A brucella antibody test strip and use

Abstract

The application belongs to the technical field of animal epidemic disease immunity detection, and particularly relates to a Brucella antibody detection test paper and application, which comprises a bottom plate, a chromatography membrane arranged on the bottom plate, one end of the chromatography membrane is provided with a sample processing layer, and the chromatography membrane is further provided with a quality control line and at least one detection line; the sample processing layer comprises chicken IgY marked latex microspheres, Protein G protein marked latex microspheres or / and Fcα / μR receptor marked latex microspheres, and the detection line comprises Brucella LPS; or the sample processing layer comprises Brucella LPS marked latex microspheres and chicken IgY marked latex microspheres, and the detection line comprises Protein G protein and Fcα / μR receptor; the application significantly improves the sensitivity of early diagnosis or infection period discrimination, and is good in specificity, repeatability and accuracy.

Description

Technical Field

[0001] This invention belongs to the field of animal disease immune detection technology, specifically relating to a Brucella antibody test strip and its application. Background Technology

[0002] Brucella ( Brucella Brucella is a Gram-negative bacterium that does not form spores, although some virulent strains can form capsules. Its virulence factors mainly include lipopolysaccharide (LPS), the Type IV secretion system (T4SS), outer membrane proteins (OMP), and the two-component compression system (TCS). LPS assists Brucella in escaping the host cell's anti-infective mechanisms during infection. Furthermore, because LPS is a major component of the Brucella outer membrane, it is widely used in Brucella detection techniques. Brucella can be classified into 6 species and 20 biotypes, including *Brucella mesenteriae* (3 biotypes, types 1, 2, and 3), *Brucella bovis* (9 biotypes, types 1-9), *Brucella suis* (5 biotypes, types 1-5), *Brucella epididymis* (1 biotype), *Brucella sarinosa* (1 biotype), and *Brucella canis* (1 biotype). *Brucella mesenteriae* and *Brucella bovis* are both smooth-skinned strains. Brucella can infect humans, livestock, and various wild animals. In humans, infection can cause symptoms such as fever, excessive sweating, joint and nerve pain, and hepatosplenomegaly. In cows, it can cause abortion and infertility. Rapid and accurate diagnostic tools are needed to scientifically carry out brucellosis prevention and control.

[0003] Current methodologies for detecting Brucella include etiological diagnostic techniques, serological diagnostic techniques, and molecular biological techniques. These mainly include bacterial culture, isolation, and identification; agglutination tests; enzyme-linked immunosorbent assays (ELISA); complement fixation tests; immunochromatography; polymerase chain reaction (PCR); and real-time quantitative PCR. Each methodology has its advantages, disadvantages, and applicable scenarios. The industry standard document WS269-2019 for Brucellosis diagnosis states that the colloidal gold immunochromatographic assay (GICA) can be used for initial screening of Brucella, and this methodology is suitable for rapid on-site detection at the livestock farming level. By combining the infection cycle of Brucella and the characteristics of positive serological antibody reactions, the early diagnostic sensitivity of the test strip can be improved by enhancing the binding of the test strip to IgM.

[0004] Current Brucella antibody detection chromatographic test strips are mostly prepared using sandwich, indirect, or competitive methods. These inventions often use extracted LPS as the capture protein. LPS is a major component of the cell wall of most Gram-negative bacteria. The LPS on the surface of Brucella is atypical LPS, which is a major component of its outer membrane. It is not only related to the survival and proliferation of Brucella itself, but also is the most important surface antigen of Brucella, and can serve as an important marker in Brucella serological diagnostic methods.

[0005] Existing research on diagnostic reagents for Brucella IgM antibodies mostly uses anti-IgM antibodies as secondary antibodies in immunoassays, participating in the binding with Brucella IgM antibodies during the reaction. For example, Liu Hanxuan et al., in their paper "Establishment and Preliminary Clinical Application of a Colloidal Gold Immunochromatographic Detection Method for Brucella Serum IgM and IgG Antibodies," used mouse anti-human IgM as a coating reagent. However, anti-IgM secondary antibodies prepared through immunization and serum purification may bind to both the Fc and Fab fragments of IgM, and IgM exhibits species specificity as an immunogen, thus affecting the detection results. Summary of the Invention

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

[0007] This invention provides a Brucella antibody test strip, including a base plate, a chromatography membrane disposed on the base plate, a sample processing layer disposed at one end of the chromatography membrane, and a control line and at least one detection line disposed on the chromatography membrane;

[0008] The sample processing layer comprises chicken IgY-labeled latex microspheres, Protein G-labeled latex microspheres, and / or Fcα / μR receptor-labeled latex microspheres; the detection line comprises Brucella LPS; or,

[0009] The sample processing layer comprises Brucella LPS-labeled latex microspheres and chicken IgY-labeled latex microspheres, and the detection line comprises Protein G protein and Fcα / μR receptor.

[0010] In one embodiment, the sample processing layer comprises chicken IgY-labeled latex microspheres, Protein G-labeled latex microspheres, and Fcα / μR receptor-labeled latex microspheres.

[0011] In one embodiment, the sample processing layer comprises chicken IgY-labeled latex microspheres, Protein G protein-labeled latex microspheres, and / or Fcα / μR receptor-labeled latex microspheres. When the detection line comprises 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.

[0012] The sample processing layer includes Brucella LPS-labeled latex microspheres and chicken IgY-labeled latex microspheres. When the detection line includes Protein G protein and Fcα / μR receptor, the Fcα / μR receptor is modified with biotin, and the Brucella LPS-labeled latex microspheres and chicken IgY-labeled latex microspheres are modified with streptavidin.

[0013] In one embodiment, there are two detection lines, and the sample processing layer includes Brucella LPS-labeled latex microspheres and chicken IgY-labeled latex microspheres. One detection line includes Protein G protein, and the other detection line (i.e., detection line T2) includes Fcα / μR receptor.

[0014] In one embodiment, the sample processing layer includes Brucella LPS-labeled latex microspheres and chicken IgY-labeled latex microspheres. The detection line includes Protein G protein. When another detection line (i.e., detection line T2) includes an Fcα / μR receptor, the Fcα / μR receptor is modified with biotin, and the Brucella LPS-labeled latex microspheres and chicken IgY-labeled latex microspheres are modified with streptavidin.

[0015] In one embodiment, the detection line further includes metal ions, which are copper ions and / or calcium ions.

[0016] 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.

[0017] In one embodiment, the chromatography membrane is a nitrocellulose membrane, and the sample processing layer includes a conjugate pad, a blood filtration membrane, and a sample pad sequentially overlapped on the chromatography 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 disposed at the other end of the chromatography membrane.

[0018] The sample treatment layer contains chicken IgY-labeled latex microspheres, Protein G-labeled latex microspheres, and / or Fcα / μR receptor-labeled latex microspheres located on the conjugate pad, and Brucella LPS-labeled latex microspheres located on the conjugate pad.

[0019] In one embodiment, the quality control line includes a goat anti-chicken IgY antibody.

[0020] In one embodiment, the sample treatment layer (specifically, the binding pad) includes a treatment solution consisting of 1% BSA, 5% sucrose, 0.1% PEG20000, 50mM Tris, 0.1% Tween-20, and 0.1% PC300.

[0021] This invention provides an application of the Brucella antibody test strip, which is used to detect the stage of Brucella infection.

[0022] The beneficial effects of this invention are:

[0023] The Fcα / μR receptor can specifically bind to the Fc fragments of IgA and IgM, exposing more of the Fab antigen-binding region, which helps improve the diagnostic specificity of IgM and the binding of the IgM variable region to Brucella LPS. Based on the above-mentioned characteristics of the Fcα / μR receptor, this invention creatively applies the recombinant Fcα / μR receptor to diagnostic reagents to improve the sensitivity and specificity of diagnostic reagents for IgM antibody binding, thereby enhancing the sensitivity of diagnostic reagents for samples in the early stages of infection.

[0024] In one embodiment, the present invention employs an indirect method to develop test strips. Microspheres are labeled with Protein G and Fcα / μR receptors, respectively, to capture IgG and IgM / IgA in infected or immune samples. The resulting complexes then bind to LPS on the T line, indicating a positive infection. This method can also detect raw milk samples, expanding the applicability of the test strips. Furthermore, to improve the diagnostic sensitivity of the reagents for early-stage infections, in addition to using the common biotin-streptavidin system, the present invention creatively coats the detection lines of the diagnostic reagents with metal ions, particularly a combination of copper and calcium ions. Through their interaction with LPS, the stability of LPS and its specific affinity for Protein G-IgG, Fcα / μR-IgM, and Fcα / μR-IgA are improved.

[0025] In this Example 1, the present invention uses an indirect method to develop a Brucella antibody test strip. The conjugate pad is coated with red latex microspheres conjugated to Brucella extract Protein G protein, Fcα / μR receptor, and chicken IgY antibody. A detection line and a control line are set on the NC membrane. The detection line is coated with LPS, and the control line is coated with goat anti-chicken IgY antibody. When Brucella LPS IgG, IgM, or IgA antibody is present in the test sample, it will first bind to the Protein G protein and Fcα / μR receptor labeled microspheres on the conjugate pad, forming a complex. This complex is laterally chromatized to the detection line on the NC membrane, where it binds a second time to the coated protein, thus forming a visible red microsphere precipitate.

[0026] The present invention has the following four advantages: (1) The Fcα / μR receptor can bind IgM and IgA with high and medium affinity, respectively, and use them 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, and the diagnostic significance of IgM is that it expands the feasibility of the test strip for detecting early-stage infection samples. (2) Compared with anti-IgM secondary antibodies, the Fcα / μR receptor can expose more Fab antigen binding domains, thereby improving the detection sensitivity of the test strip for Brucella IgM. (3) By increasing the biotin-streptavidin system and metal ion ligands, the binding amount of Brucella IgM on the test strip is increased, thereby improving the diagnostic sensitivity of early infection in samples. (4) The simple, immediate and rapid lateral flow immunochromatography technique is adopted, which is suitable for rapid on-site detection.

[0027] In Example 5, a test strip was developed using an indirect method, with two detection lines. One line (T1) captures most of the IgG antibodies, and a positive result indicates that the tested sample is in the mid-to-late stage of infection or the survival period. The other line (T2) captures most of the IgM and IgA antibodies, and a positive result indicates that the tested sample is in the early stage of infection. This method can also detect raw milk samples. By distinguishing the color development and intensity of the different detection lines, the infection stage of the tested sample can be determined, facilitating subsequent work. Furthermore, to improve the resolution sensitivity of the diagnostic reagent, in addition to using the biotin-streptavidin system, this invention creatively coats the detection lines of the diagnostic reagent with metal ions. Through their interaction with the Fcα / μR receptor, the stability of the Fcα / μR receptor and its specific affinity for IgM are improved.

[0028] In Example 5, a Brucella antibody test strip was developed using an indirect method. The conjugate pad was coated with red latex microspheres conjugated to Brucella LPS and chicken IgY antibodies. Two detection lines and one control line were set on the NC membrane. The first detection line (T1) was coated with Protein G protein, the second line (T2) was coated with Fcα / μR receptors, and the control line was coated with goat anti-chicken IgY antibody. When Brucella LPS IgG, IgM, or IgA antibodies were present in the sample, they would bind to the LPS-labeled microspheres on the conjugate pad for the first time, forming a complex. This complex would then laterally precipitate to the detection line on the NC membrane, where it would bind to the coated protein for the second time, thus forming a visible red microsphere precipitate.

[0029] Example 5 has the following three advantages: (1) Fcα / μR receptors can bind IgM and IgA with high and medium affinity, respectively, and use them as capture proteins in this 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 sample infection period is early and can distinguish between vaccine immunity. (2) Two detection lines are set up to detect IgA and IgM and 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 to eliminate cross-reactions. (3) By adding the biotin-streptavidin system and metal ion ligands, the binding sensitivity of the test strip to IgM is improved, avoiding misjudgment of the infection period caused by excessively high IgG content and low IgM binding in the sample. (4) A simple, immediate and rapid lateral flow immunochromatography technique is adopted, which is suitable for rapid on-site detection. Attached Figure Description

[0030] Figure 1 The diagram below is a schematic diagram of the structure of Embodiment 1 of the present invention. a is a schematic diagram of the front of the Brucella antibody test strip card, b is a schematic diagram of the front of the Brucella antibody test strip, and c is a schematic diagram of the side of the Brucella antibody test strip.

[0031] Figure 2 The results of Brucella antibody test strips for detecting early-infected samples are shown in (a) and (b) respectively. (a) The results of test strips prepared with Fcα / μR receptors for detecting early-infected samples are shown in (b) respectively.

[0032] Figure 3 The results of the Brucella antibody test strips for testing the same early infection sample are shown in (a) and (b) respectively. (a) is the test result of the test strip prepared after optimization in 2.1 of Example 1, and (b) is the test result of the test strip prepared without optimization in 2.1 of Example 1.

[0033] Figure 4Test strips made by adding different metal ions to the coating line of Example 1 were used to detect early infected samples. (a)-(e) are, in order, Ca 2+ and Cu 2+ Collaboration, Ca 2+ Cu 2+ Mg 2+ Zn 2+ .

[0034] Figure 5 To optimize the test strip results for different samples, (a)-(c) show the results for early to mid-stage infection, mid to late-stage infection, and negative samples, respectively.

[0035] Figure 6 The results of early infection samples detected by the test strip and anti-IgM secondary antibody test strip of Example 1 of the present invention are shown in (a)-(d), which are respectively the results of early infection samples detected by the test strip of Example 1 of the present invention at the original level, the results of early infection samples detected by the test strip of Example 1 of the present invention at a 10-fold dilution, the results of early infection samples detected by the anti-IgM secondary antibody test strip at the original level, and the results of early infection samples detected by the anti-IgM secondary antibody test strip at a 10-fold dilution.

[0036] Figure 7 This is a schematic diagram of the detection results of the Brucella antibody test strip in Example 1 of the present invention. (a)-(c) represent positive results, negative results, and invalid results, respectively.

[0037] Figure 8 The results of the Brucella antibody test strip in Example 1 of this invention are as follows: (a)-(f) are Brucella ovine M16, bovine 2308, swine S2, O157 Escherichia coli, Yersinia enterocolitica, and Salmonella antibody positive samples, respectively.

[0038] Figure 9 This is a schematic diagram of the Brucella antibody test strip structure of Embodiment 5 of the present invention. (a) is a front view of the Brucella antibody test strip, (b) is a front view of the Brucella antibody test strip, and (c) is a side view of the Brucella antibody test strip.

[0039] Figure 10 This is a schematic diagram of the Brucella antibody test strip of Embodiment 5 of the present invention, which can distinguish the infection stage. (a) is the test result of one sample in the early stage of Brucella infection, (b) is the test result of the first sample in the early to mid stage of Brucella infection, and (c) is the test result of the second sample in the early to mid stage of Brucella infection.

[0040] Figure 11The following are schematic diagrams of Brucella antibody test strip results that can distinguish the stage of infection: (a) shows the Brucella antibody test results in blood samples of animals in the early or acute stages of Brucella infection; (b) shows the Brucella antibody test results in blood samples of animals in the middle or late stages of Brucella infection or recurrent infection; (c) shows the Brucella antibody test results in raw milk samples of animals infected with Brucella; (d) shows the Brucella antibody test results in blood samples or raw milk samples of animals not infected with Brucella; and (e) shows invalid Brucella antibody test results.

[0041] In the diagram, 1 is the base plate, 2 is the nitrocellulose membrane, 3 is the absorbent filter paper, 4 is the glass cellulose membrane I, 5 is the blood filtration membrane, 6 is the glass cellulose membrane II, 7 is the quality control line, 8 is the test line, and 9 is the test line T2. Detailed Implementation

[0042] Example 1

[0043] A Brucella antibody test strip with improved sensitivity for early diagnosis, the specific structure of which is as follows: Figure 1 As shown, it includes a base plate 1, a chromatography membrane disposed on the base plate 1, a sample processing layer disposed at one end of the chromatography membrane, and a quality control line 7 and a detection line 8 disposed on the chromatography membrane.

[0044] The sample processing layer includes a conjugate pad, a blood filtration membrane 5, and a sample pad sequentially overlapped on the chromatography membrane. The conjugate pad is a glass cellulose membrane I 4, and the sample pad is a glass cellulose membrane II 6. An absorbent filter paper 3 is provided at the other end of the chromatography membrane.

[0045] Specifically, it includes: a base plate, a 4mm wide and 60mm long strip placed in the middle of the base plate, the bottom of the strip is supported by a rigid plastic base plate 1, a 25mm long solid nitrocellulose membrane 2 (i.e., chromatography membrane) is attached to the middle, a 17mm long absorbent filter paper 3 (i.e. absorbent pad) is attached to the upper end of the nitrocellulose membrane 2 and overlaps the solid nitrocellulose membrane 2 by 2mm, and a 10mm long glass cellulose membrane I 4 (i.e., conjugate pad) is successively overlapped and attached to the lower end of the solid nitrocellulose membrane 2, a 15mm long blood filtration membrane 5, and a 17mm long glass cellulose membrane II 6 (i.e., sample pad), and the strip is encased in a protective plastic shell.

[0046] The solid nitrocellulose membrane 2 is equipped with a quality control line 7 (i.e., C line) and a detection line 8 (i.e., T line).

[0047] The test line 8 is positioned close to the glass cellulose membrane I4 and is formed by spraying extracted Brucella LPS; the quality control line 7 is positioned close to the absorbent filter paper 3 and is formed by spraying sheep anti-chicken IgY.

[0048] The conjugate pad of the sample processing layer includes latex microspheres labeled with chicken IgY, latex microspheres labeled with Protein G protein, and / or latex microspheres labeled with Fcα / μR receptor. The latex microspheres labeled with chicken IgY are a different color from those labeled with Protein G protein and Fcα / μR receptor. Specifically, the latex microspheres are mainly located on the conjugate pad closest to the chromatography membrane, i.e., the glass cellulose membrane I4 is coated with blue latex microspheres labeled with chicken IgY, red latex microspheres labeled with Protein G protein, and red latex microspheres labeled with Fcα / μR receptor.

[0049] The above Brucella antibody test strips are prepared through the following steps:

[0050] 1. Labeling of latex microspheres

[0051] 1.1 Cleaning of latex microspheres

[0052] Take 12.5 μL of 400 nm latex microspheres (4% solid content) and add them to 1 mL of 10 mmol / L pH 6.2 MES (2-(N-morpholine) ethanesulfonic acid). Mix thoroughly, centrifuge at 20000 g for 10 min at 15℃, discard the supernatant, keep the precipitate, add 1 mL of 10 mmol / L pH 6.2 MES solution to resuspend and reconstitute, and disperse by ultrasonication in a water bath.

[0053] 1.2 Activation of latex microspheres

[0054] After cleaning, 3.5 μL of 10 mg / mL EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) was added and vortexed. Then, 33 μL of 10 mg / mL sulfo-NHS (N-hydroxysuccinimide) was slowly added and ultrasonically mixed. The mixture was then placed on a rotary mixer and rotated at a constant speed for 15 min. After centrifugation at 20,000 g for 10 min at 15 °C, the supernatant was discarded, and the precipitate was resuspended and reconstituted in 1 mL of 10 mmol / L pH 6.2 MES solution. The precipitate was then ultrasonically dispersed in a water bath.

[0055] 1.3 Microsphere-labeled proteins

[0056] Take the activated latex microspheres, add 50 μg of Protein G or Fcα / μR receptor or chicken IgY, mix quickly, and place at room temperature for 120 min to rotate and mix.

[0057] 1.4. Microsphere surface sealing

[0058] Add 500 μL of microsphere blocking solution to the labeled microsphere centrifuge tubes and mix by rotation at room temperature for 60 min. 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.

[0059] 1.5 Resolution of Microspheres

[0060] After complete labeling and blocking, the latex microspheres were centrifuged at 20,000 g for 10 min at 15℃ to remove unreacted and bound proteins. The supernatant was discarded, and the precipitate was collected. 1 mL of microsphere washing buffer was added, and the mixture was thoroughly mixed and centrifuged again to remove the precipitate. 500 μL of microsphere preservation solution was added to the precipitate to reconstitute it. The labeled microspheres were then sonicated to disperse them evenly, yielding labeled latex microspheres of Protein G, Fcα / μR receptor, or chicken IgY, i.e., microspheres conjugated with Protein G, microspheres conjugated with Fcα / μR receptor, and microspheres conjugated with chicken IgY. These were stored at 4℃. The microsphere washing buffer consisted of 50 mM Tris (pH 8.0 ± 0.05), 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin300. The microsphere preservation solution consisted of 25 mM Tris, 150 mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, and 0.1% ProClin 300, with the pH adjusted to 8.7 ± 0.05.

[0061] 2. Preparation of the binding pad (i.e., glass cellulose membrane I4)

[0062] The cut conjugate pads were placed in an enamel tray, and 2 mL of conjugate pad treatment solution was added to completely soak them. They were then dried in a 45℃ electric heating oven for 2 hours for later use. The microspheres coupled with Protein G, Fcα / μR receptors, and chicken IgY obtained in step 1.5 were ultrasonically dispersed in a water bath and mixed in a 1:1:1 ratio. The mixture was then pumped into a gold spraying apparatus at a flow rate of 6 μL / cm. After setting the relevant parameters, the microspheres were uniformly sprayed onto pre-treated conjugate pads cut to 5 mm wide × 30 mm long using the gold spraying apparatus. The pads were then dried in a 45℃ electric heating oven for 2 hours or overnight at 37℃, and then packaged for later use. The conjugate pad treatment solution consisted of 1% BSA, 5% sucrose, 0.1% PEG20000, 50 mM Tris, 0.1% Tween-20, and 0.1% PC300.

[0063] 3. Protein coating

[0064] In this experiment, a Sartorius CN95 nitrocellulose membrane was used and fixedly adhered to a PVC substrate. The NC membrane was equilibrated for 30 min in an environment with a humidity of 40%–60%. Goat anti-chicken IgY antibody was applied to the C-line, and extracted Brucella LPS was applied to the T-line. Both were diluted to a working concentration of 1 mg / mL with the coating buffer and then pumped into a gold-sprayed coating apparatus. The coating rate was set to 1 μL / cm. Under the action of the contact coating head, T and C-line proteins were uniformly coated onto the NC membrane. After the operation, the sheet was placed in a 45℃ electric heating oven for 2 h or dried overnight at 37℃. After drying, it was stored in an aluminum foil bag for later use. The coating buffer was 1% sucrose and 25 mM Tris (pH 7.2±0.05).

[0065] 4. Assembly of test strip cards

[0066] Cut the sample pad fiberglass into 17 mm wide pieces, the blood filtration membrane into 10 mm wide pieces, and the absorbent paper into 17 mm wide pieces. After spraying the conjugate pad with a marker and coating the NC membrane with the relevant protein, attach the pads to the PVC base plate in the following order: absorbent paper, NC membrane, conjugate pad, blood filtration membrane, and sample pad. Then, use a strip cutter to cut the pads into 4 mm wide strips. Place the strips into the appropriately sized test strip clips to assemble the test strip card, which is then ready for testing.

[0067] Example 2: Optimization of Brucella Antibody Test Strips

[0068] Biotin exhibits a very high affinity for streptavidin, and one streptavidin molecule can bind to multiple biotin molecules. This binding is specific, rapid, and stable, producing a multi-level amplification effect. Non-covalent interactions between metal ions and proteins can improve protein stability and affinity without strongly disrupting protein binding sites. Therefore, in the embodiments of this invention, these two methods improve the detection sensitivity of the test strip for early Brucella infection samples.

[0069] 2.1 Adding a biotin-streptavidin system

[0070] 100 μg of LPS was taken and biotin was added at a molar ratio of LPS to biotin of 1:100, with a total volume of 100 μL. The mixture was incubated at room temperature for 4 h, then diluted with PB buffer to 5 times the reaction volume. The solution was dialyzed at 4 °C for 72 h to obtain biotinylated LPS. The red latex microspheres were replaced with streptavidin-modified red latex microspheres, and Fcα / μR receptors were labeled according to the experimental procedures in Example 1.

[0071] 2.2 Enhancing the binding affinity between microglobulin-antibody complexes and LPS

[0072] Metal ions from magnesium chloride, copper sulfate, calcium chloride, and zinc chloride were diluted to 0.5 μM, 50 μM, 500 μM, 1 mM, and 25 mM respectively using 25 mM PB buffer at pH 5.0, 6.0, 7.0, 8.0, and 9.0, and 25 mM Tris buffer at pH 7.0, 8.0, 9.0, 10.0, and 11.0. These parameters were then paired and combined to create different experimental protocols. 1% sucrose was added to each buffer to prepare a coating solution. LPS was diluted to 1 mg / mL and coated onto the T line. Sensitivity tests were performed using samples infected with Brucella in the early stages.

[0073] Experimental results show that Cu 2+ Diluting copper sulfate to 50 μM using a pH 6.0 25 mM PB buffer significantly improved the detection sensitivity for IgM. 2+ Diluting calcium chloride to 0.5 μM with a pH 7.0 25 mM PB buffer significantly improved the detection sensitivity for IgM. Adding two other metal ions did not significantly improve the sensitivity.

[0074] Copper sulfate and calcium chloride were diluted to 0.5 μM, 50 μM, and 500 μM respectively with 25 mM PB buffer at pH 7.0. Nine different metal ion mixtures were obtained by mixing the metal ion solutions at a 1:1 ratio. 1% sucrose was added to each metal ion mixture to prepare a coating solution. LPS was diluted to 1 mg / mL and coated onto the T line. Sensitivity testing was performed using early-stage Brucella-infected samples. It was found that 0.5 μM Cu... 2+ and 0.5μM Ca 2+ There is a synergistic effect between them. Test strips made by coating LPS with a mixture of metal ions of this concentration have higher IgM detection sensitivity than test strips made with a single metal ion.

[0075] Simultaneously perform operations 2.1 and 2.2, that is, simultaneously add the biotin-streptavidin system and add 0.5 μM Cu. 2+ and 0.5μM Ca 2+ The optimized test strips were obtained.

[0076] Example 3

[0077] The test strips made using the Fcα / μR receptor (i.e., Example 1) showed higher sensitivity for detecting early Brucella infections in samples than the test strips made using IgM secondary antibodies (see Example 1). Figure 2 ).

[0078] When the same early infection sample was tested, the results were as follows: Figure 3As shown in the figure, the test line of the test strip optimized in section 2.1 is clearer than that of the unoptimized test strip.

[0079] Add an appropriate amount of Cu to the coating solution 2+ and Ca 2+ (i.e., 2.2 of Example 2) can synergistically improve the detection sensitivity of the test strip for IgM. Figure 4 ).

[0080] The optimized test strip from Example 2 (i.e., the test strip optimized in both 2.1 and 2.2) was used to test one clinical sample in the early to mid-stage of infection, one clinical serum sample in the late stage of infection, and one negative clinical sample. The results are shown in the graph. The T line is clearly visible. The experimental results are as follows: Figure 5 As shown.

[0081] The optimized test strip from Example 2 (i.e., the test strip optimized in both 2.1 and 2.2) can detect Brucella-positive samples with 1 / 10 of the original IgM antibody content, thus improving the sensitivity for early Brucella diagnosis. Figure 6 ).

[0082] Example 4: Functional Verification of Test Strip Card Products

[0083] 1. Test strip result interpretation criteria

[0084] Place 10 μL of the unknown sample directly above the sample well on the test strip card, and vertically add 60 μL of sample buffer into the well. Time the test for 8 minutes, and interpret the results according to the established method. The interpretation criteria are as follows (see diagram). Figure 7 :

[0085] Figure 7 (a) indicates that the test sample is a blood sample or a raw milk sample. After the test strip reacts, when both the control line and the test line show color, the result suggests that the sample contains Brucella IgM or IgM and IgG antibodies, and the sampled animal is a Brucella positive infection.

[0086] Figure 7 (b) indicates that the test sample is a blood sample or a raw milk sample. After the test strip reacts, if the control line develops color but the test line does not, the result indicates that the sample does not contain Brucella IgG antibodies or IgM antibodies, and the sampled animal is not infected with Brucella.

[0087] Figure 7 (c) indicates that the test sample is a blood sample or a raw milk sample. After the test strip reacts, if neither the control line nor the test line shows color, the result indicates that the test is invalid and the test strip needs to be used again.

[0088] 2. Specificity analysis of test strips

[0089] Test strips from the same batch were used to test positive samples for antibodies against different species of Brucella ovine M16, bovine 2308, swine S2, O157 Escherichia coli, Yersinia enterocolitica, and Salmonella. The results were observed after a 15-minute reaction time. The results are as follows: Figure 8 As shown, the test strip tested positive for antibodies against different species of Brucella, while the test results for samples containing interfering bacterial antibodies were all negative. This indicates that the test strip does not have significant cross-reaction with other bacterial antibodies and can specifically bind to multiple genera of Brucella.

[0090] 3. Test strip repeatability analysis

[0091] Early Brucella infection antibody-positive reference serum was diluted to three concentrations: high, medium, and low. Test strips from the same batch were randomly selected, and each serum sample was tested 10 times in parallel. The results were observed, color differences were counted, and the intra-batch coefficient of variation (CV) was calculated. Additionally, three samples were randomly selected from each of three consecutively prepared batches of test strips, and the serum samples at the different dilutions were tested. The results were observed, color differences were counted, and the inter-batch CV was calculated. The CV = (number of samples with color differences / total number of samples) × 100%. Test strips from the same batch showed consistent colorimetric development across the three reference concentrations, indicating good intra-batch repeatability. Test strips from three consecutive batches also showed consistent colorimetric development across the three reference concentrations, indicating good inter-batch repeatability.

[0092] 4. Analysis of the accuracy of test strips

[0093] Blood samples (serum, plasma, whole blood) or raw milk samples from different breeds of sheep and cattle, both negative and positive for Brucella antibodies, were collected. The presence of IgM antibodies in the samples was detected using ELISA, and samples were classified into early / acute infection and mid-to-late / recurrent infection categories. The classified clinical samples were then tested according to the instructions for the test strips, ELISA kits, and tube agglutination tests. The positive concordance rate (diagnostic sensitivity, Se) and negative concordance rate (diagnostic specificity, Sp) between the colloidal gold test strips and the ELISA kits and tube agglutination tests were evaluated, and the consistency between the test strips and the ELISA kits and tube agglutination test reagents was compared by calculating the Kappa value. A Kappa value greater than 0.75 between the test strips and the ELISA kits or tube agglutination tests was considered to indicate high consistency.

[0094] 5. Stability (Shelf Life) Analysis of Test Strips

[0095] Fifty samples from each of the three batches of test strips prepared consecutively were placed at 45℃ for accelerated thermal stability testing. The prepared test strips were then placed at a constant temperature of 45℃ for 0, 1, 3, 5, 7, 14, 21, 28, 35, 42, 56, 77, and 91 days. Positive control samples were tested. Within the set testing time, no intra-batch variation or undetectable positive control samples were observed in the test strips, and the colorimetric results were consistent, indicating good stability.

[0096] Example 5

[0097] Preparation of Brucella antibody test strips

[0098] Preparation of a Brucella antibody test strip capable of differentiating infection stages, the specific structure of which is as follows: Figure 9 As shown, it includes: a base plate, a 4mm wide and 60mm long strip placed in the middle of the base plate, the bottom of the strip is supported by a rigid plastic base plate 1, a 25mm long solid nitrocellulose membrane 2 (chromatographic membrane) is attached to the middle, a 17mm long absorbent filter paper 3 (absorbent pad) is attached to the upper end and overlaps the solid nitrocellulose membrane 2 by 2mm, a 10mm long glass cellulose membrane I 4 (conjugate pad) is attached to the lower end of the solid nitrocellulose membrane in sequence, a 15mm long blood filtration membrane 5, and a 17mm long glass cellulose membrane II 6 (sample pad), and the strip is encased in a protective plastic shell.

[0099] The solid nitrocellulose membrane 2 (chromatographic membrane) is provided with a control line 7 and a detection line 8 (i.e., T1) and a detection line T29; the detection lines are located near the binding pad, and the T1 detection line 8 is formed by spraying protein G protein; the detection line T29 is formed by spraying Fcα / μR receptor; the control line 7 is located near the absorbent filter paper 3, and the control line 7 is formed by spraying goat anti-chicken IgY.

[0100] The conjugate pad was sprayed with chicken IgY labeled with red latex microspheres and Brucella extract LPS labeled with red latex microspheres.

[0101] The above Brucella antibody test strips are prepared through the following steps:

[0102] 1. Labeling of latex microspheres

[0103] 1.1 Cleaning of latex microspheres

[0104] 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 20000 g for 10 min at 15℃, discard the supernatant, keep the precipitate, add 1 mL of 10 mmol / L pH6.2 MES solution to resuspend and reconstitute, and disperse by ultrasonication in a water bath.

[0105] 1.2 Activation of latex microspheres

[0106] After cleaning, the latex microspheres were first added to 3.5 μL of 10 mg / mL EDC and vortexed. Then, 33 μL of 10 mg / mL sulfo-NHS was slowly added and sonicated. The mixture was then placed on a rotary mixer and vortexed at a constant speed for 15 min. After centrifugation at 20,000 g for 10 min at 15 °C, the supernatant was discarded and the precipitate was retained. The precipitate was resuspended and reconstituted in 1 mL of 10 mmol / L pH 6.2 MES solution and dispersed by sonication in a water bath.

[0107] 1.3 Microsphere-labeled proteins

[0108] Take the activated latex microspheres, add 50 μg of Brucella LPS or chicken IgY, mix quickly, and place at room temperature for 120 min to rotate and mix.

[0109] 1.4. Microsphere surface sealing

[0110] Add 500 μL of microsphere blocking solution to the labeled microsphere centrifuge tubes and mix by rotation at room temperature for 60 min. 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.

[0111] 1.5 Resolution of Microspheres

[0112] After complete labeling and blocking, the latex microspheres were centrifuged at 20,000 g for 10 min at 15℃ to remove unreacted and bound proteins. The supernatant was discarded, and the precipitate was collected. 1 mL of microsphere washing buffer was added, and the mixture was thoroughly mixed and centrifuged again to remove the precipitate. 500 μL of microsphere preservation solution was added to the precipitate to reconstitute it. The labeled microspheres were then sonicated to disperse them evenly, yielding labeled LPS or chicken IgY latex microspheres, which were stored at 4℃. The microsphere washing buffer consisted of 50 mM Tris (pH 8.0 ± 0.05), 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin 300. The microsphere preservation solution consisted of 25 mM Tris, 150 mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, and 0.1% ProClin 300, adjusted to pH 8.7 ± 0.05.

[0113] 2. Preparation of the binding pad

[0114] The cut conjugate pads were placed in an enamel tray, and 2 mL of conjugate pad treatment solution was added to completely soak them. They were then dried in a 45℃ electric heating oven for 2 hours for later use. The preserved microsphere-labeled proteins were dispersed by ultrasonication in a water bath. Microspheres coupled with LPS and chicken IgY were mixed at a 2:1 ratio and pumped into a gold spraying apparatus at a flow rate of 6 μL / cm. After setting the relevant parameters, the microspheres were evenly sprayed onto pre-treated conjugate pads cut to 5 mm wide × 30 mm long using the gold spraying apparatus. The pads were then dried in a 45℃ electric heating oven for 2 hours or overnight at 37℃, and then packaged for later use. The conjugate pad treatment solution consisted of 1% BSA, 5% sucrose, 0.1% PEG20000, 50 mM Tris, 0.1% Tween-20, and 0.1% PC300.

[0115] 3. Protein coating

[0116] This experiment used a Sartorius CN95 nitrocellulose membrane, which was fixed and adhered to a PVC substrate. The membrane was equilibrated for 30 min in an environment with 40%–60% humidity. The C-line contained goat anti-chicken IgY antibody, the T1 line (detection line 8) contained Protein G protein, and the T2 line (detection line T29) contained Fcα / μR receptor. These proteins were diluted to a working concentration of 1 mg / mL with the coating buffer and then pumped into a gold-sprayed coating apparatus. The coating rate was set to 1 μL / cm. Under the action of the contact coating head, the T and C-line proteins were uniformly coated onto the NC membrane. After the operation, the membrane was placed in a 45℃ electric heating oven for 2 h or dried overnight at 37℃. After drying, it was stored in an aluminum foil bag for later use. The coating buffer was 1% sucrose and 25 mM Tris (pH 7.2 ± 0.05).

[0117] 4. Assembly of test strip cards

[0118] Cut the sample pad fiberglass into 17 mm wide pieces, the blood filtration membrane into 10 mm wide pieces, and the absorbent paper into 17 mm wide pieces. After spraying the conjugate pad with a marker and coating the NC membrane with the relevant protein, attach the pads to the PVC base plate in the following order: absorbent paper, NC membrane, conjugate pad, blood filtration membrane, and sample pad. Then, use a strip cutter to cut the pads into 4 mm wide strips. Place the strips into the appropriately sized test strip clips to assemble the test strip card, which is then ready for testing.

[0119] Example 6 Optimization of Brucella Antibody Test Strip

[0120] Biotin exhibits a very high affinity for streptavidin, and one streptavidin molecule can bind to multiple biotin molecules. This binding is specific, rapid, and stable, generating a multi-level amplification effect. Non-covalent interactions between metal ions and receptor proteins can improve the stability and affinity of receptor proteins without strongly disrupting their antibody-binding sites. Therefore, in the embodiments of this invention, these two methods improve the analytical sensitivity of the T2 line on the test strip.

[0121] 6.1. Adding a biotin-streptavidin system to specifically optimize the T2 line color development intensity.

[0122] 100 μg of Fcα / μR receptor was taken, and biotin was added at a molar ratio of Fcα / μR receptor to biotin of 1:100, with a total volume of 100 μL. The mixture was incubated at room temperature for 4 h, then diluted to 5 times the reaction volume with PB buffer and dialyzed at 4 °C for 72 h to obtain the biotinylated Fcα / μR receptor. The red latex microspheres were replaced with streptavidin-modified red latex microspheres, and LPS was labeled according to the experimental procedures in Example 1.

[0123] 6.2 Targeted optimization of the binding affinity between Fcα / μR receptor and antibody

[0124] Metal ions from ferric chloride, copper sulfate, calcium chloride, and zinc chloride were diluted to 0.05 μM, 0.5 μM, 5 μM, 50 μM, 500 μM, and 1 mM respectively using 25 mM PB buffer at pH 5, 6, 7, 8, and 9, and 25 mM Tris buffer at pH 7, 8, 9, 10, and 11. These parameters were then paired and combined to create different experimental protocols. 1% sucrose was added to each buffer to prepare a coating solution. The Fcα / μR receptor was diluted to 1 mg / mL and coated onto the T2 line. Sensitivity testing was performed using early-stage Brucella-infected samples.

[0125] The test strips developed after optimization in Example 6 (i.e., the test strips optimized simultaneously in 6.1 and 6.2) and the unoptimized test strips (i.e., the test strips from Example 5) were used to test one clinical serum sample from the early stage of infection and two samples from the early to mid-stage of infection, respectively. The color intensity of the T2 and T1 lines was observed, and the experimental results are as follows: Figure 10 As shown, in the experimental images with different numbers, that is Figure 10 In (a)-(c), the first and second test strips from left to right in each group of experimental results are the test results of the test strips before optimization, and the third and fourth test strips are the test results of the test strips after optimization. By optimizing the sensitivity of binding IgM antibody, the color intensity of T2 line has been significantly improved compared with that before optimization, making it easier to determine the difference in color intensity between T1 line and T2 line, thereby improving the inference of the infection period of the test sample.

[0126] Example 7 Functional verification of test strip products

[0127] 1. Criteria for interpreting test strip results

[0128] Place 10 μL of the unknown sample directly above the sample well on the test strip card, and vertically add 60 μL of sample buffer into the well. Time the test for 8 minutes, and interpret the results according to the established method. The interpretation criteria are as follows (see diagram). Figure 11 :

[0129] Figure 11 (a) indicates that if the sample is a blood sample, after the test strip reacts, if the control line and the T2 test line are both colored, and the T1 test line is colored or not colored, the result suggests that the sample contains Brucella IgM or IgM and IgG antibodies, and the sampled animal is in the early or acute stage of Brucella infection.

[0130] Figure 11 (b) indicates that if the sample is a blood sample, after the test strip reacts, when both the control line and the T1 test line show color, but the T2 test line does not show color, the result suggests that the sample contains Brucella IgG antibodies but does not contain IgM antibodies, and the sampled animal is in the middle or late stage of Brucella infection or a recurrent infection.

[0131] Figure 11 (c) indicates that if the sample is raw milk, after the test strip reacts, if the control line and the T1 test line are both colored, and the T2 test line is colored or not colored, the result suggests that the sample contains Brucella IgA antibody or IgA and IgG antibodies, and the sampled animal has Brucella infection.

[0132] Figure 11 (d) indicates that if the sample is a raw milk sample or a blood sample, only the control line will show color after the test strip reacts, while the T1 and T2 test lines will not show color. The result suggests that the sample does not contain Brucella antibodies and the sampled animal has not been infected with Brucella.

[0133] Figure 11 (e) indicates that if the test sample is a raw milk sample or a blood sample, after the test strip card reacts, the control line does not show color, and the T1 test line and T2 test line show color or do not show color, indicating that the test is invalid.

[0134] 2. Specificity analysis of test strips

[0135] Test strips from the same batch were used to test positive samples for antibodies against different species of Brucella ovine M16, bovine 2308, swine S2, O157 Escherichia coli, Yersinia enterocolitica, and Salmonella. After a 15-minute reaction time, the results were observed, and all were negative, indicating no significant cross-reactivity between the test strips and other bacterial antibodies. Simultaneous testing of Brucella IgM and IgG antibody-positive reference sera also showed no significant cross-reactivity between these two immunoglobulin detection methods.

[0136] 3. Test strip repeatability analysis

[0137] Brucella IgM antibody-positive reference sera and Brucella IgG antibody-positive reference sera were diluted to three concentrations: high, medium, and low. Test strips from the same batch were randomly selected, and each serum sample was tested 10 times in parallel. The results were observed, color differences were counted, and the intra-batch coefficient of variation (CV) was calculated. Additionally, three samples were randomly selected from each of the three consecutively prepared batches of test strips, and the serum samples at the different dilutions were tested. The results were observed, color differences were counted, and the inter-batch CV was calculated. The CV = (number of samples with color differences / total number of samples) × 100%. Test strips from the same batch showed consistent colorimetric results for the three reference concentrations, indicating good intra-batch repeatability. Test strips from three consecutive batches showed consistent colorimetric results for the three reference concentrations, indicating good inter-batch repeatability.

[0138] 4. Analysis of test strip accuracy

[0139] Blood samples (serum, plasma, whole blood) or raw milk samples from different breeds of sheep and cattle, both negative and positive for Brucella antibodies, were collected. The presence of IgM antibodies in the samples was detected using ELISA, and samples were classified into early / acute infection and mid-to-late / recurrent infection categories. The classified clinical samples were then tested according to the instructions for the test strips, ELISA kits, and tube agglutination tests. The positive concordance rate (diagnostic sensitivity, Se) and negative concordance rate (diagnostic specificity, Sp) between the colloidal gold test strips and the ELISA kits and tube agglutination tests were evaluated, and the consistency between the test strips and the ELISA kits and tube agglutination test reagents was compared by calculating the Kappa value. A Kappa value greater than 0.75 between the test strips and the ELISA kits or tube agglutination tests was considered to indicate high consistency.

[0140] 5. Stability (Shelf Life) Analysis of Test Strips

[0141] Fifty samples from each of the three batches of test strips prepared consecutively were placed at 45℃ for accelerated thermal stability testing. The prepared test strips were then placed at a constant temperature of 45℃ for 0, 1, 3, 5, 7, 14, 21, 28, 35, 42, 56, 77, and 91 days. Positive control samples were tested, and no intra-batch variation or undetectable positive control samples were observed. The colorimetric results were consistent, indicating good stability.

[0142] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0143] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A Brucella antibody test strip, characterized in that, Includes a base plate (1), a chromatography membrane disposed on the base plate (1), a sample processing layer disposed at one end of the chromatography membrane, and a quality control line (7) and at least one detection line disposed on the chromatography membrane; The sample processing layer comprises chicken IgY-labeled latex microspheres, Protein G-labeled latex microspheres, and Fcα / μR receptor-labeled latex microspheres; the detection line comprises Brucella LPS; or, The sample processing layer includes Brucella LPS-labeled latex microspheres and chicken IgY-labeled latex microspheres, and the detection line includes Protein G protein and Fcα / μR receptor; The quality control line (7) includes sheep anti-chicken IgY antibody.

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

3. The Brucella antibody test strip as described in claim 1, characterized in that, The detection lines are two in number. The sample processing layer includes Brucella LPS-labeled latex microspheres and chicken IgY-labeled latex microspheres. One detection line includes Protein G protein, and the other detection line includes Fcα / μR receptor.

4. The Brucella antibody test strip as described in claim 1, characterized in that, The detection line (8) also includes metal ions, which are copper ions and / or calcium ions.

5. The Brucella antibody test strip as described in claim 4, characterized in that, The metal ions are copper ions and calcium ions, with a molar ratio of 1:

1.

6. The Brucella antibody test strip as described in any one of claims 1-5, characterized in that, The chromatography membrane is a nitrocellulose membrane (2), and the sample processing layer includes a conjugate pad, a blood filtration membrane (5), and a sample pad that are sequentially overlapped on the chromatography membrane. The conjugate pad is a glass cellulose membrane I (4), and the sample pad is a glass cellulose membrane II (6). The other end of the chromatography membrane is provided with absorbent filter paper (3).

7. The Brucella antibody test strip as described in any one of claims 1-5, characterized in that, The sample processing layer includes a processing solution consisting of 1% BSA, 5% sucrose, 0.1% PEG20000, 50mM Tris, 0.1% Tween-20, and 0.1% PC300.

Images