Brucella antibody detection test paper and application

Abstract

The invention belongs to the technical field of animal epidemic disease immunodetection, and particularly relates to Brucella antibody detection test paper and application thereof.The Brucella antibody detection test paper comprises a bottom plate and a chromatography membrane arranged on the bottom plate, a sample treatment layer is arranged at one end of the chromatography membrane, and a quality control line and at least one detection line are further arranged on the chromatography membrane; the sample treatment layer comprises latex microspheres marked by chicken IgY, latex microspheres marked by Protein G protein or / and latex microspheres marked by Fc alpha / mu R receptor, and the detection line comprises brucella LPS; or, the sample treatment layer comprises latex microspheres marked by brucella LPS and latex microspheres marked by chicken IgY, and the detection line comprises Protein G protein and an Fc alpha / mu R receptor; according to the invention, the sensitivity, the specificity, the repeatability and the accuracy of early diagnosis or infection period distinguishing are obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of animal disease immunity detection, and specifically relates to a Brucella antibody detection test paper and application. Background Art

[0002] Brucella ( Brucella Brucella is a Gram-negative bacterium that does not form spores. Some virulent strains can form capsules. The main virulence factors are lipopolysaccharide (LPS), Type four secretion system (T4SS), outer membrane proteins (OMP), two-component compression system (TCS), etc. During bacterial infection, LPS helps Brucella escape the anti-infection killing mechanism of host cells. In addition, since LPS is the main component of the outer membrane of Brucella, it is widely selected for Brucella detection technology. Brucella can be divided into 6 species and 20 biotypes, including Brucella melitensis (3 biotypes, types 1, 2, and 3), Brucella bovis (9 biotypes, types 1 to 9), Brucella suis (5 biotypes, types 1 to 5), Brucella epididymis of sheep (1 biotype), Brucella sarcoma (1 biotype) and Brucella canis (1 biotype). Brucella melitensis and Brucella bovis are both smooth strains. Brucella can infect humans, livestock and a variety of wild animals. Humans will suffer from fever, sweating, joint and nerve pain, hepatosplenomegaly and other symptoms, while cows will experience miscarriage and infertility. It is necessary to cooperate with rapid and accurate disease diagnostic tools to scientifically carry out the prevention and control of brucellosis.

[0003] The current methods for detecting Brucella are divided into pathogenic diagnosis technology, serological diagnosis technology and molecular biology technology, mainly including bacterial culture isolation and identification, agglutination test, enzyme-linked immunosorbent assay, complement fixation test, immunochromatography, polymerase chain reaction, real-time fluorescence quantitative PCR, etc. Different methodologies have their own advantages and disadvantages and applicable scenarios. The WS269-2019 industry standard document for the diagnosis of brucellosis points out that the colloidal gold immunochromatography test (GICA) can be used for the initial screening of Brucella. This methodology is suitable for on-site rapid detection at the grassroots level of aquaculture. Combined with the infection cycle of Brucella and the characteristics of positive serological antibody reactions, the early diagnostic sensitivity of the test strip card can be improved by increasing the binding of the test strip card to IgM.

[0004] The current inventions of Brucella antibody detection chromatographic test strips are mostly prepared by sandwich method, indirect method and competitive method. Such inventions mostly use extracted LPS as capture protein. LPS is the main component of the cell wall of most Gram-negative bacteria. The LPS on the surface of Brucella is atypical LPS and is the main component of its outer membrane. It is not only related to the survival and proliferation of Brucella itself, but also the most important surface antigen of Brucella. It can be used as an important marker in Brucella serological diagnosis methods.

[0005] Existing research on Brucella IgM antibody diagnostic reagents mostly uses anti-IgM antibodies as immune reagent secondary antibodies to participate in the reaction and bind to Brucella IgM antibodies. For example, Liu Hanxuan et al. used mouse anti-human IgM as a coating reagent in the establishment and preliminary clinical application of Brucella serum IgM and IgG antibody colloidal gold immunochromatography detection methods. However, the anti-IgM secondary antibody prepared by immunization and purification of serum may bind to both the Fc fragment and Fab fragment of IgM, and there is species specificity of IgM as an immunogen, which affects the test results. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a Brucella antibody detection test paper and application. The present invention significantly improves the sensitivity of early diagnosis or identification of infection periods, and has good specificity, repeatability and accuracy.

[0007] The embodiment of the present invention provides a Brucella antibody detection test paper, comprising a bottom plate, a chromatographic membrane arranged on the bottom plate, a sample processing layer arranged at one end of the chromatographic membrane, and a quality control line and at least one detection line arranged on the chromatographic membrane; The sample processing layer includes latex microspheres labeled with chicken IgY, latex microspheres labeled with Protein G protein, or / and latex microspheres labeled with Fcα / μR receptor, and the detection line includes Brucella LPS; or, The sample processing layer comprises latex microspheres marked with Brucella LPS and latex microspheres marked with chicken IgY, and the detection line comprises Protein G protein and Fcα / μR receptor.

[0008] In one embodiment, the sample processing layer includes latex microspheres labeled with chicken IgY, latex microspheres labeled with Protein G protein, and latex microspheres labeled with Fcα / μR receptor.

[0009] In one embodiment, the sample processing layer includes chicken IgY-labeled latex microspheres, Protein G protein-labeled latex microspheres, or / and Fcα / μR receptor-labeled latex microspheres, and when the detection line includes Brucella LPS, the Brucella LPS is modified with biotin, and the Protein G protein-labeled latex microspheres and Fcα / μR receptor-labeled latex microspheres are modified with streptavidin; The sample processing layer comprises latex microspheres labeled with Brucella LPS and latex microspheres labeled with chicken IgY, and when the detection line comprises Protein G protein and Fcα / μR receptor, the Fcα / μR receptor is modified with biotin, and the latex microspheres labeled with Brucella LPS and latex microspheres labeled with chicken IgY are modified with streptavidin.

[0010] In one embodiment, there are two detection lines, the sample processing layer includes latex microspheres labeled with Brucella LPS and latex microspheres labeled with chicken IgY, one detection line includes Protein G protein, and the other detection line (ie, detection line T2) includes Fcα / μR receptor.

[0011] In one embodiment, the sample processing layer includes latex microspheres labeled with Brucella LPS and latex microspheres labeled with chicken IgY, the detection line includes Protein G protein, and another detection line (i.e., detection line T2) includes Fcα / μR receptors, the Fcα / μR receptors are modified with biotin, and the latex microspheres labeled with Brucella LPS and the latex microspheres labeled with chicken IgY are modified with streptavidin.

[0012] In one embodiment, the detection line also includes metal ions, and the metal ions are copper ions and / or calcium ions.

[0013] In one embodiment, the metal ions are copper ions and calcium ions, and the molar ratio of copper ions to calcium ions is 1:1.

[0014] In one embodiment, the chromatographic membrane is a nitrocellulose membrane, the sample processing layer includes a conjugate pad, a blood filter membrane and a sample pad which are sequentially overlapped on the chromatographic membrane, the conjugate pad is a glass cellulose membrane I, and the sample pad is a glass cellulose membrane II; an absorbent filter paper is provided at the other end of the chromatographic membrane.

[0015] The chicken IgY-labeled latex microspheres, Protein G-labeled latex microspheres and / or Fcα / μR receptor-labeled latex microspheres of the sample processing layer are located on the conjugate pad, and the Brucella LPS-labeled latex microspheres of the sample processing layer are located on the conjugate pad.

[0016] In one embodiment, the quality control line includes sheep anti-chicken IgY antibody.

[0017] In one embodiment, the sample treatment layer (specifically the conjugate pad) includes a treatment solution, and the treatment solution is 1% BSA, 5% sucrose, 0.1% PEG20000, 50mM Tris, 0.1% Tween-20, and 0.1% PC300.

[0018] The embodiment of the present invention provides an application of the Brucella antibody detection test paper, wherein the Brucella antibody detection test paper is used to detect the infection period of Brucella.

[0019] The beneficial effects of the present invention are: Fcα / μR receptors can specifically bind to the Fc segments of IgA and IgM, exposing more Fab antigen binding regions, which helps to improve the diagnostic specificity of IgM and the binding of IgM variable regions to Brucella LPS. Based on the above characteristics of Fcα / μR receptors, the present invention creatively applies recombinant Fcα / μR receptors to diagnostic reagents to improve the sensitivity and specificity of diagnostic reagents for IgM antibody binding, thereby enhancing the sensitivity of diagnostic reagents for early infection samples.

[0020] In one of the embodiments 1, the present invention uses an indirect method to develop a test strip, and the microspheres are labeled with Protein G protein and Fcα / μR receptors, respectively, to capture IgG and IgM / IgA in the infected sample or immune sample, respectively, and the formed complex is then combined with LPS on the T line, and the result indicates a positive infection, and can be used for testing raw milk samples, expanding the scope of application of the test strip card. In addition, in order to improve the diagnostic sensitivity of the reagent for early infection samples, in addition to using the common biotin-streptavidin system, the present invention creatively encapsulates metal ions into the detection line of the diagnostic reagent, especially using a combination of copper ions and calcium ions, through the interaction between them and LPS, the stability of LPS and the specific affinity between Protein G-IgG, Fcα / μR-IgM, and Fcα / μR-IgA are improved.

[0021] In this embodiment 1, the present invention adopts an indirect method to develop a Brucella antibody detection test strip, the conjugate pad is sprayed with red latex microspheres coupled with Brucella extracted Protein G protein, Fcα / μR receptor and chicken IgY antibody, and a detection line and a quality control line are set on the NC membrane, the detection line is coated with LPS, and the quality control line is coated with sheep anti-chicken IgY antibody. When Brucella LPS IgG, IgM or IgA antibody exists in the test sample, it will be first combined with Protein G protein and Fcα / μR receptor labeled microspheres on the conjugate pad to form a complex, and then lateral chromatography is performed to the detection line on the NC membrane, and the coating protein is secondly combined to form a red microsphere precipitation visible to the naked eye.

[0022] The present invention has the following four advantages: (1) Fcα / μR receptors can bind to IgM and IgA with high and medium affinity respectively, and they are used as capture proteins in the present invention. The diagnostic significance of IgA is to expand the detection of raw milk or other samples containing IgA antibodies, and the diagnostic significance of IgM is to expand the feasibility of the test paper card for early infection samples. (2) Compared with the anti-IgM secondary antibody, the Fcα / μR receptor can expose more Fab antigen binding domains, thereby improving the detection sensitivity of the test paper card for Brucella IgM. (3) By adding the biotin-streptavidin system and the metal ion ligand, the binding amount of the test strip to Brucella IgM is increased, thereby improving the sensitivity of the sample for early infection diagnosis. (4) The simple, instant and rapid lateral flow immunochromatography technology is adopted, which is suitable for on-site rapid detection.

[0023] In Example 5, an indirect method is used to develop a test strip, and two test lines are set, one of which (T1) can capture most of the IgG antibodies, and a positive result indicates that the sample is in the middle and late stages of infection or is expired, and the other (T2) can capture most of the IgM antibodies and IgA antibodies, and a positive result indicates that the sample is in the early stages of infection, and can be tested for raw milk samples. By distinguishing the color development and intensity of different test lines, the infection period of the sample is determined, which is convenient for guiding the development of subsequent work. In addition, in order to improve the resolution sensitivity of the diagnostic reagent, in addition to using the biotin-streptavidin system, the present invention creatively encapsulates metal ions into the test line of the diagnostic reagent, and through its interaction with the Fcα / μR receptor, the stability of the Fcα / μR receptor and the specific affinity with IgM are improved.

[0024] In Example 5, an indirect method was used to develop a Brucella antibody test strip, the conjugate pad was sprayed with red latex microspheres coupled to Brucella LPS and chicken IgY antibodies, and two test lines and one quality control line were set on the NC membrane, the first test line (T1) was coated with Protein G protein, the second line (T2) was coated with Fcα / μR receptor, and the quality control line was coated with goat anti-chicken IgY antibody. When Brucella LPS IgG, IgM or IgA antibodies are present in the test sample, they will bind to the LPS labeled microspheres on the conjugate pad for the first time to form a complex, and then lateral chromatography to the test line on the NC membrane, and bind to the coated protein for the second time, thereby forming a red microsphere precipitate visible to the naked eye.

[0025] Example 5 has the following three advantages: (1) Fcα / μR receptors can bind to IgM and IgA with high and medium affinity respectively, and they are used as capture proteins in the present invention. The diagnostic significance of IgA is that it expands the detection of raw milk or other samples containing IgA antibodies. The diagnostic result of IgM indicates that the infection period of the sample is early, and it can distinguish vaccine immunity. (2) Two detection lines are set up to detect IgA and IgM, IgG antibodies respectively. It is necessary to ensure that there is no mutual interference between these immunoglobulins, and the two types of detection are integrated on the same test strip, and the cross-reactivity in the reaction needs to be eliminated. (3) By adding the biotin-streptavidin system and the metal ion ligand, the binding sensitivity of the test strip to IgM is improved, avoiding the misjudgment of the infection period caused by too high IgG content in the sample and too low IgM binding. (4) A simple, instant and rapid lateral flow immunochromatography technology is adopted, which is suitable for on-site rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a structural schematic diagram of Example 1 of the present invention, a is a front schematic diagram of a Brucella antibody detection test paper card, b is a front schematic diagram of a Brucella antibody detection test paper strip, and c is a side schematic diagram of a Brucella antibody detection test paper strip.

[0027] Figure 2 These are the results of Brucella antibody detection test paper cards for early infection samples. (a) is the result of Fcα / μR receptor-made test paper cards for early infection samples. (b) is the result of anti-IgM secondary antibody-made test paper cards for early infection samples.

[0028] Figure 3 The results of Brucella antibody detection test strips for the same early infection sample, (a) is the test result of the test strip obtained after the optimization in 2.1 of Example 1, and (b) is the test result of the test strip obtained without the optimization in 2.1 of Example 1.

[0029] Figure 4The test paper cards prepared by adding different metal ions to the coated wire of Example 1 to detect early infection samples, (a)-(e) are Ca 2+ and Cu 2+ Synergistic, Ca 2+ , Cu 2+ Mg 2+ 、Zn 2+ .

[0030] Figure 5 The results of the optimized test strips for different samples are shown in Figure 2. (a)-(c) are samples from the early and middle stages of infection, samples from the middle and late stages of infection, and negative samples, respectively.

[0031] Figure 6 The test paper card and the anti-IgM secondary antibody test paper card of Example 1 of the present invention detect the results of early infection samples, (a)-(d) are respectively the results of the test paper card of Example 1 of the present invention detecting the original early infection samples, the results of the test paper card of Example 1 of the present invention detecting the early infection samples diluted 10 times, the results of the anti-IgM secondary antibody test paper card detecting the original early infection samples, and the results of the anti-IgM secondary antibody test paper card detecting the early infection samples diluted 10 times.

[0032] Figure 7 This is a schematic diagram of the test results of the Brucella antibody detection test strip in Example 1 of the present invention, (a)-(c) are positive results, negative results, and invalid results respectively.

[0033] Figure 8 The specific detection results of the Brucella antibody detection test strips of Example 1 of the present invention, (a)-(f) are Brucella sheep M16, cattle 2308, pig S2, O157 Escherichia coli, Yersinia enterocolitica, and Salmonella antibody positive samples respectively.

[0034] Fig. 9 The structure diagram of the Brucella antibody detection test paper card of Example 5 of the present invention is shown in Figure 5. (a) is a front view of the Brucella antibody detection test paper card, (b) is a front view of the Brucella antibody detection test paper strip, and (c) is a side view of the Brucella antibody detection test paper strip.

[0035] Fig.10 The schematic diagram of the structure of the Brucella antibody detection test strip that can distinguish the infection period according to Example 5 of the present invention. (a) is the test result of a sample of early Brucella infection, (b) is the test result of the first sample of early and middle Brucella infection, and (c) is the test result of the second sample of early and middle Brucella infection.

[0036] Fig.11Schematic diagram of Brucella antibody test strip test results that can distinguish the infection period, (a) is a schematic diagram of Brucella antibody test results in blood samples of animals with early or acute Brucella infection, (b) is a schematic diagram of Brucella antibody test results in blood samples of animals with mid-to-late or recurrent Brucella infection, (c) is a schematic diagram of Brucella antibody test results in raw milk samples of Brucella infected animals, (d) is a schematic diagram of Brucella antibody test results in blood samples or raw milk samples of animals without Brucella infection, and (e) is a schematic diagram of invalid Brucella antibody test results.

[0037] In the figure, 1 is a base plate, 2 is a nitrocellulose membrane, 3 is a water-absorbing filter paper, 4 is a glass cellulose membrane I, 5 is a blood filter membrane, 6 is a glass cellulose membrane II, 7 is a quality control line, 8 is a detection line, and 9 is a detection line T2. DETAILED DESCRIPTION

[0038] Example 1 A Brucella antibody test strip with improved early diagnosis sensitivity, the specific structure is as follows Figure 1 As shown, it includes a bottom plate 1, a chromatographic membrane arranged on the bottom plate 1, a sample processing layer is arranged at one end of the chromatographic membrane, and a quality control line 7 and a detection line 8 are also arranged on the chromatographic membrane.

[0039] The sample processing layer includes a conjugate pad, a blood filter membrane 5 and a sample pad which are overlapped on the chromatographic membrane in sequence. The conjugate pad is a glass cellulose membrane I4, and the sample pad is a glass cellulose membrane II6. The other end of the chromatographic membrane is provided with a water-absorbing filter paper 3.

[0040] Specifically, it includes: a card bottom plate, a 4mm wide and 60mm long strip is placed in the middle of the bottom plate, the bottom of the strip is supported by a hard plastic bottom plate 1, a 25mm long solid phase nitrocellulose membrane 2 (i.e., chromatographic membrane) is adhered to the middle, a 17mm long absorbent filter paper 3 (i.e., absorbent pad) is adhered to the upper end of the nitrocellulose membrane 2 and overlaps with the solid phase nitrocellulose membrane 2 by 2mm, a 10mm long glass cellulose membrane I 4 (i.e., conjugate pad), a 15mm long blood filter membrane 5, and a 17mm long glass cellulose membrane II 6 (i.e., sample pad) are adhered to the lower end of the solid phase nitrocellulose membrane 2 in sequence, and the strip is covered with a protective plastic shell.

[0041] A quality control line 7 (ie, C line) and a detection line 8 (ie, T line) are provided on the solid phase nitrocellulose membrane 2 .

[0042] The detection line 8 is arranged close to the glass cellulose membrane Ⅰ 4 and is formed by spraying the extracted Brucella LPS; the quality control line 7 is arranged close to the absorbent filter paper 3 and is formed by spraying the sheep anti-chicken IgY.

[0043] The conjugate pad of the sample processing layer includes chicken IgY-labeled latex microspheres, Protein G-labeled latex microspheres, or / and Fcα / μR receptor-labeled latex microspheres, and the chicken IgY-labeled latex microspheres are different in color from the Protein G-labeled latex microspheres and the Fcα / μR receptor-labeled latex microspheres. Specifically, the latex microspheres are mainly located on the conjugate pad closest to the chromatographic membrane, that is, the glass cellulose membrane Ⅰ4 is sprayed with chicken IgY labeled with blue latex microspheres, Protein G protein labeled with red latex microspheres, and Fcα / μR receptors labeled with red latex microspheres.

[0044] The above-mentioned Brucella antibody detection reagent strip is prepared by the following steps: 1. Labeling of latex microspheres 1.1. Cleaning of latex microspheres Take 12.5 μL of 400 nm latex microspheres (4% solid content) and add them to 1 mL of 10 mmol / L pH6.2 MES (2-(N-morpholino)ethanesulfonic acid) and mix thoroughly. Centrifuge at 20000 g for 10 min at 15°C, discard the supernatant, retain the precipitate, add 1 mL of 10 mmol / L pH 6.2 MES solution to resuspend and dissolve, and disperse by ultrasonication in a water bath.

[0045] 1.2. Activation of latex microspheres After washing, the latex microspheres were first added with 3.5 μL 10 mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and vortexed, then 33 μL 10 mg / mL sulfo-NHS (N-hydroxysuccinimide) was slowly added and ultrasonically mixed, and the microspheres were placed on a rotating mixer for uniform rotation and mixing for 15 min, then centrifuged at 15°C and 20,000 g for 10 min, the supernatant was discarded, the precipitate was retained, 1 mL 10 mmol / L pH 6.2 MES solution was added for re-suspending and dissolution, and ultrasonic dispersion was performed in a water bath.

[0046] 1.3 Microsphere-labeled proteins Take the activated latex microspheres, add 50 μg of Protein G or Fcα / μR receptor or chicken IgY, mix quickly, place at room temperature, and rotate to mix for 120 minutes.

[0047] 1.4. Microsphere surface sealing Add 500 μL of microsphere blocking solution to the labeled microsphere centrifuge tube, and rotate and mix at room temperature for 60 minutes. The microsphere blocking solution is borate buffer (5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0 ± 0.05), 1% BSA (bovine serum albumin), and 0.24% ethanolamine.

[0048] 1.5. Redissolution of microspheres The latex microspheres after complete labeling and sealing were centrifuged at 15°C and 20000 g for 10 min to remove unreacted and bound proteins, discard the supernatant, retain the precipitate, add 1 mL of microsphere washing solution to mix thoroughly, centrifuge again to precipitate, add 500 μL of microsphere preservation solution to the precipitate to re-dissolve, and disperse the labeled microspheres evenly by ultrasound to obtain latex microspheres of labeled Protein G or Fcα / μR receptor or chicken IgY, that is, microspheres coupled to Protein G, microspheres coupled to Fcα / μR receptor, microspheres coupled to chicken IgY, and stored at 4°C for use. The microsphere washing solution is 50 mM Tris (PH 8.0±0.05), 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin300. The microsphere storage solution is 25mM Tris, 150mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300, and the pH is adjusted to 8.7±0.05.

[0049] 2. Preparation of conjugate pad (i.e., glass cellulose membrane I4) Place the cut conjugate pad in an enamel tray, add 2 mL of conjugate pad treatment solution to soak it completely, and place it in a 45°C electric blast constant temperature drying oven to dry for 2 hours for use. 1.5 The obtained microspheres coupled to Protein G, microspheres coupled to Fcα / μR receptors, and microspheres coupled to chicken IgY were dispersed in a water bath ultrasound, mixed in a ratio of 1:1:1, and pumped into the gold spray film instrument at a spray volume of 6 μL / cm. After setting the relevant parameters, the gold spray film instrument was used to evenly spray the microspheres on the pre-treated conjugate pad cut to 5 mm wide × 30 mm long, placed in a 45°C electric blast constant temperature drying oven to dry for 2 hours or 37°C overnight, packaged, and used. The conjugate pad treatment solution is 1% BSA, 5% sucrose, 0.1% PEG20000, 50mM Tris, 0.1% Tween-20, and 0.1% PC300.

[0050] 3. Protein coating In this experiment, Sartorius CN95 nitrocellulose membrane was used, which was fixed and pasted on the PVC bottom plate, and the NC membrane was balanced in a humidity environment of 40%~60% for 30 min. The C line is sheep anti-chicken IgY antibody, and the T line is the extracted Brucella LPS. After being diluted to a working concentration of 1 mg / mL with the membrane coating solution, they were pumped into the gold spray membrane instrument, and the streaking volume was set to 1 μL / cm. Under the action of the contact point membrane head, the T and C line proteins were evenly coated on the NC membrane. After the operation was completed, the sheet was placed in a 45℃ electric blast constant temperature drying oven for 2 h or 37℃ overnight. After drying, it was placed in an aluminum foil bag for storage. The membrane coating solution is 1% sucrose, 25mM Tris (pH 7.2±0.05).

[0051] 4. Assembly of test strip card Cut the sample pad glass fiber into 17 mm width, the blood filter membrane into 10 mm width, and the absorbent paper into 17 mm width. After spraying the conjugate pad with markers and coating the NC membrane with relevant proteins, stick the pads to the PVC base plate in the order of absorbent paper, NC membrane, conjugate pad, blood filter membrane, and sample pad, and then cut them into 4 mm wide strips with a strip cutter. Place the narrow strips into the test strip buckle card of the appropriate width, assemble into a test strip card, and then perform relevant tests.

[0052] Example 2 Optimization of Brucella Antibody Detection Test Strips There is extremely high affinity between biotin and streptavidin, and a molecule of streptavidin can be combined with multiple biotin molecules, and this combination has specificity, and reacts rapidly, combines stably, can produce multi-stage amplification effect.Metal ion and protein can improve protein stability and affinity by some non-covalently bound interactions without strongly destroying protein binding sites.Therefore the present invention improves the detection sensitivity of test strips to Brucella early infection samples by these two schemes in an embodiment.

[0053] 2.1. Add biotin-streptavidin system Take 100 μg LPS, add biotin at a molar ratio of LPS to biotin of 1:100, the total volume is 100 μL, incubate at room temperature for 4 h, dilute to 5 times the volume of the reaction solution with PB buffer, and dialyze at 4 ° C for 72 h to obtain biotinylated LPS. Replace the red latex microspheres with streptavidin-modified red latex microspheres, and label the Fcα / μR receptor according to the experimental steps in Example 1.

[0054] 2.2. Improving the binding affinity of microglobulin-antibody complex to LPS The metal ions in magnesium chloride, copper sulfate, calcium chloride and zinc chloride were diluted to 0.5μM, 50μM, 500μM, 1mM and 25mM with 25mM PB buffer at pH 5.0, 6.0, 7.0, 8.0 and 9.0 and 25mM Tris buffer at pH 7.0, 8.0, 9.0, 10.0 and 11.0, respectively. The above parameters were paired and combined to set up different experimental schemes. 1% sucrose was added to different buffers to make coating solution. LPS was diluted to 1 mg / mL and coated on the T line. Sensitivity test was carried out using early samples infected with Brucella.

[0055] The experimental results show that Cu 2+ When copper sulfate was diluted to 50 μM using pH 6.0 25 mM PB buffer, the detection sensitivity of IgM was greatly improved. 2+ When calcium chloride was diluted to 0.5 μM using pH 7.0 25 mM PB buffer, the detection sensitivity of IgM was greatly improved, while the addition of the other two metal ions did not significantly improve the sensitivity.

[0056] Copper sulfate and calcium chloride were diluted to 0.5 μM, 50 μM, and 500 μM with pH 7.0 25 mM PB buffer, respectively. The metal ion solutions of different concentrations were mixed at a ratio of 1:1 to obtain 9 different metal ion mixtures. 1% sucrose was added to the metal ion mixture to prepare the coating solution. LPS was diluted to 1 mg / mL and coated on the T line. The sensitivity test was performed using early samples infected with Brucella. It was found that 0.5 μM Cu 2+ and 0.5 μM Ca 2+ There is a synergistic effect between them. The test paper card prepared by using the metal ion mixture of this concentration as the coating liquid to coat LPS has a higher IgM detection sensitivity than the test paper card prepared by single metal ion.

[0057] Perform the steps 2.1 and 2.2 simultaneously, i.e., add the biotin-streptavidin system and 0.5 μM Cu 2+ and 0.5 μM Ca 2+ , and obtain the optimized test strip.

[0058] Example 3 The test paper card made of Fcα / μR receptor (ie, Example 1) has a higher sensitivity for detecting early samples of Brucella infection than the test paper card made of IgM secondary antibody (see Figure 2 ).

[0059] The same early infection sample was tested and the results were as follows Figure 3 As shown, it can be seen that the test line of the test strip optimized by 2.1 is clearer than that of the test strip that has not been optimized.

[0060] Add appropriate amount of Cu in the coating solution 2+ and Ca 2+ (i.e., 2.2 of Example 2) can synergistically improve the detection sensitivity of the test paper card to IgM ( Figure 4 ).

[0061] The test results of using the optimized test strip of Example 2 (i.e., the test strip optimized by 2.1 and 2.2 at the same time) to test 1 clinical sample in the early and middle stages of infection, 1 clinical serum sample in the late stage of infection, and 1 negative clinical sample are shown in the graph. The T line is clearly colored. The experimental results are as follows Figure 5 shown.

[0062] The test strip card optimized in Example 2 (i.e., the test strip optimized in 2.1 and 2.2 at the same time) can detect Brucella positive samples with 1 / 10 of the original IgM antibody content, thus improving the sensitivity of early diagnosis of Brucella ( Figure 6 ).

[0063] Example 4 Functional verification of test paper card products 1Test strip result judgment standard Pipette 10μL of unknown sample to be tested onto the top of the sample well of the test paper card, and vertically drip 60μL of sample buffer into the sample well. Time for 8 minutes and judge the test result according to the result judgment method. The result judgment standard is as follows, and the schematic diagram is shown in Figure 7 : Figure 7 (a) indicates that the test sample is a blood sample or a raw milk sample. After the test card reacts, when both the quality control line and the test line are colored, the result indicates that the sample contains Brucella IgM or IgM and IgG antibodies, and the sampled animal is positive for Brucella infection.

[0064] Figure 7 (b) means that the test sample is a blood sample or a raw milk sample. After the test card reacts, when the quality control line shows color and the test line does not show color, the result indicates that the sample does not contain Brucella IgG antibodies and IgM antibodies, and the sampled animal is not infected with Brucella.

[0065] Figure 7 (c) means that the test sample is a blood sample or a raw milk sample. After the test card reacts, when both the quality control line and the test line do not show color, the result indicates that the test is invalid and the test card needs to be retested.

[0066] 2Test strip specificity analysis Take the same batch of test strips to detect the positive samples of antibodies of different species of Brucella sheep M16, cattle 2308, pig S2, O157 Escherichia coli, enterocolitica Yersinia, and Salmonella. After 15 minutes of reaction time, observe the test results. The test results are as follows Figure 8 As shown, the test strips tested antibody-positive samples of different species of Brucella, and the test results were positive, while the test results of interfering bacterial antibody samples were all negative, indicating that there was no significant cross-reaction between the test strips and other bacterial antibodies, and that they could specifically bind to multiple genera of Brucella.

[0067] 3. Repeatability analysis of test strips The positive reference serum for early antibody of Brucella infection was diluted to three concentrations: high, medium and low. Test strips from the same batch were randomly selected to perform 10 parallel repeated tests on each serum sample, and the test results were observed. The color difference was counted and the intra-batch coefficient of variation was calculated. Three samples of serum samples with different dilutions were randomly selected from each of the three batches of test strips prepared continuously to test the serum samples. The test results were observed, the color difference was counted and the inter-batch coefficient of variation was calculated. Coefficient of variation CV = (number of test samples with color difference / total number of test samples) × 100%. The test strips developed in the same batch showed consistent colorimetry for the three gradient reference samples, and the intra-batch repeatability was good. The test strips developed in three consecutive batches showed consistent colorimetry for the three gradient reference samples, and the inter-batch repeatability was good.

[0068] 4Test strip accuracy analysis Blood samples (serum, plasma, whole blood) or raw milk samples with negative and positive Brucella antibodies from different sheep and cattle species were collected. The ELISA method was used to detect whether the samples contained IgM antibodies, and the samples were classified according to early or acute infection and mid-late or recurrent infection. The classified clinical samples were tested according to the operating instructions of the test strips, ELISA kits and test tube agglutination tests. The positive coincidence rate (diagnostic sensitivity Se) and negative coincidence rate (diagnostic specificity Sp) of the colloidal gold test strips with the ELISA kits and test tube agglutination tests were evaluated, and the consistency between the test strips and the ELISA kits and test tube agglutination test reagents was compared by calculating the Kappa value. The Kappa values ​​between the test strips and the ELISA kits or test tube agglutination tests were all greater than 0.75, and it was considered that there was a high consistency between the two.

[0069] 5. Test Strip Stability (Shelf Life) Analysis 50 samples of three batches of test strips prepared continuously were taken out and placed at 45°C for thermal accelerated stability test. The prepared test strips were placed at a constant temperature of 45°C for 0, 1, 3, 5, 7, 14, 21, 28, 35, 42, 56, 77, and 91 days to detect positive quality control products. Within the set test time, the test strips did not produce intra-batch differences or the positive quality control products could not be detected. The colorimetric results were consistent and the stability was good.

[0070] Example 5 Preparation of Brucella antibody test strips Preparation of a Brucella antibody test strip that can distinguish infection periods, the specific structure is as follows Fig. 9 As shown, it includes: a card bottom plate, a 4mm wide and 60mm long strip is placed in the middle of the bottom plate, the bottom of the strip is supported by a hard plastic bottom plate 1, a 25mm long solid phase nitrocellulose membrane 2 (chromatographic membrane) is adhered to the middle, a 17mm long absorbent filter paper 3 (absorbent pad) is adhered to the upper end and overlaps with the solid phase nitrocellulose membrane 2 by 2mm, a 10mm long glass cellulose membrane I 4 (conjugate pad), a 15mm long blood filter membrane 5, and a 17mm long glass cellulose membrane II 6 (sample pad) are adhered to the lower end of the solid phase nitrocellulose membrane in sequence, and the strip is covered with a protective plastic shell.

[0071] A quality control line 7, a detection line 8 (i.e., T1), and a detection line T29 are provided on the solid phase nitrocellulose membrane 2 (chromatographic membrane); the detection line is arranged close to the conjugate pad, and the T1 detection line 8 is formed by spraying the Protein G protein; the detection line T29 is formed by spraying the Fcα / μR receptor; the quality control line 7 is arranged close to the absorbent filter paper 3, and the quality control line 7 is formed by spraying the sheep anti-chicken IgY.

[0072] The conjugate pad was sprayed with red latex microspheres labeled chicken IgY and red latex microspheres labeled Brucella extracted LPS.

[0073] The above-mentioned Brucella antibody detection reagent strip is prepared by the following steps: 1. Labeling of latex microspheres 1.1. Cleaning of latex microspheres Take 12.5 μL of 400 nm latex microspheres (4% solid content) and add them to 1 mL of 10 mmol / L pH6.2 MES, mix thoroughly, centrifuge at 15℃ and 20000 g for 10 min, discard the supernatant, keep the precipitate, add 1 mL of 10 mmol / L pH6.2 MES solution to resuspend and dissolve, and disperse by water bath ultrasound.

[0074] 1.2. Activation of latex microspheres After washing, 3.5 μL of 10 mg / mL EDC was added to the latex microspheres and vortexed to mix. 33 μL of 10 mg / mL sulfo-NHS was slowly added and ultrasonically mixed. The microspheres were placed on a rotating mixer and rotated at a constant speed for 15 min. The microspheres were then centrifuged at 15°C and 20,000 g for 10 min. The supernatant was discarded and the precipitate was retained. 1 mL of 10 mmol / L pH 6.2 MES solution was added to resuspend and dissolve the microspheres, and then ultrasonically dispersed in a water bath.

[0075] 1.3 Microsphere-labeled proteins Take the activated latex microspheres, add 50 μg of Brucella LPS or chicken IgY, mix quickly, place at room temperature, and rotate to mix for 120 minutes.

[0076] 1.4. Microsphere surface sealing Add 500 μL of microsphere blocking solution to the labeled microsphere centrifuge tube, and rotate and mix at room temperature for 60 minutes. The microsphere blocking solution is borate buffer (5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0 ± 0.05), 1% BSA, and 0.24% ethanolamine.

[0077] 1.5. Redissolution of microspheres The latex microspheres after complete labeling and sealing were centrifuged at 15°C and 20,000 g for 10 min to remove unreacted and bound proteins, discard the supernatant, retain the precipitate, add 1 mL of microsphere washing solution to mix thoroughly, centrifuge again to precipitate, add 500 μL of microsphere preservation solution to the precipitate to re-dissolve, and disperse the labeled microspheres evenly by ultrasound to obtain the labeled LPS or chicken IgY latex microspheres, which were stored at 4°C for use. The microsphere washing solution is 50 mM Tris (PH 8.0±0.05), 0.5% BSA, 0.05% Tween-20, 0.03% ProClin 300. The microsphere preservation solution is 25 mM Tris, 150 mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300, and the pH is adjusted to 8.7±0.05.

[0078] 2. Preparation of conjugate pad Place the cut conjugate pad in an enamel tray, add 2mL of conjugate pad treatment solution to soak it completely, and place it in a 45℃ electric blast constant temperature drying oven for 2h for use. After the stored microsphere labeled protein is dispersed by water bath ultrasound, the microspheres coupled to LPS and microspheres coupled to chicken IgY are mixed in a ratio of 2:1, and pumped into the gold spray film instrument at a spray volume of 6 μL / cm. After setting the relevant parameters, the gold spray film instrument is used to evenly spray the microspheres on the pre-treated conjugate pad cut to 5 mm wide × 30 mm long, and placed in a 45℃ electric blast constant temperature drying oven for 2 h or 37℃ overnight, packaged, and used. The conjugate pad treatment solution is 1% BSA, 5% sucrose, 0.1% PEG20000, 50mM Tris, 0.1% Tween-20, and 0.1% PC300.

[0079] 3. Protein coating In this experiment, Sartorius CN95 nitrocellulose membrane was used, which was fixed and pasted on the PVC bottom plate, and the NC membrane was balanced in a humidity environment of 40%~60% for 30 min. The C line is sheep anti-chicken IgY antibody, the T1 line (i.e., detection line 8) is Protein G protein, and the T2 line (i.e., detection line T29) is Fcα / μR receptor. After being diluted to a working concentration of 1 mg / mL with the membrane coating solution, they were pumped into the gold spray membrane instrument, and the streaking volume was set to 1 μL / cm. Under the action of the contact point membrane head, the T and C line proteins were evenly coated on the NC membrane. After the operation was completed, the sheet was placed in a 45℃ electric heating blast constant temperature drying oven for 2 h or 37℃ overnight. After drying, it was placed in an aluminum foil bag for storage. The membrane coating solution is 1% sucrose, 25mM Tris (pH7.2±0.05).

[0080] 4. Assembly of test strip card Cut the sample pad glass fiber into 17 mm width, the blood filter membrane into 10 mm width, and the absorbent paper into 17 mm width. After spraying the conjugate pad with markers and coating the NC membrane with relevant proteins, stick the pads to the PVC base plate in the order of absorbent paper, NC membrane, conjugate pad, blood filter membrane, and sample pad, and then cut them into 4 mm wide strips with a strip cutter. Place the narrow strips into the test strip buckle card of the appropriate width, assemble into a test strip card, and then perform relevant tests.

[0081] Example 6 Optimization of Brucella Antibody Detection Test Strips Biotin and streptavidin have a very high affinity, and one molecule of streptavidin can bind to multiple biotin molecules. This binding is specific, and the reaction is rapid, the binding is stable, and a multi-stage amplification effect can be produced. The metal ions and the receptor protein can improve the stability and affinity of the receptor protein without strongly destroying the antibody binding site of the receptor protein through some non-covalent binding interactions. Therefore, the present invention improves the analytical sensitivity of the test strip T2 line through these two schemes in the embodiment.

[0082] 6.1. Add biotin-streptavidin system to optimize T2 line color intensity Take 100 μg of Fcα / μR receptor, add biotin at a molar ratio of 1:100 to Fcα / μR receptor, the total volume is 100 μL, incubate at room temperature for 4 h, dilute to 5 times the volume of the reaction solution with PB buffer, and dialyze at 4°C for 72 h to obtain biotinylated Fcα / μR receptor. Replace the red latex microspheres with streptavidin-modified red latex microspheres, and label LPS according to the experimental steps in Example 1.

[0083] 6.2. Targeted optimization of Fcα / μR receptor-antibody binding affinity The metal ions in ferric chloride, copper sulfate, calcium chloride and zinc chloride were diluted to 0.05μM, 0.5μM, 5μM, 50μM, 500μM and 1mM with 25mM PB buffer at pH 5, 6, 7, 8 and 9 and 25mM Tris buffer at pH 7, 8, 9, 10 and 11, respectively. The above parameters were paired and combined to set up different experimental schemes. 1% sucrose was added to different buffers to make coating solution. The Fcα / μR receptor was diluted to 1mg / mL and coated on the T2 line. Sensitivity test was carried out using early samples infected with Brucella.

[0084] The test strips prepared after optimization in Example 6 (i.e., the test strips after optimization in 6.1 and 6.2) and the test strips before optimization (i.e., the test strips in Example 5) were used to test one clinical serum sample in the early stage of infection and two clinical serum samples in the early and middle stages of infection, respectively, and the color intensity of the T2 line and the T1 line was observed. The experimental results are shown in FIG. Fig.10 As shown, in the experimental pictures with different numbers, Fig.10 In (a)-(c), the first and second test strips from left to right in each set of experimental results are the test strip test results before optimization, and the third and fourth test strips are the test strip test results after optimization. By optimizing the sensitivity of the combined IgM antibody, the color intensity of the T2 line has been significantly improved compared with before optimization, making it easier to determine the difference in color intensity between the T1 line and the T2 line, thereby improving the inference of the infection period to which the test sample belongs.

[0085] Example 7 Functional verification of test paper card products 1. Test strip result judgment criteria Pipette 10μL of unknown sample to be tested onto the top of the sample well of the test paper card, and vertically drip 60μL of sample buffer into the sample well. Time for 8 minutes and judge the test result according to the result judgment method. The result judgment standard is as follows, and the schematic diagram is shown in Fig.11 : Fig.11 (a) means that if the test sample is a blood sample, after the test paper card reacts, when the quality control line and the T2 test line are colored, and the T1 test line is colored or not colored, the result indicates: the sample contains Brucella IgM or IgM and IgG antibodies, and the sampling animal is in the early stage of Brucella infection or acute infection.

[0086] Fig.11 (b) means that if the test sample is a blood sample, after the test card reacts, when the quality control line and the T1 test line are colored, but the T2 test line is not colored, the result indicates that the sample contains Brucella IgG antibodies but no IgM antibodies, and the sampled animal is in the middle or late stage of Brucella infection or recurrent infection.

[0087] Fig.11 (c) means that if the test sample is a raw milk sample, after the test paper card reacts, when the quality control line and the T1 test line are colored, and the T2 test line is colored or not colored, the result indicates: the sample contains Brucella IgA antibodies or IgA and IgG antibodies, and the sampled animal is already infected with Brucella.

[0088] Fig.11 (d) means that if the test sample is a raw milk sample or a blood sample, after the test paper card reacts, only the quality control line will show color, and the T1 test line and the T2 test line will not show color. The result indicates that the sample does not contain Brucella antibodies and the sampled animal is not infected with Brucella.

[0089] Fig.11 (e) means that if the test sample is a raw milk sample or a blood sample, after the test paper card reacts, the quality control line does not show color, the T1 test line and the T2 test line show color or do not show color, and the result indicates that the test is invalid.

[0090] 2. Test strip specificity analysis The test strips developed in the same batch were used to test the positive samples of Brucella sheep M16, cattle 2308, pig S2, O157 Escherichia coli, enterocolitica Yersinia, and Salmonella antibodies of different species. The test results were observed after 15 minutes of reaction time. The test results were all negative, indicating that there was no significant cross-reaction between the test strips and other bacterial antibodies. The results of the simultaneous detection of Brucella IgM antibody positive reference serum and Brucella IgG antibody positive reference serum also showed that there was no significant cross-reaction between the two immunoglobulin tests.

[0091] 3. Repeatability analysis of test strips Brucella IgM antibody positive reference serum and Brucella IgG antibody positive reference serum were diluted to three concentrations: high, medium, and low. Test strips from the same batch were randomly selected to perform 10 parallel repeated tests on each serum sample, and the test results were observed. The color difference was counted and the intra-batch coefficient of variation was calculated. Three samples of serum samples with different dilutions were randomly selected from each of the three batches of test strips prepared continuously to test the serum samples. The test results were observed, the color difference was counted, and the inter-batch coefficient of variation was calculated. Coefficient of variation CV = (number of test copies with color difference / total number of test copies) × 100%. The test strips developed in the same batch showed consistent colorimetry for the three gradient reference products, and the intra-batch repeatability was good. The test strips developed in three consecutive batches showed consistent colorimetry for the three gradient reference products, and the inter-batch repeatability was good.

[0092] 4. Test strip accuracy analysis Blood samples (serum, plasma, whole blood) or raw milk samples with negative and positive Brucella antibodies from different sheep and cattle species were collected. The ELISA method was used to detect whether the samples contained IgM antibodies, and the samples were classified according to early or acute infection and mid-late or recurrent infection. The classified clinical samples were tested according to the operating instructions of the test strips, ELISA kits and test tube agglutination tests. The positive coincidence rate (diagnostic sensitivity Se) and negative coincidence rate (diagnostic specificity Sp) of the colloidal gold test strips with the ELISA kits and test tube agglutination tests were evaluated, and the consistency between the test strips and the ELISA kits and test tube agglutination test reagents was compared by calculating the Kappa value. The Kappa values ​​between the test strips and the ELISA kits or test tube agglutination tests were all greater than 0.75, and it was considered that there was a high consistency between the two.

[0093] 5. Test strip stability (shelf life) analysis 50 samples of three batches of test strips prepared continuously were taken out and placed at 45°C for thermal accelerated stability test. The prepared test strips were placed at a constant temperature of 45°C for 0, 1, 3, 5, 7, 14, 21, 28, 35, 42, 56, 77, and 91 days to detect positive quality control products. The test strips did not produce intra-batch differences or the positive quality control products could not be detected. The color development of the test results was consistent and the stability was good.

[0094] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0095] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.

Claims

1. A Brucella antibody test strip, characterized in that: It comprises a bottom plate (1), a chromatographic membrane arranged on the bottom plate (1), a sample processing layer being arranged at one end of the chromatographic membrane, and a quality control line (7) and at least one detection line being arranged on the chromatographic membrane; The sample processing layer includes latex microspheres labeled with chicken IgY, latex microspheres labeled with Protein G protein, or / and latex microspheres labeled with Fcα / μR receptor, and the detection line includes Brucella LPS; or, The sample processing layer comprises latex microspheres marked with Brucella LPS and latex microspheres marked with chicken IgY, and the detection line comprises Protein G protein and Fcα / μR receptor.

2. Brucella antibody detection test paper as claimed in claim 1, is characterized in that, The sample processing layer comprises latex microspheres marked with chicken IgY, latex microspheres marked with Protein G protein and latex microspheres marked with Fcα / μR receptor.

3. Brucella antibody detection test paper as claimed in claim 1, is characterized in that, The sample processing layer includes chicken IgY-labeled latex microspheres, Protein G protein-labeled latex microspheres or / and Fcα / μR receptor-labeled latex microspheres. When the detection line (8) includes Brucella LPS, the Brucella LPS is modified with biotin, and the Protein G protein-labeled latex microspheres and Fcα / μR receptor-labeled latex microspheres are modified with streptavidin. The sample processing layer includes latex microspheres labeled with Brucella LPS and latex microspheres labeled with chicken IgY, and when the detection line (8) includes Protein G protein and Fcα / μR receptor, the Fcα / μR receptor is modified with biotin, and the latex microspheres labeled with Brucella LPS and the latex microspheres labeled with chicken IgY are modified with streptavidin.

4. Brucella antibody detection test paper as claimed in claim 1, is characterized in that, There are two detection lines, the sample processing layer includes latex microspheres labeled with Brucella LPS and latex microspheres labeled with chicken IgY, one detection line includes Protein G protein, and the other detection line includes Fcα / μR receptor.

5. Brucella antibody detection test paper as claimed in claim 1, is characterized in that, The detection line (8) also includes metal ions, and the metal ions are copper ions and / or calcium ions.

6. Brucella antibody detection test paper as claimed in claim 5, characterized in that, The metal ions are copper ions and calcium ions, and the molar ratio of copper ions to calcium ions is 1:

1.

7. A Brucella antibody test paper as described in any one of claims 1-6, characterized in that, The chromatographic membrane is a nitrocellulose membrane (2), and the sample processing layer comprises a conjugate pad, a blood filter membrane (5) and a sample pad which are sequentially overlapped and arranged on the chromatographic membrane, the conjugate pad is a glass cellulose membrane I (4), and the sample pad is a glass cellulose membrane II (6); a water-absorbing filter paper (3) is arranged at the other end of the chromatographic membrane.

8. A Brucella antibody test paper as described in any one of claims 1-6, characterized in that, The quality control line (7) includes sheep anti-chicken IgY antibody.

9. as any one of claim 1-6 described Brucella antibody detection test paper, it is characterized in that, The sample treatment layer includes a treatment solution, which includes 1% BSA, 5% sucrose, 0.1% PEG20000, 50 mM Tris, 0.1% Tween-20, and 0.1% PC300.

10. An application of a Brucella antibody test paper as described in any one of claims 1 to 9, characterized in that: The Brucella antibody test paper is used to detect the infection period of Brucella.

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