A kit for detecting brucellosis and its application

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a kit for detecting brucellosis and its application. The kit includes an enzyme-linked immunosorbent assay (ELISA) plate coated with a capture antibody, HRP-labeled antibody, antigen standards, washing buffer, chromogenic solution, and stop solution. The heavy chain amino acid sequence of the anti-CTSC protein monoclonal antibody CTSC-1 in the capture antibody is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2. The heavy chain amino acid sequence of the anti-CTSC protein monoclonal antibody CTSC-2 in the HRP-labeled antibody is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4. The kit for detecting brucellosis provided by this invention has high specificity, can specifically detect the content of CTSC protein in urine, has high sensitivity, and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a kit for detecting brucellosis and its application. Background Technology

[0002] Brucellosis, commonly known as brucellosis, is a zoonotic infectious disease caused by Brucella bacteria. The pathogen primarily invades the host through the mucous membranes of the digestive tract, respiratory tract, genitourinary tract, and intact skin. The main symptoms of brucellosis include prolonged fever, excessive sweating, joint pain, and hepatosplenomegaly. The acute phase primarily presents with fever, excessive sweating, fatigue, arthritis, and orchitis; secondary symptoms include headache, neuralgia, and lymphadenopathy. In summary, brucellosis is a serious infectious disease that threatens human health and livestock development. Understanding the background technologies related to its pathogen, source of infection, clinical manifestations, diagnostic techniques, treatment, and prevention allows for a better understanding and control of this disease.

[0003] Currently, there is no effective treatment for brucellosis. Therefore, early detection and treatment of brucellosis, as well as large-scale screening for brucellosis, are of great significance. Commonly used methods for detecting brucellosis include the Standard Agglutination Test (SAT), Rose Bengal Plate Test (RBPT), Antiglobulin Test (AHG or Coombs Test), and Complement Fixation Test (CFT). However, due to the cross-referencing of results, cumbersome and time-consuming procedures, and low specificity and sensitivity, these methods are prone to missed diagnoses and are rarely used in practice. Therefore, developing a new detection method that can rapidly, sensitively, specifically, and accurately detect Brucella is crucial for the prevention and treatment of this disease. ELISA is an easy-to-operate, highly specific, and sensitive method with promising applications.

[0004] To achieve the above objectives, the present invention provides a kit for detecting brucellosis and its application. Summary of the Invention

[0005] The first objective of this invention is to provide a kit for detecting brucellosis, the kit comprising an enzyme-linked immunosorbent assay (ELISA) plate coated with a capture antibody and an HRP-labeled antibody working solution; both the capture antibody and the HRP-labeled antibody are monoclonal antibodies against the CTSC protein; the heavy chain amino acid sequence of the anti-CTSC protein monoclonal antibody CTSC-1 in the capture antibody is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of the anti-CTSC protein monoclonal antibody CTSC-2 in the HRP-labeled antibody is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0006] The second objective of this invention is to provide an application of a kit for detecting brucellosis.

[0007] One of the objectives of this invention is achieved through the following technical solution: A kit for detecting brucellosis, the kit comprising an ELISA plate coated with a capture antibody and an HRP-labeled antibody working solution; wherein both the capture antibody and the HRP-labeled antibody are monoclonal antibodies against the CTSC protein; The heavy chain amino acid sequence of the anti-CTSC protein monoclonal antibody CTSC-1 in the capture antibody is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of the anti-CTSC protein monoclonal antibody CTSC-2 in the HRP-labeled antibody is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0008] Furthermore, the kit is an ELISA kit.

[0009] Furthermore, the kit also includes antigen standards, washing solution, colorimetric solution, and stop solution.

[0010] Furthermore, the antigen standard is recombinant CTSC protein; the washing solution is PBST; the chromogenic solution is TMB; and the stop solution is sulfuric acid.

[0011] Furthermore, the concentration of the colorimetric solution is 2 mg / L-4 mg / L; the concentration of the stop solution is 2 M-4 M.

[0012] Furthermore, the detection steps are as follows: (1) Centrifuge the sample to be tested and collect the supernatant; (2) Remove the ELISA plate coated with capture antibody, bring it to room temperature, wash the plate with washing solution and spin dry, add the supernatant of the sample to be tested in step (1), incubate at room temperature and then wash the plate with washing solution. (3) Add the HRP-labeled antibody working solution to the system in step (2), incubate at room temperature for 1-3 hours, and then wash the plate with washing solution; (4) Add the color developing solution to the system after the reaction in step (3), develop the color at room temperature in the dark for 10-30 minutes, and then add the stop solution to terminate the reaction; (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and calculate the results based on the standard curve.

[0013] Furthermore, the sample to be tested is urine.

[0014] The second objective of this invention is achieved by the following technical solution: The above-described kit for detecting brucellosis is used to detect the CTSC protein content in urine.

[0015] Compared with the prior art, the main advantages of the present invention are as follows: The kit for brucellosis detection provided by this invention can detect the CTSC protein content in urine. It has high specificity and sensitivity, and is simple to operate. It can effectively determine the risk of brucellosis in the tested population and has good application prospects. Attached Figure Description

[0016] Figure 1 Standard curve for a kit specifically designed for the detection of CTSC protein; Figure 2 This is a schematic diagram illustrating the usage process of a kit for detecting brucellosis. Figure 3 The ROC curve for detecting CTSC protein in urine using the kit of this invention. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0018] The amino acid sequence of the recombinant CTSC protein used in the embodiments of this invention is as follows: SEQ ID NO.5: FQVGSSGSQRDVNCSVMGPQKVVYLQKLDTAYDDDDLGNSGHFTIIYNQFEIVLNDYKWFAFFKYKEE.

[0019] Example 1 Preparation of anti-CTSC protein monoclonal antibody: (1) Immunization in mice: Recombinant CTSC protein was used as the immunogen and emulsified with an equal volume of complete Freund's adjuvant. Eight healthy female mice were selected and injected subcutaneously at multiple sites in the abdomen. The initial immunization dose was 100 μg / mouse. Three weeks after the first immunization, a second immunization was performed. Recombinant CTSC protein was mixed with an equal volume of incomplete Freund's adjuvant, emulsified, and injected. The immunization dose was 50 μg / mouse. Four weeks after the second immunization, a third immunization was performed. The immunization dose was 50 μg / mouse. After the third injection, high-titer antibodies were ensured to be produced in the mice.

[0020] (2) Hybridoma cell fusion: Mice with the highest antibody titer were sacrificed, and spleens were aseptically removed and crushed in a petri dish to prepare a spleen cell suspension. Spleen cells and myeloma cells SP2 / 0 were mixed at a ratio of 5:1, using 50% polyethylene glycol as a fusion agent, and the fusion time was 2 minutes. Five days after the spleen cells and myeloma cells were fused, the medium was replaced with HAT selective medium for further culture. The supernatant of the hybridoma cell culture wells was detected by indirect ELISA to screen for positive hybridoma cells. The screened positive hybridoma cells were subcloned three times using the limiting dilution method, and the hybridoma cells with the highest sensitivity and specificity were selected for expansion culture.

[0021] (3) Preparation and purification of monoclonal antibodies: 0.5 mL of liquid paraffin was injected into healthy female mice. Seven days later, hybridoma cells obtained from screening were injected into the peritoneum. Ascites fluid was collected after the mice's abdomens swelled. The collected mouse ascites fluid was centrifuged at 3000 rpm for 15 min to remove cellular components. The ascites fluid was filtered through a 0.2 μm filter. The collected ascites fluid was purified by immunochromatography to obtain purified monoclonal antibodies.

[0022] (4) Analysis of monoclonal antibody sequences: The activity of the purified monoclonal antibodies was accurately measured. The two monoclonal antibodies with the highest activity were named CTSC-1 and CTSC-2, and their variable region sequences were determined, as shown in Table 1. The heavy chain amino acid sequence of monoclonal antibody CTSC-1 is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of monoclonal antibody CTSC-2 is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4.

[0023] Example 2 Preparation of a kit for brucellosis detection: (1) Preparation of ELISA plates coated with capture antibody: The monoclonal antibody CTSC-1 prepared in Example 1 was selected as the capture antibody. A 96-well ELISA plate was used as the solid-phase carrier, and the capture antibody was diluted to a certain concentration using carbonate coating buffer. 100 μL of the diluted capture antibody was added to the ELISA plate, sealed with tape, and incubated overnight at 4°C to allow the capture antibody to fully bind to the plate. After incubation, the liquid in the wells was discarded. The ELISA plate was washed three times with PBST to remove unbound capture antibody. 200 μL / well of 1% BSA blocking buffer was added, and the plate was blocked at room temperature for 2 h. The liquid in the wells was discarded, the plate was patted dry, sealed, and stored at 4°C.

[0024] (2) Preparation of HRP-labeled antibody working solution: Weigh 5 mg of HRP dry powder and dissolve it in 1 mL of distilled water to prepare an HRP solution. Use CTSC-2 as the HRP-labeled antibody. Dialyze the monoclonal antibody CTSC-2 in 50 mM carbonate buffer for 24 h, changing the buffer twice during the process. After dialysis, adjust the concentration of the monoclonal antibody CTSC-2 to 2 mg / mL. Mix 0.2 mL of freshly prepared 0.1 M NaIO4 solution with 1 mL of HRP solution and stir at room temperature in the dark for 30 min to oxidize the glycosyl group of HRP to an aldehyde group. Dialyze overnight in 10 mM NaAc buffer at 4℃. Put the aldehyde-modified HRP solution into a dialysis bag and dialyze in sodium acetate buffer at 4℃ overnight. After dialysis, adjust the pH of the dialysate to 9.0 with carbonate buffer. Then, immediately add an equal volume of monoclonal antibody CTSC-2, and gently stir at room temperature in the dark for 2 hours. Next, add 0.1 mL of freshly prepared 4 mg / L NaBH4, mix well, and incubate at 4°C for 2 hours to obtain a stable enzyme-labeled antibody. Place the above sample into 0.15 M pH 7.4 PBS and dialyze overnight at 4°C to remove unbound antibody and other impurities. Centrifuge the dialysate to remove the precipitate; the supernatant is the HRP-labeled antibody working solution.

[0025] (3) Standard curve determination of the kit: Remove the ELISA plate coated with capture antibody, allow it to return to room temperature, wash the plate three times with PBST washing buffer and agitate dry. Add 100 μL of recombinant CTSC protein antigen at different dilutions (10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL), incubate at room temperature for 2 h, wash the plate three times with PBST washing buffer and agitate dry. Add 100 μL of HRP-labeled antibody working solution to the reaction wells, incubate at room temperature for 2 h, wash the plate three times with PBST washing buffer and agitate dry. Add 100 μL of 2 mg / L TMB chromogenic reagent to the reaction wells, incubate at room temperature in the dark for 30 min, and add 100 μL of 2M sulfuric acid to terminate the reaction. A dual-wavelength assay was performed using an ELISA reader to measure the OD values ​​at a reference wavelength of 450 nm and 630 nm. The OD value at 450 nm was then subtracted from the OD value at 630 nm. A standard curve was plotted with the standard concentration on the x-axis (ng / mL) and OD 450 - OD 630 on the y-axis. The standard curve was then plotted based on the results, as follows: Figure 1 As shown. The detection range of the kit for brucellosis detection in this invention is 20-100 ng / mL, and the standard curve is y=0.0495x+0.2856, R0. 2 =0.9979.

[0026] (4) Preparation of ELISA kit: The enzyme-labeled plate coated with capture antibody prepared in step (1), the working solution of HRP-labeled antibody prepared in step (2), the antigen standard CTSC protein, the washing solution PBST, the colorimetric solution TMB, and the stop solution sulfuric acid are packaged separately to form a kit for brucellosis detection according to the present invention.

[0027] Experimental Example 1 Urine sample testing: Urine samples were collected from 15 healthy individuals (numbered 1-15) and brucellosis patients (numbered 16-30) using sterile tubes. After centrifugation at 6000 rpm for 20 min, the supernatant was collected as the test sample and tested using the kit prepared in Example 2.

[0028] Twenty minutes before the experiment, remove the ELISA plate coated with CTSC-1 antibody, allow it to reach room temperature, wash it three times with PBST buffer, and agitate dry. Add 100 μL of the sample to be tested, incubate at room temperature for 2 hours, wash the plate three times with PBST buffer, and agitate dry. Add 100 μL of HRP-labeled antibody working solution to the reaction wells, incubate at room temperature for 2 hours, wash the plate three times with PBST buffer, and agitate dry. Add 100 μL of 2 mg / L TMB chromogenic solution to the reaction wells, incubate at room temperature in the dark for 30 minutes, and stop the reaction by adding 100 μL of 2 M sulfuric acid. Figure 2 The diagram shows the specific detection process of this kit. A microplate reader was used for dual-wavelength detection, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm. The OD value at 450 nm was subtracted from the OD value at 630 nm, and the CTSC protein content was calculated using a standard curve. The results are shown in Table 2.

[0029] The results are shown in Table 2, which presents the test results of urine samples from healthy individuals and patients with brucellosis. Table 2 shows that the CTSC protein content in the urine of all 15 brucellosis patients exceeded 20 ng / mL, resulting in a positive result. In contrast, the urine of healthy volunteers contained trace amounts of CTSC, below the detection limit of the kit, resulting in a negative result. This demonstrates that the kit of the present invention can use urine as a sample to detect brucellosis, meeting the needs of basic research and clinical diagnosis.

[0030] Experimental Example 2 ROC curves were used to evaluate the diagnostic value of the kit for brucellosis. Another 100 urine samples were collected for independent validation, including 50 urine samples from healthy individuals as the control group and 50 urine samples from patients with brucellosis as the observation group. The CTSC protein content in all samples was measured according to the procedures outlined in Experiment Example 1. ROC curves were plotted based on the results, and AUC values ​​were calculated to evaluate the diagnostic value of CTSC protein in brucellosis. The results are as follows: Figure 3 As shown.

[0031] The results are as follows Figure 3 As can be seen, the AUC value of the kit of this invention for detecting urinary CTSC protein in the diagnosis of brucellosis is 0.927. An AUC between 0.7 and 0.9 indicates that the model has good classification ability. This shows that CTSC protein can specifically distinguish between brucellosis patients and healthy individuals. By detecting the level of CTSC protein in urine, the purpose of screening for brucellosis can be achieved, demonstrating good application prospects.

[0032] In conclusion, CTSC protein can serve as a urinary marker for diagnosing brucellosis. By detecting the level of CTSC protein in urine, the purpose of screening for brucellosis can be achieved. It has the advantages of high sensitivity and high accuracy, and can provide an important reference for the early diagnosis of brucellosis.

[0033] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A kit for Brucellosis detection, characterized in that, The kit includes an ELISA plate coated with capture antibody and an HRP-labeled antibody working solution; both the capture antibody and the HRP-labeled antibody are monoclonal antibodies against the CTSC protein. The heavy chain amino acid sequence of the anti-CTSC protein monoclonal antibody CTSC-1 in the capture antibody is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2; the heavy chain amino acid sequence of the anti-CTSC protein monoclonal antibody CTSC-2 in the HRP-labeled antibody is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.

4.

2. A kit for detection of brucellosis according to claim 1, characterized in that, The kit is an ELISA kit.

3. A kit for detection of Brucellosis as claimed in claim 1 wherein, The kit also includes antigen standards, washing solution, colorimetric solution, and stop solution.

4. A kit for detection of brucellosis according to claim 3, characterized in that, The antigen standard is recombinant CTSC protein; the washing solution is PBST; the chromogenic solution is TMB; and the stop solution is sulfuric acid.

5. A kit for detection of Brucellosis as claimed in claim 4 wherein, The concentration of the colorimetric reagent is 2 mg / L-4 mg / L; the concentration of the stop solution is 2 M-4 M.

6. The kit for detection of brucellosis according to claim 1, characterized in that, The detection steps are as follows: (1) Centrifuge the sample to be tested and collect the supernatant; (2) Remove the ELISA plate coated with capture antibody, bring it to room temperature, wash the plate with washing solution and spin dry, add the supernatant of the sample to be tested in step (1), incubate at room temperature and then wash the plate with washing solution. (3) Add the HRP-labeled antibody working solution to the system in step (2), incubate at room temperature for 1-3 hours, and then wash the plate with washing solution; (4) Add the color developing solution to the system after the reaction in step (3), develop the color at room temperature in the dark for 10-30 minutes, and then add the stop solution to terminate the reaction; (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm, and calculate the results based on the standard curve.

7. A kit for detection of brucellosis according to claim 6, characterized in that, The sample to be tested was urine.

8. Use of a kit for the detection of brucellosis according to any one of claims 1 to 7, characterized in that, Used to detect the level of CTSC protein in urine.

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