sera and 12 brucellosis negative sera) were identified and provided by the National Animal Brucellosis Specialty Laboratory of the China Animal Health and Epidemiology Center. All the above-mentioned brucellosis positive serum samples were confirmed by both RBT and a commercially available indirect ELISA antibody detection kit (ID-VET, France); negative serum samples were obtained from national brucellosis-free zones. Positive sera for *Escherichia coli* O157:H7, *Mycobacterium tuberculosis*, *Vibrio cholerae*, *Legionella*, *Salmonella*, foot-and-mouth disease virus (FMD) types O and A, bovine viral diarrhea virus, ovine contagious pleuropneumonia, goatpox virus, and peste des petits ruminants virus were identified and collected or prepared by the National Animal Brucellosis Laboratory of the China Animal Health and Epidemiology Center. Eighty-one clinical sheep serum samples and ninety-six bovine serum samples were collected by the National Animal Brucellosis Laboratory of the China Animal Health and Epidemiology Center. Magnetic microparticle coating kits and acridine ester labeling kits were purchased from Boyuan Nuoxin (Beijing) Biotechnology Co., Ltd. Recombinant Streptococcal protein G (SPG) was purchased from Beijing Baisaisi Technology Co., Ltd. Bacterial lipopolysaccharide extraction kits were purchased from Beijing Bio-Lab Technology Co., Ltd. Fetal bovine serum (FBS) was purchased from Gibco and used for system optimization and dilution of bovine standard positive sera. The fully automated chemiluminescence immunoassay analyzer Flash 200 was provided by Shenzhen Lvshiyuan Biotechnology Co., Ltd.

[0029] 2LPS extraction

[0030] Brucella abortus strain A19 was cultured in tryptone soybean broth (TSB) to the exponential growth phase. The culture was then heat-inactivated at 80°C for 2 h. Bacterial cells were collected by centrifugation, and LPS was extracted using a bacterial lipopolysaccharide extraction kit. The samples were loaded onto a 12.5% ​​polyacrylamide gel for SDS-PAGE and silver staining analysis. Finally, the LPS was lyophilized and stored at -80°C.

[0031] Coupling of 3LPS and Magnetic Beads

[0032] The coupling of LPS with magnetic beads was performed according to the following steps: 1 mg of magnetic beads were washed and resuspended in 900 μL of binding buffer (0.1 M HEPES buffer, pH 8.0), followed by the addition of 0.1 mg of LPS and 550 μL of catalytic reagent solution, and incubated at 37 °C for 18 h on a rotary oscillator. The reaction was then blocked with 100 μL of blocking reagent and further incubated at 37 °C for 2 h. After washing with washing buffer, the LPS-MPs were dissolved in PBS and stored at 4 °C.

[0033] Coupling of 4AE and SPG

[0034] The labeling of SPG and AE was performed according to the following steps: 0.1 mg SPG was dissolved in AE labeling buffer 4. After ultrafiltration, 100 μL of SPG with a concentration of 1 mg / mL was obtained. Then, 4 μL of AE solution was added, and the reaction was carried out at 25°C in the dark for 30 min. The reaction was blocked with 4 μL of blocking reagent, and incubated with gentle shaking at 25°C in the dark for 30 min. Finally, AE labeling buffer 5, glycerol, and AE labeling protectant were added to the reaction solution to achieve a final concentration of AE-SPG of 0.5 mg / mL.

[0035] 5. Optimization of the reaction system

[0036] To obtain the optimal reaction conditions for the Bru-CLIA detection method of this invention, five key parameters were optimized. Bovine standard positive serum was serially diluted with FBS to obtain a series of dilutions with concentrations of 500 IU / mL, 125 IU / mL, 31.25 IU / mL, 7.81 IU / mL, and 1.95 IU / mL, which were used to optimize the reaction system. FBS was used as a negative control during the reaction system optimization.

[0037] 5.1. Optimization of LPS-MPs Dilution

[0038] Magnetic microparticles coated with LPS-MPs at concentrations of 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL were reacted with serially diluted bovine standard positive serum and FBS. Each concentration was tested in triplicate, and the average value was used to calculate the P / N ratio.

[0039] 5.2. Optimization of AE-SPG dilution

[0040] AE-SPG at dilutions of 1:5000, 1:10000, 1:15000, and 1:20000 was reacted with serially diluted bovine standard positive serum and FBS. Each dilution was performed in triplicate, and the average value was used to calculate the P / N ratio.

[0041] 5.3. Optimization of serum sample dilution

[0042] Serum samples were diluted 5, 10, 20 and 40 times with reaction diluent. Each dilution was repeated three times. The average value was taken and the P / N ratio was calculated.

[0043] 5.4. Optimization of the reaction dilution solution

[0044] The following reaction dilutions were used to react with serially diluted bovine standard positive serum and FBS: Buffer 1 (0.02M PB, pH 7.4), Buffer 2 (0.02M Tris, pH 7.4), Buffer 3 (0.02M PB + 0.9% NaCl), Buffer 4 (0.02M Tris, pH 7.4 + 0.9% NaCl), Buffer 5 (0.02M PB, pH 7.4 + 0.9% NaCl + 0.1% Tween 20), Buffer 6 (0.02M PB, pH 7.4 + 0.9% NaCl + 0.2% Triton), and Buffer 7 (1% sodium caseinate). Each dilution was tested in triplicate, and the average value was used to calculate the P / N ratio.

[0045] 5.5 Reaction Procedure Optimization

[0046] The following four procedures were used for the reaction: Procedure 1: Add 50 μL LPS-MPs, 5 μL diluted serum sample, and 50 μL reaction diluent to the reaction tube, incubate at 37°C for 10 minutes, wash with TBST, then add 50 μL LAE-SPG, 100 μL pre-activation buffer, and 100 μL activation buffer sequentially, incubate again at 37°C for 10 minutes, and detect relative luminescent units (RLU). Procedure 2: Use 100 μL LPS-MPs, 5 μL diluted serum sample, and 100 μL reaction diluent, with other steps the same as Procedure 1. Procedure 3: Add 50 μL LPS-MPs, 5 μL diluted serum sample, 50 μL reaction diluent, 50 μL LAE-SPG, 100 μL pre-activation buffer, and 100 μL activation buffer to the reaction tube, incubate at 37°C for 10 minutes, and directly detect RLU. Reaction Procedure 4: Add 100 μL LPS-MPs, 5 μL diluted serum sample, 100 μL reaction diluent, 100 μL LAE-SPG, 100 μL pre-activation solution, and 100 μL activation solution to the reaction tube. The other steps are the same as in Procedure 3.

[0047] 6. Qualitative Analysis

[0048] Using Bru-CLIA, 33 sheep serum samples and 23 bovine serum samples with clear backgrounds were tested. The test data were entered into GraphPadPrism version 10.0.0 and ROC curve analysis was performed to calculate cut-off values ​​and generate interactive scatter plots.

[0049] 7. Evaluation of sensitivity, repeatability and specificity

[0050] 7.1 Sensitivity

[0051] First, bovine standard positive serum was diluted to 500 IU / mL using FBS. Then, the diluted bovine serum (500 IU / mL) and sheep standard positive serum (400 IU / mL) were serially diluted from 1:2 to 1:1024. The RLU values ​​of each dilution were simultaneously determined using Bru-CLIA, and the highest serum dilution with an RLU value exceeding the cut-off value was defined as the limit of detection for this method.

[0052] 7.2 Repeatability

[0053] Five sheep serum samples and five bovine serum samples with different RLU values ​​were selected. Each sample was tested in three replicates. The mean, standard deviation (SD), and coefficient of variation (CV%) of the RLU were calculated for each sample.

[0054] 7.3 Specificity

[0055] Bru-CLIA was used to detect positive sera for Escherichia coli O157:H7, Mycobacterium tuberculosis, Vibrio cholerae, Legionella, Salmonella, foot-and-mouth disease virus type O and A, bovine viral diarrhea virus, ovine infectious pleuropneumonia, goatpox virus, and peste des petits ruminants virus, and to assess whether there was cross-reactivity between this method and sera of common bovine and ovine diseases.

[0056] 8. Comparison of compliance rates

[0057] Eighty-one sheep serum samples and 96 bovine serum samples were simultaneously tested using Bru-CLIA and ID-VET ELISA commercial kits, and the concordance rate of the two test results was compared.

[0058] result

[0059] 1. Optimization results of reaction conditions

[0060] 1.1 Optimization of LPS-MPs and AE-SPG concentrations

[0061] The working concentrations of LPS-MPs and AE-SPG are key parameters affecting reaction sensitivity and specificity. Results showed that the P / N ratio reached its maximum value when the concentration of LPS-MPs was 0.1 mg / mL. Figure 1 A); When the dilution ratio of AE-SPG is 1:10000, the P / N ratio reaches its maximum value ( Figure 1 B). Therefore, in subsequent experiments, the concentration of LPS-MPs was selected as 0.1 mg / mL, and the dilution ratio of AE-SPG was 1:10000.

[0062] 1.2 Optimization of serum sample dilution

[0063] The P / N ratio reaches its maximum value when the serum sample is diluted to 1:5. Figure 1 C). Therefore, a serum sample dilution ratio of 1:5 was chosen for subsequent experiments.

[0064] 1.3 Optimization of the reaction diluent

[0065] Seven types of reaction diluents were tested, including Buffer 1 (0.02M PB, pH 7.4), Buffer 2 (0.02M Tris, pH 7.4), Buffer 3 (0.02M PB + 0.9% NaCl), Buffer 4 (0.02M Tris, pH 7.4 + 0.9% NaCl), Buffer 5 (0.02M PB, pH 7.4 + 0.9% NaCl + 0.1% Tween 20), Buffer 6 (0.02M PB, pH 7.4 + 0.9% NaCl + 0.2% Triton), and Buffer 7 (1% Sodium Caseinate). Serum samples were diluted with these diluents. Figure 1As shown in Figure D, serum diluted with 0.02 M PB (pH = 7.4) exhibited the highest P / N ratio compared to serum diluted with other reaction diluents. Therefore, 0.02 M PB (pH = 7.4) was chosen for subsequent experiments.

[0066] 1.4 Optimization of the reaction procedure

[0067] Under the above-mentioned preferred operating conditions, four reaction procedures were compared. For example... Figure 1 As shown in Figure E, reaction program 1 exhibits a higher P / N ratio than other programs. Therefore, reaction program 1 was selected for subsequent experiments.

[0068] 2. Qualitative Analysis and Evaluation Results

[0069] Qualitative analysis was performed on 33 sheep serum samples and 23 bovine serum samples. Results for the sheep serum samples showed that, at a cut-off value of 86075, the diagnostic sensitivity was 100% and the diagnostic specificity was 95%. Figure 2 (A and 2B). For bovine serum sample testing, the results showed that when the cut-off value was 89487, the diagnostic sensitivity was 100% and the diagnostic specificity was 100%. Figure 2 (C and 2D). The areas under the curve (AUC) for detecting Brucella antibodies in sheep and bovine serum were 0.9962 and 1, respectively (P < 0.0001). An AUC value greater than 0.9 indicates good diagnostic performance. These results demonstrate that the established Bru-CLIA method has good accuracy, especially for the detection of Brucella antibodies in bovine serum.

[0070] Sensitivity, specificity, and repeatability results of 3Bru-CLIA

[0071] The established Bru-CLIA assay was used to detect serially diluted serum samples. The results showed that the limits of detection of Bru-CLIA for serially diluted sheep and bovine standard serum samples were 1:512 and 1:256, respectively, which are approximately equivalent to 1 IU / mL and 2 IU / mL (Table 1).

[0072] Table 1. Analytical Sensitivity Tests for Bru-CLIA

[0073]

[0074] a. sheep serum; b. bovine serum.

[0075] The results showed that Bru-CLIA detected no cross-reactivity in positive sera for Escherichia coli O157:H7, Mycobacterium tuberculosis, Vibrio cholerae, Legionella, Salmonella, type O and type A foot-and-mouth disease virus, bovine viral diarrhea virus, goatpox virus, and peste des petits ruminants virus (Table 2), indicating good specificity.

[0076] Table 2. Analytical specificity test for Bru-CLIA

[0077]

[0078]

[0079] The coefficients of variation (CVs) for intra-assay and inter-assay repeatability tests of 10 Brucella sera with different RLU values ​​were 0.9%–4.2% and 0.3%–8.1%, respectively (Table 3). All results were less than 10%, indicating that Bru-CLIA has good repeatability.

[0080] Table 3. Bru-CLIA Analytical Repeatability Tests

[0081]

[0082] a. sheep serum; b. bovine serum.

[0083] 4. Compliance Rate Test Results

[0084] To evaluate the performance of the established Bru-CLIA method in detecting Brucella antibodies in clinical serum, the concordance rates of Bru-CLIA and commercially available kits were compared. As shown in Table 4, the concordance rates of Bru-CLIA with the commercially available ID-VET ELISA kit were 87.65% and 93.75%, respectively, in detecting Brucella antibodies in sheep and bovine serum. Therefore, Bru-CLIA shows promise as a diagnostic method with potential applications in the clinical diagnosis of brucellosis.

[0085] Table 4. Comparison of concordance rates between Bru-CLIA and ELISA

[0086]

[0087] a. sheep serum; b. bovine serum.

[0088] Serological testing plays a crucial role in the diagnosis of brucellosis in humans and animals. Although pathogen isolation and nucleic acid detection technologies are equally clinically significant, in most endemic areas of brucellosis, serological diagnostic methods (such as RBT and ELISA) are preferred due to their ease of implementation in resource-constrained clinical settings. RBT is commonly used for initial screening of brucellosis; it is quick and simple to perform, but its sensitivity is relatively low. ELISA offers both good sensitivity and specificity, but it is expensive and requires highly trained technicians. CLIA combines highly sensitive chemiluminescence assays with a highly specific immune response, and when used with appropriate instruments, it enables fully automated testing, reducing human intervention and making results more reliable, making it suitable for widespread adoption at the grassroots level. Currently, CLIA is widely used in human medical diagnosis and is showing a trend of replacing ELISA.

[0089] This invention employs a chemiluminescence method based on magnetic microparticles. Magnetic beads possess a larger surface area, superparamagnetism, and excellent biocompatibility, enabling automated operation of the detection process, reducing manual intervention, and effectively improving throughput and result consistency. Experimental data show that the sensitivity of Bru-CLIA is approximately twice that of commercially available ID-VET ELISA kits. Detection time, a key indicator for evaluating the efficacy of diagnostic methods, directly determines the practical application efficiency of the detection system. The Bru-CLIA developed in this study uses acridil ester as a signal probe. This substance is a typical flash chemiluminescent label, and its signal acquisition speed is significantly superior to traditional enzyme labels such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). Based on this, Bru-CLIA can complete the entire detection process within 40 minutes, and its simple operation can meet the needs of batch detection of large numbers of samples. Meanwhile, Bru-CLIA showed no cross-reactivity with positive sera of Escherichia coli O157:H7, Mycobacterium tuberculosis, Vibrio cholerae, Legionella, Salmonella, as well as O and A type foot-and-mouth disease virus, bovine viral diarrhea virus, goatpox virus, and peste des petits ruminants virus, confirming that the method has good specificity.

[0090] This invention provides the first evaluation and analysis of the application of Bru-CLIA in the clinical diagnosis of brucellosis. Results showed that the cut-off values ​​for detecting Brucella antibodies in sheep and bovine serum were 86075 and 89487, respectively, with AUCs of 0.9962 and 1, indicating excellent diagnostic accuracy. For sheep and bovine serum samples, the concordance rates between Bru-CLIA and the commercial ID-VET ELISA kit were 87.65% and 93.75%, respectively. Compared to ELISA, Bru-CLIA exhibits higher sensitivity and faster speed in Brucella antibody detection, providing important technical support for epidemiological surveys of brucellosis in livestock, evaluation of immunization efficacy, and quantitative studies of standard sera.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for detecting Brucella antibodies in livestock, characterized in that, include: The apparatus comprises coated magnetic beads, a signal tracer, and a reaction diluent; the coated magnetic beads are magnetic beads coated with Brucella-specific lipopolysaccharide antigen, with a dilution concentration of 0.05–0.15 mg / mL; the signal tracer is acridinium-labeled recombinant streptococcal protein G, with a dilution ratio of 1:8000–12000; and the reaction diluent is phosphate buffer, with a concentration of 0.01–0.03 M and a pH of 7.3–7.

5.

2. The reagent kit according to claim 1, characterized in that, The kit also includes: pre-activation solution and activation solution.

3. The reagent kit according to claim 1, characterized in that, The lipopolysaccharide was extracted from Brucella bovis strain A19 after inactivation through culture.

4. The reagent kit according to claim 1, characterized in that, The method for preparing the coated magnetic beads is as follows: after washing the magnetic beads, resuspend them in binding buffer, add Brucella-specific lipopolysaccharide and catalytic reagent solution, incubate, block, incubate again, and wash.

5. The reagent kit according to claim 1, characterized in that, The signal tracer is prepared as follows: recombinant streptococcal protein G is dissolved in acridinium ester labeling buffer 4, and after ultrafiltration, a recombinant streptococcal protein G solution is obtained; then, acridinium ester solution is added for reaction in the dark, blocked, incubated, and acridinium ester labeling buffer 5, glycerol and acridinium ester labeling protectant are added to the reaction solution.

6. The kit according to any one of claims 1 to 5 is used for detecting Brucella antibodies in the serum of livestock.

7. The application according to claim 6, characterized in that, The method of application is as follows: (1) Mix and incubate the coated magnetic beads, serum sample and reaction dilution solution; (2) After washing the incubated solution system, add the signal tracer, pre-activation solution and activation solution, mix and incubate again, detect and judge the results.

8. The application according to claim 7, characterized in that, The volume ratio of the coated magnetic beads, serum sample, and dilution buffer is 50–100:5:50–100.

9. The application according to claim 7, characterized in that, The incubation temperature in steps (1) and (2) is 36-38°C and the incubation time is 8-12 min.

10. The application according to claim 7, characterized in that, The volume ratio of the signal tracer, pre-excitation solution and excitation solution in step (2) is 8-12:8-20:8-20.

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