Brucella antibody detection kit and its preparation method and application

Abstract

This invention discloses a Brucella lipopolysaccharide monoclonal antibody, the heavy chain variable region sequence of which is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.2. This invention also discloses a kit for detecting Brucella antibodies, the kit comprising an effective amount of monoclonal antibody and Brucella lipopolysaccharide, as well as matching detection reagents. The Brucella antibody detection kit prepared by this invention has good specificity and sensitivity, and is suitable for field applications of Brucella.

Description

Technical Field

[0001] This invention belongs to the field of viral disease diagnostic technology, specifically relating to Brucella antibody detection kits, their preparation methods, and applications. Background Technology

[0002] Brucella is a group of short, Gram-negative bacilli that most easily infect animals such as cattle, sheep, and pigs, causing infectious abortions in females. Humans can be infected through contact with infected animals or by consuming infected animals and their dairy products. The genus Brucella includes six species and 20 biotypes: sheep, cattle, pig, rodent, sheep, and canine Brucella. In China, the most prevalent species are sheep (Br. melitensis), cattle (Br. Bovis), and pig (Br. suis), with ovine brucellosis being the most common.

[0003] Brucellosis is a chronic infectious disease caused by Brucella bacteria, affecting both humans and animals, primarily impacting the reproductive system. In sheep, infection is characterized by abortion in ewes and orchitis in rams. This disease is widespread, infecting not only various livestock but also easily spreading to humans.

[0004] Brucella bacteria primarily infect livestock. Clinical symptoms in livestock are often subtle. However, pregnant animals are highly susceptible to abortion or stillbirth, as their amniotic fluid, placenta, and secretions contain large amounts of Brucella, making them particularly infectious. The bacteria are also present in their fur, urine, feces, and milk. Shedding can last for more than three months. Humans can become infected through contact with livestock, ingestion of contaminated milk and meat, inhalation of contaminated dust, or the bacteria entering the conjunctiva of the eye. The disease typically affects individuals over 30 years of age. After entering the human body through damaged skin and mucous membranes, the digestive tract, or the respiratory tract, the bacteria are first phagocytosed by phagocytes, then enter the lymph nodes, where they can sometimes survive and multiply, forming foci of infection. After approximately 2-3 weeks, they can enter the bloodstream, causing bacteremia. Subsequently, new infectious foci form within the reticuloendothelial system, such as the liver, spleen, and bone marrow. These foci can repeatedly breach cells and enter the bloodstream, causing bacteremia and acute clinical symptoms again. This manifests as a fever period averaging 2-3 weeks, with recurring fevers every 3 days to two weeks, exhibiting a wavy pattern, hence the name "undulating fever." Simultaneously, Brucella bacteria, containing endotoxins and the bacteria themselves, can cause allergic reactions in humans, leading to various allergic lesions. Bone and joint lesions typically occur around six months after infection, although in a few cases, earlier. Brucella osteomyelitis is a localized manifestation of hematogenous Brucella infection in the bones and joints.

[0005] Therefore, Brucella poses a significant threat to human health and is difficult to eradicate. Thus, there is a need to develop a rapid test strip for detecting Brucella antibodies, which would also facilitate self-testing by frontline production workers or other personnel. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, one objective of this invention is to provide a monoclonal antibody against Brucella lipopolysaccharide and a method for preparing the same; another objective of this invention is to provide a Brucella antibody detection kit prepared using Brucella lipopolysaccharide and monoclonal antibody prepared according to this invention.

[0007] Therefore, the present invention discloses a Brucella lipopolysaccharide monoclonal antibody, wherein the monoclonal antibody can specifically bind Brucella lipopolysaccharide; the heavy chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO.1, and the light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO.2.

[0008] On the other hand, the present invention also discloses a kit for detecting Brucella antibodies, the kit comprising an effective amount of the aforementioned monoclonal antibody and Brucella lipopolysaccharide; and a matching detection reagent.

[0009] Preferably, the kit of the present invention is a colloidal gold test strip, the test strip comprising a PVC base plate, on which a sample pad, a latex microsphere pad, a nitrocellulose membrane and absorbent paper are sequentially fixed; the latex microsphere pad of the test strip is latex microsphere labeled with Brucella lipopolysaccharide; the surface of the nitrocellulose membrane of the test strip is marked with a detection line and a control line, wherein the control line is the monoclonal antibody and the detection line is Brucella lipopolysaccharide.

[0010] Preferably, the test strip of the present invention further includes a sample diluent.

[0011] Preferably, the sample diluent of the present invention is a 1×PBS solution.

[0012] Preferably, the test strip of the present invention is suitable for detecting Brucella lipopolysaccharide antibodies in serum and milk.

[0013] Preferably, the serum described in this invention is bovine serum, sheep blood, or pig serum.

[0014] Furthermore, this invention also discloses the application of the aforementioned Brucella lipopolysaccharide monoclonal antibody in the preparation of a Brucella detection kit.

[0015] The monoclonal antibody and Brucella lipopolysaccharide prepared in this invention have good specificity and sensitivity, and are suitable for preparing various Brucella diagnostic reagents, such as colloidal gold, ELISA, chemiluminescence and other detection kits.

[0016] The Brucella antibody detection kit (colloidal gold test strip) provided by this invention is suitable for the detection of Brucella antibodies in serum and milk. It exhibits high specificity, high sensitivity, good stability, and fast detection speed, making it ideal for early screening of Brucella, particularly suitable for on-site and self-infection detection, and also for epidemiological surveys. This kit can be used for the detection of various animal samples, such as cattle, sheep, and pigs. Those skilled in the art should understand that although this application only tested three animal samples, this kit can also be used for the detection of samples from other animals or humans. Attached Figure Description

[0017] Figure 1 A schematic diagram of the test strip assembly, where A: sample pad, B: latex microsphere pad, C: test line, D: control line, and E: absorption pad.

[0018] Figure 2 Diagram illustrating the test strip detection and result interpretation process. Detailed Implementation

[0019] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0020] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0021] Example 1: Preparation of Brucella lipopolysaccharide

[0022] The outer membrane of Brucella bacteria is mainly composed of lipopolysaccharide (LPS). The rough-type Brucella lacks the LPS-O chain structure found in the smooth-type. The O chain is an antigenic determinant of the bacterial cell, consisting of a repeating oligosaccharide structure composed of 3-5 glycosyl groups. The composition and structure of these glycosyl groups vary among different species and even among different groups of the same species, thus determining its species and type specificity. As the most abundant antigenic structure on the surface of Brucella, antibodies produced by the body's immune response to it are the main detection antibodies in various diagnostic methods for brucellosis. Therefore, extracting pure LPS antigen is crucial for detecting Brucella. The Brucella LPS used in this study was provided by a collaborating company (Hangzhou Yimino Biotechnology Co., Ltd.), which will not be elaborated upon here.

[0023] Example 2: Preparation and purification of Brucella lipopolysaccharide monoclonal antibody

[0024] Vaccine preparation and animal immunization: The prepared Brucella lipopolysaccharide was emulsified with an equal mass of Freund's complete adjuvant and injected subcutaneously at multiple sites into 6-8 week old BALB / c mice (100 μg / mouse). Two weeks later, the mixture was emulsified with incomplete Freund's adjuvant and immunized subcutaneously at multiple sites (100 μg / mouse). This process was repeated two weeks later. After the third immunization, antibody titers were measured. A booster immunization (100 μg / mouse) was administered intraperitoneally three days before cell fusion.

[0025] The ELISA titer for mouse serum (indirect ELISA detection) was 1:10. 5 Spleen cells were collected from the mice. The method for collecting spleen cells was as follows: Three days after a booster immunization, BALB / c mice were euthanized by cervical dislocation. After soaking the carcass in 75% alcohol for 5 minutes, the spleen of the BALB / c mice was aseptically collected and placed in a sterile Petri dish containing RPMI 1640 (containing 100U of triple antibody). After washing twice with RPMI 1640, the spleen was transferred to a 200-mesh copper grid. The spleen was then crushed with a sterile agitator, gradually squeezing the spleen cells into the liquid phase. After repeatedly dispersing the cells by pipetting, the cells were filtered through a 200-mesh copper grid and transferred to a 50ml sterile centrifuge tube. The cells were centrifuged at 1000rpm at room temperature for 10 minutes, the supernatant was discarded, and the cells were resuspended in RPMI 1640, washed once, and then stained with trypan blue for counting (viable cell count >90%). The cells were then ready for use.

[0026] The cell fusion method is as follows: Myeloma cells in logarithmic growth phase are placed in a 50ml centrifuge tube and centrifuged at 1000rpm for 10min at room temperature. The supernatant is discarded, and the cells are washed once with RPMI 1640 for counting. Myeloma cells and spleen cells are mixed at a ratio of 1:10 and centrifuged at 1000rpm for 10min at room temperature. The supernatant is aspirated, and the cells are washed once with RPMI 1640. The 50ml centrifuge tube is placed in a 37℃ water bath. 1ml of pre-warmed 50% PEG 1450 solution (37℃) is added within 1min for fusion. After standing for 1min, RPMI 1640 is added gradually to terminate the fusion. The cells are centrifuged at 1000rpm for 10min at room temperature, the supernatant is discarded, and 50ml of complete RPMI 1640 containing 20% ​​newborn calf serum (HAT) is added. The fused cells are then added to 96-well plates, 100μl per well. The culture plates were placed in a 37°C, 5% CO2 incubator and cultured for 15 days. After that, they were replaced with complete RPMI 1640 containing 20% ​​calf serum with HT.

[0027] Monoclonal screening: Hybridoma cells that tested positive by ELISA were cloned using a limiting dilution method. Cell lines with high antibody titers and good morphology were selected and further cloned using the limiting dilution method until monoclonal cells were obtained. These cells were then expanded and preserved. Finally, one relatively good positive hybridoma cell line was obtained, which was designated as 1A2.

[0028] Ascites preparation: 6-8 week old BALB / c mice were intraperitoneally injected with 1 ml of sterile liquid paraffin (0.5 ml per mouse). Seven days later, each mouse was injected with 1 × 10⁻⁶ hybridoma cells. 6 Seven days later, ascites fluid was extracted from the mice and centrifuged at 1000 rpm for 10 minutes at 2–8°C. The supernatant was collected and divided into 5 ml tubes, which were then stored at -20°C for later use.

[0029] Antibody purification: Take 10 ml of ascites fluid, thaw it, centrifuge at 1000 rpm for 10 minutes at 4℃, collect the supernatant, add 40 ml of acetate buffer (0.06 mol / L, pH 4.5), mix thoroughly with magnetic stirring, then add 330 μl of caprylic acid dropwise while stirring at room temperature. Mix for 30 minutes at room temperature, then incubate at 2–8℃ for 2 hours. Centrifuge at 10000 rpm for 30 minutes at 2–8℃, collect the supernatant, record the volume of the supernatant, and slowly add the same volume of saturated ammonium sulfate solution stored at 2–8℃ over 30 minutes on ice. Incubate at 2–8℃ for 16 hours, then centrifuge at 5000 rpm for 30 minutes at 2–8℃, collect the precipitate. Dissolve the precipitate with 5 ml of PBS, then dialyze with 1 L of PBS for 16 hours, changing the dialysate at least 3 times during this period. Filter the dialyzed monoclonal antibody through a 0.22 μm microporous membrane for sterilization, and aliquot into centrifuge tubes, 0.1 ml / tube. The concentration of the monoclonal antibody was detected using a BCA kit, and the result was 2.18 mg / ml.

[0030] Example 3 Systematic Identification of Monoclonal Antibodies

[0031] Antibody specificity test: The monoclonal antibody was tested against bovine viral diarrhea virus, streptococcus, and Escherichia coli to determine its specificity. The results showed that the monoclonal antibody was negative in all tests, indicating that the monoclonal antibody has good specificity.

[0032] Determination of the variable region sequence of monoclonal antibody: The heavy chain variable region and light chain variable region of the prepared monoclonal antibody were determined according to the method of Chinese invention patents (CN 111393525B, CN113354734A). After sequencing, the sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0033] SEQ ID NO.1:

[0034] NEESGGGLSVYPLGSMKLSCVASRDCGCKPWVRQSPEKFIFPPKPKDAEIRLKSNTCV VTHYAESVDVEVHISRDDSKNSVYLQMNNLRPEDTGIYYCTPIYSPFAYWGQ GTLVTVSAAK.

[0035] SEQ ID NO.2:

[0036] DRYTVMTQSHKFMSTVCFLNRVSITDINVKDVSTQNGVYQQKPQDSKKLLIYSASYR YMSSTDRFTGVKSFNRFTTCEATHKVQAEDSMSSTCQQHYSSPERQNGTKKDE RADAAPTVSI.

[0037] Example 4: Detection of Brucella antibodies (latex method)

[0038] 4.1 Preparation of test strips

[0039] 4.1.1 Preparation of nitrocellulose membrane: The nitrocellulose membrane (purchased from Sartorius) was attached to the corresponding position on the PVC substrate (Hangzhou Ruijian). Brucella lipopolysaccharide was diluted to 1 mg / ml with coating buffer (PBS buffer). The scribing position and height of the scribing machine were adjusted to form a T-line, which is the test line, close to the end of the latex microsphere pad. The monoclonal antibody prepared in Example 2 was diluted to 1 mg / ml with coating buffer. The scribing position and height of the scribing machine were adjusted to form a C-line, which is the control line, close to the absorbent pad. The distance between the two lines was 5-8 mm. The membrane was dried in a 37°C oven for 20 hours, then sealed in an aluminum foil bag containing desiccant and stored at room temperature for later use.

[0040] 4.1.2 Preparation of Latex Pads: Brucella lipopolysaccharide was added to latex microspheres at a concentration of 1 mg / ml (purchased from Shanghai Huizhi), and labeled for 2 hours. BSA was added to a final concentration of 1%, and the mixture was blocked for 1 hour. The mixture was centrifuged at 12000 rpm for 30 minutes at 4°C, the supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in 1 ml of preservation solution (0.05 M Tris buffer containing 1% BSA, pH 8.0) and sonicated for 1 minute. The resuspended latex microspheres labeled with Brucella lipopolysaccharide were evenly spread on the treated latex microsphere pads, dried in a 37°C oven for 20 hours, sealed in aluminum foil bags, and stored at room temperature for later use.

[0041] 4.1.3 Sample pad preparation: Immerse the sample pad (purchased from Tongcheng Paper Industry, 300mm×20mm) in the sealing solution (10% BSA solution) for 30 minutes, then dry it in an oven at 37℃ for 20 hours, seal it in an aluminum foil bag, and store it at room temperature for later use.

[0042] 4.1.4 Assembly: such as Figure 1 As shown, the sample pad, latex microsphere pad, and absorbent pad are sequentially attached to the corresponding positions on the PVC base plate to which the nitrocellulose membrane has been attached, so that the latex microsphere pad and absorbent pad are in partial contact with the nitrocellulose membrane, and the sample pad is in partial contact with the latex microsphere pad, thus forming a large plate.

[0043] 4.1.5 Packaging: Cut the large plate into 3mm wide test strips using a strip cutter, attach them to the outer shell, and seal them in an aluminum foil bag. Each bag contains one test strip, one pipette, and one packet of desiccant. Store at room temperature away from light for 18 months.

[0044] 4.1.6 Preparation of sample dilution buffer: 1×PBS buffer, quantitatively dispensed into dilution tubes, 980 μL / tube, and stored at room temperature for later use.

[0045] 4.2 Test strip testing

[0046] 4.2.1 Sample processing:

[0047] Collect fresh whole blood, allow it to stand to separate serum or centrifuge to obtain plasma samples, ensuring the samples are free of turbidity or precipitation. Add 20 μL to a diluent tube, mix well, and set aside.

[0048] If the sample is milk, 20 μL of the sample can be directly added to the diluent tube and mixed well for later use.

[0049] 4.2.2 Operating procedures (e.g.) Figure 2 As shown):

[0050] Take out a test card bag and tear it open, take out the test card, and place it horizontally on the operating platform;

[0051] Add 2-3 drops of diluted sample to sample well “S”;

[0052] Observe the results within 5-10 minutes; results are invalid after 15 minutes.

[0053] 4.2.3 Judgment (e.g.) Figure 2 As shown):

[0054] *Positive (+): The presence of red bands on both the control C line and the test T line indicates the presence of Brucella antibodies in the sample.

[0055] *Negative (-): If no color develops on the T line, it indicates that the sample does not contain Brucella antibodies.

[0056] *Invalid: No quality control C line or white board appears, indicating incorrect operation or a faulty test card. Please test again.

[0057] 4.3 Performance verification of the test strip

[0058] 4.3.1 Specificity test

[0059] Three specific samples (positive serum for bovine viral diarrhea virus, positive serum for streptococcus, and positive serum for Escherichia coli) were tested using three batches of test strips. All three batches of test strips showed negative results, indicating a negative detection rate of 100%, as shown in Table 1.

[0060] Five Brucella antibody-negative serum samples and five Brucella antibody-negative breast milk samples were tested using three batches of test strips. All three batches of test strips showed negative results, indicating a negative detection rate of 100%. See Table 1 for details.

[0061] Table 1 Specificity Detection Results

[0062]

[0063] Note: "+" indicates a positive test result, and "-" indicates a negative test result.

[0064] 4.3.2 Sensitivity Test

[0065] The test results of three batches of test strips on three positive samples (Table 2) show that the limits of detection for the three positive serum samples were 1:800, 1:200, and 1:400, respectively. This indicates that the test strips have good sensitivity.

[0066] Table 2 Sensitivity test results

[0067]

[0068] Note: "+" indicates a positive test result, and "-" indicates a negative test result.

[0069] 4.3.3 Repeatability Evaluation

[0070] All three batches of test strips were tested according to the instructions for strongly positive serum, positive serum, weakly positive serum, and negative serum samples. The results are shown in Table 3. As can be seen from the table, the three batches of test strips prepared in our laboratory showed consistent intra-batch and inter-batch results in detecting Brucella antibody strong positive serum, positive serum, weakly positive serum, and negative serum samples, indicating that the Brucella antibody detection test strips prepared by the laboratory method have good repeatability.

[0071] Table 3 Repeatability test results

[0072]

[0073]

[0074] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A Brucella lipopolysaccharide monoclonal antibody, characterized in that, The monoclonal antibody specifically binds to Brucella lipopolysaccharide; the heavy chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO.1, and the light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO.

2.

2. A kit for detecting Brucella antibodies, characterized in that, The kit includes an effective amount of the monoclonal antibody and Brucella lipopolysaccharide as described in claim 1, as well as matching detection reagents.

3. The reagent kit according to claim 2, characterized in that, The kit is a colloidal gold test strip. The test strip includes a PVC base plate, on which a sample pad, a latex microsphere pad, a nitrocellulose membrane, and absorbent paper are sequentially fixed. The latex microsphere pad of the test strip is latex microsphere-labeled Brucella lipopolysaccharide. The surface of the nitrocellulose membrane of the test strip is marked with a detection line and a control line, wherein the control line is the monoclonal antibody as described in claim 1, and the detection line is Brucella lipopolysaccharide.

4. The reagent kit according to claim 3, characterized in that, The test strip also includes a sample diluent.

5. The reagent kit according to claim 4, characterized in that, The sample diluent is a 1×PBS solution.

6. The reagent kit according to claim 3, characterized in that, The test strip described is suitable for detecting Brucella lipopolysaccharide antibodies in serum and milk.

7. The reagent kit according to claim 6, characterized in that, The serum in question is bovine serum, sheep blood, and pig serum.

8. The use of the Brucella lipopolysaccharide monoclonal antibody as described in claim 1 in the preparation of a Brucella detection kit.

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