Test strip for identifying and detecting Brucella bovine / sheep infection antibody and vaccine immune antibody based on colloidal gold method

Abstract

The invention relates to the technical field of veterinary biological products, in particular to a test strip for identifying and detecting a bovine / sheep brucella infection antibody and a vaccine immune antibody based on a colloidal gold method. The invention provides a test strip for identifying and detecting a brucella infection antibody and a vaccine (BA0711 strain) immune antibody based on a colloidal gold method. The NC membrane of the test strip is coated with a C line of protein G (Protein G), a T1 line of lipopolysaccharide and a T2 line of cold shock protein A. And a gold pad of the test strip is coated with a gold nanoflower-labeled anti-bovine or goat IgG secondary antibody. The gold nanoflower is used as a carrier, detection is convenient and rapid, result interpretation can be completed without the help of related instruments and equipment, and the detection sensitivity is higher. According to the technical scheme, the technical problems of non-ideal detection accuracy, complicated operation and the like of a method for identifying and detecting the bovine / sheep Brucella infection antibody and the vaccine immune antibody in the prior art can be solved, and the method is suitable for clinical popularization and application.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and specifically relates to a test strip for differentiating and detecting antibodies against bovine / sheep Brucella infection and vaccine-induced antibodies based on the colloidal gold method. Background Art

[0002] Brucellosis is a zoonotic infectious disease caused by Brucella, which is mainly transmitted to humans through contact with infected animals or their products. This disease causes significant economic losses to the livestock industry and seriously threatens human health. Therefore, it is of great significance to timely and accurately detect whether animals are infected with Brucella or have developed immunity through vaccination for the prevention and control of this disease.

[0003] Traditional Brucella detection methods include bacterial culture, serological detection (tube agglutination, rose bengal plate agglutination, indirect hemagglutination, ELISA), etc. However, these methods have disadvantages such as complex operation, long time consumption, and low sensitivity (high false positive rate). The ELISA method is cumbersome to operate, time-consuming, and requires specific instrument equipment, which is not conducive to rapid detection in farms or pastoral areas. In recent years, colloidal gold technology has been widely used in the biomedical field due to its advantages such as simplicity, rapidity, and strong specificity. However, existing colloidal gold test strips for detection often have difficulty in distinguishing antibodies produced by natural infection from those induced by vaccination due to the lack of corresponding labeled antigens, which limits their application in clinical diagnosis and animal quarantine.

[0004] Therefore, there is an urgent need for a differential immunoassay method with high sensitivity, point-of-care testing, convenient operation, and wide applicability. Summary of the Invention

[0005] The purpose of the present invention is to provide a test strip for differentiating and detecting antibodies against bovine / sheep Brucella infection and vaccine-induced antibodies based on the colloidal gold method, so as to solve the technical problems in the prior art that the methods for differentiating and detecting antibodies against bovine / sheep Brucella infection and vaccine (strain BA0711) - induced antibodies have unsatisfactory detection accuracy, cumbersome operation, high requirements for equipment, and cannot achieve rapid detection. In this technical solution, the antibodies induced by vaccination specifically refer to the antibodies induced by vaccination with the vaccine prepared from strain BA0711. According to the concept of an ideal vaccine for animal Brucella, a stable gene - deleted strain (strain BA0711) was obtained through a virulence (with both expression regulation functions) gene deletion technology. This strain has been experimentally proven to have good immunogenicity, safety, and stable genotype, and has obtained the "New Veterinary Drug Certificate of Class III for Live Brucella Vaccine (strain BA0711)": (2023) New Veterinary Drug Certificate No. 50. However, there is no corresponding diagnostic method available to determine whether the antibodies produced by cattle and sheep after immunization with this vaccine are vaccine - induced antibodies or infection - induced antibodies.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A test strip for differentiating and detecting antibodies against bovine / sheep Brucella infection and vaccine immune antibodies based on the colloidal gold method, comprising a gold pad and a nitrocellulose membrane arranged in sequence along the sample flow direction. The gold pad is distributed with a gold nanoflower-labeled anti-IgG secondary antibody for binding to the Fc end of IgG-class antibodies; on the nitrocellulose membrane, a T1 line, a T2 line, and a C line are arranged in sequence along the sample flow direction; wherein, O-chain lipopolysaccharide is fixed on the T1 line; cold shock protein A of Brucella cell surface antigen is fixed on the T2 line; and cell surface protein G derived from group G streptococcus is fixed on the C line.

[0008] Furthermore, the O-chain lipopolysaccharide includes at least one of lipopolysaccharides from smooth-type Brucella weak strains of bovine, ovine, or swine species;

[0009] Preferably, the Brucella weak strain includes at least one of Brucella abortus weak strain A19, Brucella melitensis strain M5, and Brucella suis strain S2;

[0010] Preferably, the content of LPS on the T1 line is 0.4 - 0.8 μg / cm, and further preferably, the content of LPS on the T1 line is 0.5 μg / cm;

[0011] Preferably, the O-chain lipopolysaccharide is obtained by the following method: taking the inactivated bovine or ovine Brucella liquid after fermentation, centrifuging to collect the bacterial cells; treating the bacterial cells with phenol, discarding the lower phenolic phase after centrifugation, adding cold methanol to the upper liquid for incubation after filtration, and repeatedly resuspending the obtained precipitate with water, centrifuging to collect the supernatant and mixing; then adding trichloroacetic acid to the collected and mixed supernatant, centrifuging and taking the supernatant; and finally obtaining O-chain lipopolysaccharide with a purity > 90% after dialysis.

[0012] Furthermore, the amino acid sequence of the cold shock protein A is as shown in SEQ ID NO.2;

[0013] Preferably, the dosage of cold shock protein A on the T2 line is 0.4 - 0.8 μg / cm, and further preferably, the content of cold shock protein A on the T2 line is 0.5 μg / cm.

[0014] Furthermore, the gold nanoflower-labeled anti-IgG secondary antibody is formed by a gold nanoflower-labeled secondary antibody against bovine or ovine IgG; the dosage ratio of the gold nanoflower to the secondary antibody against IgG is 5:0.150 - 0.250;

[0015] Preferably, the gold nanoflower binds to the secondary antibody against bovine or ovine anti-IgG through ionic and hydrophobic interactions;

[0016] Preferably, the gold nanorods are reacted with anti-bovine or anti-sheep anti-IgG secondary antibody in a borate buffer solution containing bovine serum albumin and polyethylene glycol, blocked with bovine serum albumin, filtered and centrifuged to obtain the gold nanorod-labeled anti-IgG secondary antibody.

[0017] Further, the gold nanorods are prepared by the following method: chloroauric acid and hydroxylamine hydrochloride are heated to boiling under the condition of pH 9.0 - 10.0, then sodium citrate is added, and the reaction continues. After the reaction ends and cools down, the gold nanorods are obtained after washing with water;

[0018] Preferably, a 0.01% - 0.02% chloroauric acid solution and a 1 - 2 μM hydroxylamine hydrochloride solution are mixed at a volume ratio of 100:0.3 - 100:0.2 and heated to boiling; then 10 - 12 ml of a 0.1% - 0.15% sodium citrate solution is added, and the reaction continues for 10 - 15 minutes before ending the reaction;

[0019] Preferably, the particle size of the gold nanorods is 25 - 30 nm.

[0020] Further, the dosage of protein G on the C line is 1 - 1.5 μg / cm.

[0021] Further, a test strip for differentiating and detecting antibodies against Brucella bovis / ovis infection and vaccine immune antibodies based on the colloidal gold method includes a sample pad, a gold pad, a nitrocellulose membrane, and a water absorption pad arranged in sequence along the sample flow direction; the sample pad, the gold pad, the nitrocellulose membrane, and the water absorption pad are all fixed on a polyvinyl chloride bottom plate; there are overlaps between the sample pad and the gold pad, between the gold pad and the nitrocellulose membrane, and between the nitrocellulose membrane and the water absorption pad;

[0022] Preferably, the test strip is cut and then loaded into a test strip cartridge to form a test strip card; the test strip card is encapsulated in an aluminum foil bag.

[0023] Further, it is used for detecting Brucella antibodies in the body fluids of cattle or sheep, and the body fluids include at least one of blood, milk, saliva, urine, and semen;

[0024] After dropping the body fluid of cattle or sheep onto the sample pad, when the T1 line, the T2 line, and the C line all show color simultaneously, it is judged as positive for antibodies against Brucella infection;

[0025] When the T1 line and the C line show color simultaneously while the T2 line does not show color, it is judged as positive for immune antibodies of the live Brucella vaccine strain BA0711;

[0026] When the T1 line and the T2 line do not show color and the C line shows color, it is judged as negative for Brucella antibodies.

[0027] The present technical solution also provides a method for preparing a test strip for differentiating and detecting antibodies against bovine / sheep Brucella infection and vaccine immune antibodies based on the colloidal gold method, comprising the following steps carried out in sequence:

[0028] S1 Raw material preparation

[0029] Preparing gold nanoflower-labeled anti-IgG secondary antibody: adding gold nanoflowers to a borate buffer containing bovine serum albumin and polyethylene glycol, mixing with anti-bovine or anti-sheep anti-IgG secondary antibody, and obtaining the gold nanoflower-labeled anti-IgG secondary antibody after incubation;

[0030] Preparing O-chain lipopolysaccharide: taking the inactivated bovine or sheep Brucella liquid after fermentation, centrifuging to collect the bacterial cells; treating the bacterial cells with phenol, discarding the lower phenolic phase after centrifugation, adding cold methanol to the upper liquid for incubation after filtration, and resuspending the obtained precipitate with water multiple times, centrifuging to collect the supernatant and mixing; then adding trichloroacetic acid to the collected and mixed supernatant, centrifuging and taking the supernatant; and obtaining O-chain lipopolysaccharide with a purity > 90% after dialysis;

[0031] Preparing cold shock protein: integrating the nucleotide sequence shown in SEQ ID NO.1 into an empty expression vector and transferring it into a receptor bacterium to obtain an engineered bacterium; after inducing the expression of the engineered bacterium and purifying the protein, obtaining cold shock protein A;

[0032] S2 Preparation of test strip components

[0033] Preparation of the gold pad: after the glass fiber membrane is blocked, spraying the gold nanoflower-labeled anti-IgG secondary antibody into it to obtain the gold pad;

[0034] Preparation of the T1 line, T2 line, and C line on the nitrocellulose membrane: fixing O-chain lipopolysaccharide on the T1 line, fixing cold shock protein A on the T2 line, and fixing protein G on the cell surface on the C line;

[0035] S3 Assembly of the test strip

[0036] Sequentially arranging a sample pad, a gold pad, a nitrocellulose membrane, and a water absorption pad in the sample flow direction.

[0037] The present technical solution also provides an application of a discrimination system for preparing antibodies against bovine / sheep Brucella infection and vaccine immune antibodies according to the above test strip. When the test sample meets and contacts the gold nanoflower-labeled anti-IgG secondary antibody in the gold pad, the IgG-class antibodies in the test sample specifically bind to the gold nanoflower-labeled anti-IgG secondary antibody; subsequently, the test sample continues to move on the test strip and passes through the T1 line, T2 line, and C line in sequence;

[0038] If there are antibodies produced by bovine / sheep Brucella infection in the sample to be tested, the antibodies produced by bovine / sheep Brucella infection will bind to the O-chain lipopolysaccharide immobilized on the T1 line, and will also bind to the cold shock protein A on the T2 line, and form a stable binding complex with the cell surface protein G on the C line; then color reactions will occur on the T1 line, T2 line and C line;

[0039] If the sample to be tested contains antibodies produced by the live Brucella vaccine strain BA0711, the antibodies produced by the live Brucella vaccine strain BA0711 bind to the O-chain lipopolysaccharide on the T1 line, and bind to the cell surface protein G on the C line; the live Brucella vaccine strain BA0711 lacks cold shock protein A and cannot cause the immunized animals to produce corresponding antibodies, and the antibodies produced by the live Brucella vaccine strain BA0711 cannot bind to the cold shock protein A on the T2 line; then the T1 and C lines show color, and the T2 line does not show color;

[0040] If the sample to be tested does not contain Brucella-related antibodies, the IgG antibodies in the sample to be tested bind to the cell surface protein G on the C line; then the C line shows color, but the T1 and T2 lines do not show color.

[0041] This technical solution also provides an application of gold nanoflowers in the preparation of immunochromatographic test strips. The gold nanoflowers are used to label the anti-IgG secondary antibody to form a gold nanoflower-labeled anti-IgG secondary antibody. The gold nanoflower-labeled anti-IgG secondary antibody is embedded in the gold pad of the test strip. Using the above-mentioned label can more sensitively detect the antibodies produced by immunization with the BA0711 vaccine.

[0042] The technical principle and beneficial effects of this technical solution are as follows:

[0043] The present invention provides a test strip for colloidal gold method-based differential detection of Brucella infection antibodies and vaccine (strain BA0711) immune antibodies in animal (cattle, sheep) blood samples. The test strip includes a polyvinyl chloride bottom plate (PVC bottom plate), a sample pad, a gold pad, a nitrocellulose membrane (NC membrane) and a water absorption pad. The sample pad, the gold pad, the NC membrane and the water absorption pad are sequentially bonded to the PVC bottom plate in the sample flow direction. The NC membrane is coated with a quality control line (C line) and detection lines (T1 and T2). The quality control line is coated with bovine (or ovine) protein A. The detection line T1 is coated with lipopolysaccharide (LPS), and the detection line T2 is coated with cold shock protein (cspA). The gold pad is coated with a gold nanoflower-labeled anti-bovine or anti-sheep IgG secondary antibody. The present invention uses gold nanoflowers as a carrier, which is convenient and fast for detection, and the result can be judged without the aid of relevant instrument equipment, and the detection sensitivity is higher. Applying this method to immunoassay can solve the problems of cumbersome and time-consuming conventional enzyme-linked immunosorbent assay (ELISA) differential detection, and has the advantages of low cost, high sensitivity and short detection time, and is suitable for clinical promotion and application. Brief Description of the Drawings

[0044] Figure 1 It is the electron microscope image of the gold nanorose in Method 1 of Example 1.

[0045] Figure 2 It is the electron microscope image of the gold nanorose in Method 2 of Example 1.

[0046] Figure 3 It is the ultraviolet absorption peak diagram before and after the gold nanorose (prepared by Method 2) in Example 1 is labeled with IgG.

[0047] Figure 4 It is the SDS-PAGE diagram of the cspA protein in Example 3.

[0048] Figure 5 It is the schematic structural diagram of a test strip for differentiating and detecting antibodies against bovine / sheep Brucella infection and vaccine immune antibodies based on the colloidal gold method in Example 7.

[0049] Figure 6 It is the result determination diagram for detecting various samples in Example 8 (A: Positive for Brucella antibody; B: Negative for Brucella antibody; C: Detection fails; D: Positive for antibody against live Brucella vaccine (strain BA0711)). Detailed Description of the Embodiments

[0050] The present invention will be further described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can all be obtained from commercial channels.

[0051] Example 1: Preparation of Gold Nanorose-Labeled Anti-IgG Secondary Antibody

[0052] (1) Preparation of Gold Nanoroses

[0053] Method 1 (two-step method):

[0054] First step: Prepare a gold nanoseed solution. Take 50 ml of 10 mM chloroauric acid solution and add it to a 200 ml round-bottom flask. Stir evenly at 1500 rpm on a magnetic stirrer and heat to boiling. Quickly add 5 ml of 38.8 mM sodium citrate solution, continue heating and stirring vigorously for 20 min, then remove the heat source, and continue stirring for 30 min until cooled to room temperature to obtain the gold nanoseed solution.

[0055] Step 2: Preparation of gold nanoflowers: 50 μl of the gold nanoseed solution, 50 μl of AgNO3 solution (1 mM), 20 μl of hydroxylamine hydrochloride solution (0.04 M) and 250 μl of hydroquinone (50 mM) were added to 10 ml of 0.5 mM chloroauric acid solution (containing 5 μl of 1 M HCl), stirred evenly, and fully reacted on a shaker at 100 rpm for 10 min to obtain gold nanoflowers, which were then stored at 4 °C for future use. The electron microscopy morphology is shown in Figure 2. Figure 1 Electron microscopy showed that it was an irregular polygon with a particle size of about 30 nm, which was used for subsequent experiments.

[0056] Method 2 (one-step method):

[0057] First, add 100 ml of 0.01 wt% HAuCl4 (chloroauric acid solution) and 300 μl of 2 μM hydroxylamine hydrochloride solution into a 250 ml glass reactor (the actual operation can be as follows: 0.01%-0.02% chloroauric acid solution and 1-2 μM hydroxylamine hydrochloride solution are mixed in a volume ratio of 100:0.3-100:0.2), adjust the pH value to 10.0 (optional range 9.0-10.0) with 0.1 mol / L sodium hydroxide solution, stir at 1500 rpm on a magnetic stirrer, heat to boiling, and quickly add 12 ml of 0.1% sodium citrate solution (the actual operation can be as follows: 0.01%-0.02% chloroauric acid solution and 0.1%-0.15% sodium citrate solution The volume ratio is: 100:10-12), continue stirring and heating for 10-15 minutes, and cool to room temperature after the reaction is completed to obtain gold nanoflowers. After centrifugation (speed 8000r / min), wash with ultrapure water 2-3 times, disperse in ultrapure water and store for later use. The particle size of the gold nanoflower is 25-30nm. The obtained gold nanoflower is stored at 4°C for later use. The ultraviolet absorption graph is as shown in Figure 3 The electron microscopy morphology is shown in Figure 2 Its ultraviolet absorption peak is 520nm, and electron microscopy shows that it is thorn-shaped, with uniform particles and a particle size of about 30nm (the particle size of the gold nanoflower prepared according to this scheme is 25-30nm), which can be used for subsequent experiments.

[0058] (2) Gold nanoflower labeled anti-IgG secondary antibody

[0059] Take anti-bovine IgG secondary antibody as an example: take 1 ml of commercial anti-bovine IgG secondary antibody and add it to a pre-treated dialysis bag with a molecular weight cutoff of 50kD, dialyze it in 20 ml of normal saline for 12 hours, and continue to dialyze it in 20 ml of boric acid buffer (0.2M, pH9.0) for 12 hours to remove more salt in the secondary antibody solution.

[0060] Take 5 ml of the gold nanoflowers prepared by the above Method 1 and Method 2 (concentration: 1 mg / ml) respectively, and add them to 5 ml of 200 mM (pH 9.0) borate buffer (containing 2% BSA and 5% PEG 20000). Adjust the pH of the solution to 9.0 with an appropriate amount of 25 mM K2CO3. Add 150 - 250 μg of anti-bovine IgG secondary antibody (the dosage ratio of gold nanoflowers to anti-IgG secondary antibody is 5:0.150 - 0.250), and react on a shaker at 100 - 120 rpm for 30 - 60 min at 30°C. Block with 5% BSA at 30°C for 30 min. After blocking, filter the gold-labeled complex solution through a 0.22 μm filter membrane, centrifuge at 4°C and 1500 rpm for 10 min, carefully collect the supernatant, centrifuge at 4°C and 10000 rpm (8000 - 10000 rpm can be selected) for 30 min, collect the precipitate and dilute the gold-labeled secondary antibody (gold nanoflower-labeled anti-IgG secondary antibody, gold flower-labeled anti-IgG secondary antibody, AuNF-Ab2) with an appropriate volume of diluent (20 mM Na2B4O7 (pH 9.0 buffer), 1% BSA, 2% sucrose, 0.1% Triton-100, 0.02% preservative), and measure its ultraviolet absorption OD 570nm Control it to be about 2.0 and store it at 4°C for later use. The coupling of anti-sheep IgG secondary antibody and gold nanoflowers adopts the same method. For samples from different animal sources, the corresponding species' anti-IgG secondary antibodies need to be used. These anti-IgG secondary antibodies are all commercially available antibodies and can be directly purchased as existing products. The technical key point of the gold nanoflower-labeled anti-IgG secondary antibody obtained in this step lies in the production of special gold nanoflowers. Figure 3 Shows the ultraviolet absorption peak diagrams before and after the coupling of the gold nanoflowers prepared by Method 2 and rabbit anti-sheep IgG secondary antibody.

[0061] Example 2: Preparation of lipopolysaccharide (LPS, O-chain lipopolysaccharide)

[0062] This technical solution uses the hot phenol-water method for the extraction of LPS (World Organization For Animal Health Terrestrial Manual [M]. Chapter 3.1.4). Inactivate 50 ml of freshly cultured Brucella abortus strain A19 (CVCC70202) by heating in a water bath at 65 °C for 20 min. Centrifuge the inactivated and qualified Brucella broth at 8000 g for 20 minutes and collect the precipitate. Add sterile water according to the wet weight of the cells at a ratio of 1:3 (W / W), mix well and heat to 66 °C, then add an equal volume of phenol solution. After continuous stirring for 15 minutes, place it at 4 °C and centrifuge at 10000 g for 15 minutes. Aspirate and discard the lower brownish-red phenol phase, and filter the upper layer of liquid with a commercial filter paper. Add 3 volumes of cold methanol (2 - 8 °C) containing 1% saturated sodium acetate, incubate at 4 °C for 2 hours, centrifuge at 10000 g for 10 minutes, and discard the supernatant. Resuspend the obtained precipitate with distilled water at 1 / 2 volume of the original aqueous phase, centrifuge at 10000 g for 10 minutes at 4 °C. Collect the supernatant and store it at 2 - 8 °C. Repeat the above operations for the precipitate part to collect the supernatant and mix it with the above supernatant. Add a final concentration of 5% trichloroacetic acid, stir at room temperature for 15 minutes, centrifuge at 10000 g for 15 minutes, and discard the precipitate. Dialyze the supernatant with distilled water (for 12 hours), change the liquid 2 - 3 times during this period (the volume of each liquid change is at least 4000 ml), and collect the content after dialysis to obtain purified LPS. Take 2 ml of Escherichia coli LPS standard solutions with different concentrations, add 1 ml of 80% phenol solution and 5 ml of concentrated sulfuric acid respectively, mix well and let it stand at room temperature for 30 minutes, then measure the OD 490 nm and plot the standard curve. Treat the purified LPS antigen in the same way, measure its OD 490 nm, calculate the content and purity of the antigen in the sample, and the purity should not be less than 90%.

[0063] In addition to LPS from Brucella abortus strain A19 (CVCC70202), LPS (O-chain lipopolysaccharide) from Brucella melitensis strain M5 (CVCC18) or Brucella suis strain S2 (CVCC70502) can also be used for the preparation of test strips, and the preparation method of LPS is the same. Extract and prepare LPS from strains of different species sources for the preparation and detection of the T1 line of this test strip, and there is no significant difference in the detection effect on clinical samples (see Example 11 for reference).

[0064] Example 3: Preparation of cspA (cold shock protein A of Brucella cell surface antigen)

[0065] According to the cold shock protein gene sequence of Brucella (strain BA0711) published by NCBI (GenBank: >NC_003317.1:c519153-518938), 6 His tags were added before its stop codon TGA, and this sequence was inserted into the pET-32a vector for expression.

[0066] The specific deoxynucleotide sequence is (SEQ ID NO.1):

[0067] ATGGCACAGACCGGACAGGTCAAATTCTTCAACACCGAAAAAGGTTTCGGTTTCATCAAGCCCGATGATGGCGGCGCGGACATCTTCGTGCATATTTCTGCAGTTCAGGCTTCTGGCCTGCCAGGCCTTGCTGACAATCAGAAGGTTTCCTATGAAACGGAACCAGATCGTCGTGGAAAAGGCCCCAAGGCCGTGAACATCACCATTACCGGCCATCATCATCATCATCATTGA.

[0068] After its construction was completed, it was induced by IPTG and purified to obtain a protein with a molecular weight of about 9 kDa. As Figure 4 shown, Lane M: Marker (1.7 - 40 kDa); 1: Before induction; 2: After induction; 3 - 4: Flow-through solutions with imidazole elution concentrations of 25 mM and 50 mM respectively; 5 - 7: Flow-through solutions with imidazole elution concentrations of 100 mM, 150 mM, and 200 mM respectively. Among them, the samples in Lanes 5 - 7 are the cold shock protein cspA, and its amino acid sequence is (SEQ ID NO.2):

[0069] MKRRRQGKGTDPMAQTGQVKFFNTEKGFGFIKPDDGGADIFVHISAVQASGLPGLADNQKVSYETEPDRRGKGPKAVNITITG.

[0070] The obtained cspA protein was placed at -20 °C for standby. The purity of the purified cspA was not less than 80%.

[0071] Example 4: Pretreatment of the gold pad and sample pad

[0072] Cut glass fiber membranes with specifications of 300×7 mm (gold pad) and 300×15 mm (sample pad), soak them in the blocking solution (10 mM PBS (pH 7.4), 3.5% sucrose, 2% BSA, 1.5% tween - 20, 0.02% preservative) for 30 min, take them out and dry them at 56 °C in an oven for standby.

[0073] Example 5: Preparation of Gold Pad

[0074] Place the gold pad in Example 4 on a gold spraying instrument and set the following parameters: X: 5.00 mm; Y: 8.00 mm, 2 pumps at 5.00 μl / cm; Z: 9.80 mm; length: 120 mm, speed: 100 mm / s. Use the gold flower label - anti - IgG secondary antibody (bovine or sheep) prepared in Example 1 and perform intermittent spraying (interval duration 2 - 3 min) onto the gold pad according to the above parameters (spraying volume 5.00 μl / cm, spraying speed mm / s), and air - dry it naturally in a dryer.

[0075] Example 6: Preparation of T1 Line, T2 Line, and C Line

[0076] Set the gold spraying instrument parameters as follows: X: 5.00 mm, 1 pump at 0.80 μl / cm; Y: 45.00 mm; Z: 5.00 mm, 3 pumps at 0.80 μl / cm; length: 100 mm, speed 50 mm / s. Before scribing, first attach a 200×700 mm NC membrane (nitrocellulose membrane) to a PVC bottom plate (polyvinyl chloride bottom plate). Dilute the LPS prepared in Example 2 with a diluent (10 mM PBS (pH 7.4), 0.5% sucrose, 0.1% tween - 20, 0.02% preservative) to 0.1 mg / ml, and then intermittently spray (interval duration 2 - 3 min) the diluted LPS onto the standardized T1 line at 0.4 - 0.6 μg / cm. Dilute the cspA prepared in Example 3 with a diluent (10 mM PBS (pH 7.4), 0.5% sucrose, 0.1% tween - 20, 0.02% preservative) to 0.1 mg / ml, and then intermittently spray (interval duration 2 - 3 min) the diluted cspA onto the standardized T2 line at 5 μl / cm (the dosage of cspA can be 0.4 - 0.6 μg / cm); Dilute the commercially available protein G from group G streptococcus (protein G, which has the ability to specifically bind to the Fc segment of antibodies) with a diluent (10 mM PBS (pH 7.4), 0.5% sucrose, 0.1% tween - 20, 0.02% preservative) to 0.2 mg / ml, and then use it to standardize the C line (the spraying method is the same as before, at 5 μl / cm; the dosage of protein G can be 0.8 - 1.2 μg / cm). The intervals between the T1 line, T2 line, and C line are all 1.0 cm. After drying their respective marked lines, place them in a 5% BSA solution for blocking, block at 4°C for 12 - 14 h, after the blocking is completed, rinse once in PBS at pH 7.0 (about 5 - 10 s), and place it in a dryer to air - dry naturally. After the marking lines are completed and air - dried naturally, add a desiccant and seal it in a self - sealing bag.

[0077] Example 7: Assembly of Test Strip

[0078] First, stick the NC membrane in the middle of the PVC bottom plate. Then, paste a gold pad at the front end of the NC membrane (the front end in the order of material flow), and a glass fiber absorbent pad at the back end. There is an overlap of 2 mm between the NC membrane and the gold pad, and also an overlap of 2 mm between the NC membrane and the absorbent pad. Preferably, the NC membrane is placed below the overlapping part of the gold pad or the absorbent pad. At the end of the gold pad away from the NC membrane, a sample pad is pasted, and there is an overlap of 2 mm between the sample pad and the gold pad. Preferably, the gold pad is placed below the overlapping part of the sample pad. Finally, a layer of filter paper is added to the end of the sample pad for loading samples. The assembled semi-finished product is put into a cutting machine for cutting, and the cutting parameter is set to 4.00 mm, that is, the width of the test strip is 4.00 mm. The cut test strip is loaded into a pre-prepared test strip card and sealed with a pressing machine. The prepared test strip card is packaged in an aluminum foil bag, sealed with a sealing machine, and marked with the date and batch number, and stored at room temperature for later use. For the structural schematic diagram of the test strip, see Figure 5 。

[0079] Example 8: Detection of clinical samples - I

[0080] The gold nanoflowers prepared by the above Method 1 and Method 2 were respectively used for clinical effect detection (the test strips were prepared by the aforementioned optimal method, and the test strips were used for sample detection to explore the influence of different gold nanoflowers on the detection effect of the test strips; when preparing the gold nanoflower-labeled anti-IgG secondary antibody, the dosage ratio of the gold nanoflower to the anti-IgG secondary antibody was 5:0.150 - 0.250. In this example, in order to better compare the effect differences of the gold nanoflowers prepared by different preparation processes, the ratio of 5:0.200 (that is, 1 mg of gold nanoflower was mixed with 0.2 mg of anti-sheep IgG secondary antibody) was used for both test strips; for the preparation methods of the two test strips, except for the different sources of the gold nanoflowers, other operation steps were kept consistent). The clinical samples were bovine or ovine body fluids, including but not limited to blood, milk, saliva, urine, semen, etc. Taking ovine blood as an example: 10 ovine blood samples with known background information (see Table 1 for details) were taken and detected by colloidal gold method, cELISA, RBT, and CFT respectively. To evaluate the sensitivity of the gold nanoflowers prepared by Method 1 and Method 2 and to evaluate the sensitivity of the colloidal gold method compared with other detection methods, the experimental results are shown in Table 2.

[0081] Table 1: Clinical sample situation

[0082] Animal number BA0711 vaccine antibody Brucella antibody Sheep 1# Positive Positive Sheep 2# Positive Positive Sheep 3# Positive Positive Sheep 4# Positive Positive Sheep 5# Positive Positive Sheep 6# Negative Positive Sheep 7# Negative Positive Sheep 8# Negative Positive Sheep 9# Negative Positive Sheep 10# Negative Positive

[0083] Table 2: Detection results for the samples in Table 1

[0084]

[0085]

[0086] Note: cELISA: competitive ELISA; RBT: Rose Bengal plate agglutination; CFT: complement fixation test.

[0087] Through the above detections, it was found that: the detection results of the gold nanoflowers prepared by Method 2 were consistent with the actual information of the samples, and the detection sensitivity was significantly better than that of the gold nanoflowers prepared by Method 1. Specifically, the detection result of the sheep blood sample No. 5 using the test strip prepared by Method 1 was negative for the BA0711 vaccine antibody, which did not conform to the actual sample situation. However, the detection result of the sheep blood sample No. 5 using the test strip prepared by Method 2 was correct. It may be due to the difference in the surface structure of the gold nanoflowers, resulting in different binding effects between the nanoflowers and the anti-IgG secondary antibody, and then the color development effect of the T2 line, ultimately leading to the difference in sensitivity in the actual detection.

[0088] In most of the existing methods for preparing gold nanoflowers, chloroauric acid and sodium citrate are first heated to boiling for reaction, and the gold nanoparticles obtained by the reduction method are used as seeds. Then, reducing substances such as ethanolamine and hydroxylamine hydrochloride are added to reduce chloroauric acid to prepare gold nanoflower nanoparticles (for example, Method 1 mentioned above, Chinese Patent CN105903948A, a gold nanoflower nanoparticle and its preparation method). However, for the special immunochromatographic test strip of this scheme (aiming to distinguish between bovine / sheep Brucella infection antibodies and vaccine immune antibodies), there is no research in the existing technology on which kind of gold nanoflowers are more applicable. The inventor found through a large number of studies that the test strip prepared by using the one-step method of this scheme to prepare gold nanoflowers has a more sensitive ability to detect bovine / sheep Brucella infection antibodies, which was also unexpected before the inventor's experiment. There are many technical links involved in the immunochromatographic test strip (substances sprayed in structures such as the T1 line, T2 line, C line, and gold pad, and the pretreatment of each component), and there is no report in the existing technology on which one or which ones are the key points affecting the effect of the special test strip of this scheme. The inventor found through a large number of studies that the conjugation of gold nanoflowers with anti-IgG secondary antibody is a key point with a significant impact, and explored a preparation method of gold nanoflowers with very excellent effects for the test strip of this scheme, obtaining unexpected technical effects.

[0089] The colloidal gold method of this patent, the conventional competitive ELISA method, Rose Bengal plate agglutination method, and complement fixation test method in the existing technology were used for comparative detection of 10 clinical samples. The results showed that the colloidal gold method of this patent had higher sensitivity than competitive ELISA, Rose Bengal plate agglutination, and complement fixation test.

[0090] Example 9: Detection of Clinical Samples II

[0091] The paper strip formed by the gold nanoflowers prepared by Method 2 is used for detecting clinical samples. The clinical samples are bovine or ovine body fluids, including but not limited to blood, milk, saliva, urine, semen, etc. Taking bovine blood as an example: 2 - 3 drops of the sample to be tested are added to the sample pad, and under the action of chromatography, it flows towards the absorbent pad (passing through the gold pad, T1 line, T2 line, and C line in sequence).

[0092] When the blood sample passes through the gold pad, the Fc end of the IgG - type antibody (Ab1) contained in the sample will bind to the Fab end of the gold - flower - labeled anti - bovine / anti - ovine IgG secondary antibody (AuNF - Ab2), forming an AuNF - Ab2@Ab1 complex. This complex continues to flow towards the absorbent pad under the action of chromatography.

[0093] When it passes through the T1 line (labeled with LPS), if the sample to be tested contains Brucella antibody IgG (BAb1), then its Fab end will bind to the LPS on the T1 line through antigen - antibody reaction, forming an AuNF - Ab2@BAb1@LPS complex, and then the T1 line will show color. At this time, it is judged as positive for Brucella antibody. If the sample to be tested does not contain Brucella antibody, when the sample passes through the T1 line, it cannot bind to the LPS on the T1 line and cannot make the T1 line show color. At this time, it is judged as negative for Brucella antibody.

[0094] When it passes through the T2 line (labeled with cold shock protein cspA), if the sample to be tested contains Brucella infection antibody (BAb1), then the Fab end of BAb1 will bind to the cspA on the T2 line through antigen - antibody reaction, forming an AuNF - Ab2@BAb1@cspA complex, and then the T2 line will show color, and it is judged as positive for Brucella infection antibody. If the sample to be tested only contains the antibody of Brucella live vaccine (strain BA0711), at this time the T1 line shows color, and because of the deletion of the cspA gene of this vaccine, the antibody corresponding to the cspA antigen cannot be produced, and then it cannot bind to the cold shock protein cspA on the T2 line, and the T2 line cannot show color. That is, when the T1 line shows color and the T2 line does not show color, it is judged as positive for the antibody of Brucella live vaccine (strain BA0711).

[0095] Whether the above - mentioned clinical samples contain Brucella infection antibody (BAb1) or not, they all contain IgG - type antibodies. This antibody can bind to Protein G on the C line to form an AuNF - Ab2@Ab1@ProteinG complex, and then the C line will show color. When the sample flows towards the absorbent pad and passes through the C line under the action of chromatography, the C line will show color, and it is judged that the detection is established; if the C line does not show color, it is judged that the detection is not established.

[0096] That is, the judgment criteria are as follows:

[0097] In the case of C line showing color: When both T1 line and T2 line show color simultaneously, it is judged as positive for Brucella infection antibody;

[0098] When T1 line shows color while T2 line does not show color, it is judged as positive for live Brucella vaccine (strain BA0711);

[0099] When T1 does not show color, it is judged as negative for Brucella antibody.

[0100] If the C line does not show color, it indicates that the test strip is not available and the test result is not valid. For the result judgment diagrams of various samples, see Figure 6 。

[0101] Example 10: Detection of clinical samples III

[0102] Using the test strip described in this patent (preparing gold nanoflowers by method two), 5 bovine Escherichia coli O157 positive sera, 5 bovine Salmonella typhimurium positive sera, 5 bovine Streptococcus positive sera, 5 bovine Brucella antibody negative sera, 5 A19 immunized bovine sera (Brucella antibody positive), and 5 BA0711 vaccine immunized bovine sera (BA0711 vaccine antibody positive) were detected respectively. The sample information is shown in Table 3 in detail, and the test results are shown in Table 4 in detail.

[0103] Table 3: Sample information

[0104]

[0105]

[0106] Table 4: Test results for the samples in Table 3

[0107]

[0108]

[0109] From the above results, it can be seen that the test strips prepared by this patent have a detection rate of 100% for both 5 Brucella antibody negative sera and 5 BA0711 vaccine immunized bovine sera (BA0711 vaccine antibody positive). There is no cross - reaction with Escherichia coli O157 positive serum, Salmonella typhimurium positive serum, and Streptococcus positive serum.

[0110] Example 11: Detection of clinical samples IV

[0111] The lipopolysaccharides (O-chain lipopolysaccharides, LPS) of Brucella abortus strain A19 (CVCC70202), Brucella melitensis strain M5 (CVCC18), and Brucella suis strain S2 (CVCC70502) were extracted respectively according to the above methods. After extraction, the gold nanoflowers prepared by Method 2 were used to obtain test strips according to the optimal preparation method of the aforementioned test strips. The test strips and the aforementioned detection methods were used to detect 30 sheep blood and bovine blood samples with known clinical sample background information (see Table 5 for details). The differences in the detection of bovine / sheep source samples by LPS from Brucella abortus strain A19 (CVCC70202), Brucella melitensis strain M5 (CVCC18), and Brucella suis strain S2 (CVCC70502) were evaluated, and the test results are shown in Table 6.

[0112] Table 5: Clinical sample situation

[0113]

[0114]

[0115] Table 6: Detection results for the samples in Table 5 (Strain A19, Strain M5, and Strain S2 respectively represent the test strips prepared using LPS from these cell strains)

[0116]

[0117]

[0118] From the above results, it can be seen that for the source of LPS labeled on the T1 line of the test strip prepared in this patent, there are no obvious differences in the detection of 30 sheep blood and bovine blood samples with known clinical sample background information by Brucella abortus strain A19 (CVCC70202), Brucella melitensis strain M5 (CVCC18), and Brucella suis strain S2 (CVCC70502), and the detection results are all consistent with the actual information of the clinical samples.

[0119] The above are only examples of the present invention. Common knowledge such as specific structures and characteristics known in the art are not described in detail herein. It should be noted that for those skilled in the art, without departing from the structure of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to explain the content of the claims.

Claims

1. A test strip for identifying and detecting bovine / sheep Brucella infection antibodies and vaccine immune antibodies based on the colloidal gold method, comprising a gold pad and a nitrocellulose membrane arranged in sequence according to the sample flow direction, characterized in that: The gold pad is provided with an anti-IgG secondary antibody labeled with gold nanoflowers for binding to the Fc end of IgG antibodies; the nitrocellulose membrane is provided with T1 line, T2 line, and C line in sequence according to the sample flow direction; Among them, O-chain lipopolysaccharide is fixed on the T1 line; the cold shock protein A of the Brucella surface antigen is fixed on the T2 line; and the cell surface protein G from group G streptococcus is fixed on the C line.

2. A test strip for detecting bovine / sheep Brucella infection antibodies and vaccine immune antibodies based on colloidal gold method according to claim 1, characterized in that: The O-chain lipopolysaccharide includes at least one of the lipopolysaccharides of the smooth-type attenuated strain of Brucella from bovine, ovine or porcine species; Preferably, the attenuated strain of Brucella includes at least one of the attenuated strain A19 of Brucella bovis, the strain M5 of Brucella melitensis and the strain S2 of Brucella suis; Preferably, the content of LPS on the T1 line is 0.4-0.8 μg / cm, and further preferably, the content of LPS on the T1 line is 0.5 μg / cm; Preferably, the O-chain lipopolysaccharide is obtained by the following method: taking the fermented Brucella bovis or Brucella melitensis liquid for inactivation treatment, and collecting the bacteria by centrifugation; treating the bacteria with phenol, discarding the lower phenol phase after centrifugation, filtering the upper liquid, adding cold methanol for incubation, resuspending the obtained precipitate with water for multiple times, centrifuging and collecting the supernatant and mixing; Then, trichloroacetic acid is added to the collected mixed supernatant for treatment, and then the supernatant is obtained by centrifugation; and after dialysis, O-chain lipopolysaccharide with a purity of more than 90% is obtained.

3. A test strip for detecting bovine / sheep Brucella infection antibodies and vaccine immune antibodies based on colloidal gold method according to claim 1, characterized in that: The amino acid sequence of the cold shock protein A is shown in SEQ ID NO.2; Preferably, the amount of cold shock protein A on the T2 line is 0.4-0.8 μg / cm, and more preferably, the amount of cold shock protein A on the T2 line is 0.5 μg / cm.

4. A test strip for detecting bovine / sheep Brucella infection antibodies and vaccine immune antibodies based on colloidal gold method according to claim 1, characterized in that: The gold nanoflower-labeled anti-IgG secondary antibody is formed by gold nanoflower-labeled anti-cow or sheep IgG secondary antibody; the usage ratio of gold nanoflower and anti-IgG secondary antibody is 5:0.150-0.250; Preferably, the gold nanoflowers bind to the anti-bovine or anti-goat IgG secondary antibody through ionic and hydrophobic interactions; Preferably, the gold nanoflowers are reacted with an anti-bovine or goat anti-IgG secondary antibody in a borate buffer containing bovine serum albumin and polyethylene glycol, blocked with bovine serum albumin, filtered and centrifuged to obtain the gold nanoflower-labeled anti-IgG secondary antibody.

5. A test strip for detecting bovine / sheep Brucella infection antibodies and vaccine immune antibodies based on colloidal gold method according to claim 4, characterized in that: The gold nanoflowers are prepared by the following method: chloroauric acid and hydroxylamine hydrochloride are heated to boiling at a pH of 9.0-10.0, and then sodium citrate is added to continue the reaction, and after the reaction is completed, the gold nanoflowers are cooled and washed with water to obtain the gold nanoflowers; Preferably, 0.01%-0.02% chloroauric acid solution and 1-2 μM hydroxylamine hydrochloride solution are mixed in a volume ratio of 100:0.3-100:0.2 and heated to boiling; then 10-12 ml of 0.1%-0.15% sodium citrate solution is added, and the reaction is continued for 10-15 minutes before the reaction is terminated; Preferably, the particle size of the gold nanoflowers is 25-30 nm.

6. A test strip for identifying and detecting bovine / sheep Brucella infection antibodies and vaccine immune antibodies based on the colloidal gold method according to any one of claims 1-5, characterized in that: The amount of cell surface protein G on line C was 1-1.5 μg / cm.

7. A test strip for identifying and detecting bovine / sheep Brucella infection antibodies and vaccine immune antibodies based on colloidal gold method according to claim 6, characterized in that: The invention comprises a sample pad, a gold pad, a nitrocellulose membrane and a water-absorbing pad which are arranged in sequence according to the flow direction of the sample; the sample pad, the gold pad, the nitrocellulose membrane and the water-absorbing pad are all fixed on a polyvinyl chloride bottom plate; there is overlap between the sample pad and the gold pad, between the gold pad and the nitrocellulose membrane, and between the nitrocellulose membrane and the water-absorbing pad; Preferably, the test strips are cut and placed in a test paper card box to form a test paper card; the test paper card is packaged in an aluminum foil bag.

8. A test strip for identifying and detecting bovine / sheep Brucella infection antibodies and vaccine immune antibodies based on the colloidal gold method according to claim 6, characterized in that: It is used for testing Brucella antibodies in bodily fluids of cattle or sheep, wherein the bodily fluids include at least one of blood, milk, saliva, urine and semen; After dropping the bovine or sheep body fluid onto the sample pad, when the T1 line, T2 line, and C line are colored at the same time, it is judged as positive for Brucellosis infection antibodies; When the T1 line and the C line are colored at the same time but the T2 line is not colored, it is judged that the immune antibody of the Brucella live vaccine BA0711 strain is positive; When the T1 and T2 lines did not show color and the C line showed color, it was judged as Brucella antibody negative.

9. A method for preparing a test strip for detecting bovine / sheep Brucella infection antibodies and vaccine immune antibodies based on colloidal gold method according to claim 6, characterized in that: The method includes the following steps in sequence: S1 Raw material preparation Preparation of gold nanoflower-labeled anti-IgG secondary antibody: adding gold nanoflowers to a borate buffer containing bovine serum albumin and polyethylene glycol, and mixing with anti-bovine or goat anti-IgG secondary antibody, and obtaining gold nanoflower-labeled anti-IgG secondary antibody after incubation; Preparation of O-chain lipopolysaccharide: inactivate the fermented Brucella bovis or Brucella melitensis liquid, collect the bacteria by centrifugation; treat the bacteria with phenol, discard the lower phenol phase after centrifugation, filter the upper liquid, add cold methanol for incubation, resuspend the obtained precipitate with water several times, collect the supernatant by centrifugation and mix; Then, trichloroacetic acid is added to the collected mixed supernatant for treatment, and then the supernatant is obtained by centrifugation; and then O-chain lipopolysaccharide with a purity of more than 90% is obtained after dialysis; Preparation of cold shock protein: integrating the nucleotide sequence shown in SEQ ID NO.1 into an empty expression vector, and transferring it into a recipient bacterium to obtain an engineered bacterium; After inducing the expression of engineered bacteria and purifying the protein, cold shock protein A was obtained; Preparation of S2 test strip elements Preparation of gold pad: After the glass cellulose membrane is blocked, the anti-IgG secondary antibody labeled with gold nanoflowers is sprayed into it to obtain the gold pad; Preparation of T1 line, T2 line and C line on nitrocellulose membrane: O-chain lipopolysaccharide is fixed on T1 line, cold shock protein A is fixed on T2 line, and cell surface protein G is fixed on C line; Assembly of S3 test strips The sample pad, gold pad, nitrocellulose membrane and absorbent pad were arranged in sequence according to the sample flow direction.

10. The use of the test strip according to claim 6 in preparing a system for identifying bovine / sheep Brucella infection antibodies and vaccine immune antibodies, characterized in that: When the sample to be tested meets and contacts the anti-IgG secondary antibody labeled with the gold nanoflowers in the gold pad, the IgG antibodies in the sample to be tested specifically bind to the anti-IgG secondary antibody labeled with the gold nanoflowers; then, the sample to be tested continues to move on the test strip, passing through the T1 line, T2 line and C line in sequence; If there are antibodies produced by bovine / sheep Brucella infection in the sample to be tested, the antibodies produced by bovine / sheep Brucella infection will react with the O-chain lipopolysaccharide fixed on the T1 line, and will also bind to the cold shock protein A on the T2 line, and form a stable binding complex with the cell surface protein G on the C line; then the T1 line, T2 line and C line will all show color reaction; If the sample to be tested contains antibodies produced by Brucellosis Live Vaccine BA0711 strain, the antibodies produced by Brucellosis Live Vaccine BA0711 strain bind to the O-chain lipopolysaccharide of the T1 line and to the cell surface protein G of the C line; Brucellosis Live Vaccine BA0711 strain lacks cold shock protein A and cannot make the immunized animal produce corresponding antibodies, and the antibodies produced by Brucellosis Live Vaccine BA0711 strain cannot bind to the cold shock protein A of the T2 line; then the T1 and C lines will develop color, and the T2 line will not develop color; If the sample to be tested does not contain Brucella-related antibodies, the IgG antibodies in the sample to be tested will bind to the cell surface protein G of line C; then line C will develop color, but lines T1 and T2 will not.

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