A method for protein chip detection of brucellosis

Abstract

This application discloses a method for protein chip detection of brucellosis, relating to the technical field of brucellosis detection. The method involves spotting Brucella synergistic composite antigens for multi-index combined detection onto a pretreated solid-phase carrier, drying, and then washing. A blocking agent is then added for blocking, followed by washing to obtain a protein chip. An initial sample is then collected, diluted, centrifuged, and the supernatant is collected. The supernatant is added to the protein chip site, incubated with shaking, washed, and then a secondary antibody is added for further incubation and washing. Finally, a fluorescence scanner is used to read the signal, subtract the background, and determine whether the sample is positive or negative. This method has a wide detection range, strong specificity, and high sensitivity, and has a very broad application prospect.

Description

Technical Field

[0001] The present application relates to the technical field of brucellosis detection, and in particular to a method for protein chip detection of brucellosis. Background Art

[0002] Currently commonly used brucellosis detection methods are limited by the use of a single antigen, resulting in a narrow detection range and an inability to cover infection detection for multiple strains, such as bovine, ovine, and human species. Furthermore, the solid-phase carrier surface of common protein chips often does not bind firmly enough to the antigen protein loaded thereon, which can easily cause the antigen protein to fall off, significantly reducing detection accuracy. Accuracy, repeatability, and sensitivity are all crucial in protein chip detection methods. However, while some current brucellosis protein chip detection methods often have high accuracy, they struggle to balance other performance indicators. Therefore, the development of new detection methods is needed. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this application provides a method for protein chip detection of brucellosis. The method involves spotting a Brucella synergistic composite antigen for multi-indicator combined detection onto a pretreated solid-phase carrier, drying it, and washing it. A blocking agent is then added for blocking, followed by washing to obtain a protein chip. An initial sample is then collected, diluted, centrifuged, and the supernatant is collected. The supernatant is applied to the protein chip site, incubated with shaking, washed, and then a secondary antibody is added for further incubation and washing. Finally, a fluorescence scanner is used to read the signal, subtract the background, and determine whether the test is positive or negative. This method has a wide detection range, strong specificity, and high sensitivity, and has broad application prospects.

[0004] To achieve the above objectives, this application adopts a technical solution comprising the following steps:

[0005] Spotting Brucella synergistic complex antigens on the pretreated solid phase support, followed by drying and washing with detergent;

[0006] Add a blocking agent, then block and wash with a detergent to obtain a protein chip;

[0007] Collecting an initial sample, diluting the initial sample with a diluent, centrifuging it, and taking the supernatant;

[0008] Add the quantitative supernatant sample to the protein chip site, incubate with shaking, wash with detergent, then add the secondary antibody, incubate, and wash;

[0009] Fluorescence signals were read using a fluorescence scanner, and the blank background was subtracted to determine whether the test was positive or negative. The infection status was then analyzed based on the IgG / IgM subtype.

[0010] The concentration of the Brucella synergistic composite antigen is 1-20 μg / mL, and the composite antigen includes a first component and a second component; the first component includes an S-LPS antigen, a BP26 antigen, and a VirB12 antigen; the second component includes any one of OMP25, OMP31, OMP2b, OMP16, OMP19, and an outer membrane protein multi-epitope chimeric antigen; wherein the outer membrane protein multi-epitope chimeric antigen includes a fusion or chimera of any four of OMP25, OMP31, OMP2b, OMP16, and OMP19.

[0011] In a possible implementation, the amount of the supernatant sample added to the protein chip site is 50 μL.

[0012] In one possible implementation, the pretreatment of the solid support comprises the following steps:

[0013] Soak the solid phase support in a 1-5% mass concentration NaOH solution or KOH solution for 5-10 minutes, take it out and wash it with pure water;

[0014] The cleaned solid phase support is immersed in hydrogen peroxide with a mass concentration of 5-15% for 10-20 minutes, taken out and washed with pure water to obtain a hydroxylated solid phase support;

[0015] The hydroxylated solid phase support is cyclically immersed and pulled in an ethanol solution of an aldehyde silane coupling agent using an immersion and pulling coating machine;

[0016] After the cycle is completed, the solid phase support is dried in a vacuum oven at 110-120° C. for 1-2 hours to obtain a pretreated solid phase support.

[0017] In a possible implementation, the pretreated solid support includes any one of an aluminum oxynitride sheet, a glass sheet, or a silicon sheet surface-treated with an aldehyde-silane coupling agent.

[0018] In a possible implementation, the aldehyde-based silane coupling agent includes any one of trimethoxysilyl propionaldehyde, triethoxysilyl propionaldehyde, triethoxysilyl butyraldehyde, and triethoxysilyl undecanealdehyde.

[0019] In a possible implementation, in the ethanol solution of the aldehyde silane coupling agent, the mass ratio of the aldehyde silane coupling agent to ethanol is (1-5): (95-99).

[0020] In one possible implementation, the dipping and pulling cycles are performed 20 to 50 times, wherein each dipping time is 1 to 3 minutes, and each pulling rate is 2 to 10 mm / s. In a specific implementation of this application, the dipping and pulling cycles are preferably performed 20 times, wherein each dipping time is 1 minute, and each pulling rate is 2 mm / s.

[0021] In a possible implementation, the detergent is a PBS solution containing 0.05-0.1% Tween-20 by mass; the concentration of the PBS solution is 0.1 M, and the pH is 7.4.

[0022] In one possible implementation, the blocking agent includes any one of a first blocking agent and a second blocking agent; wherein the first blocking agent includes a mixture of 0.1M Tris-HCl buffer at pH 8.0, PEG-20000 (mass concentration 0.5%) and Tween-20 (mass concentration 0.1%); and the second blocking agent includes a mixture of gelatin (mass concentration 1%), glycine (0.1M), Tris-HCl buffer at pH 7.4 (0.1M) and Tween-20 (mass concentration 0.05%).

[0023] In one possible implementation, the initial sample includes any one of human and animal serum and plasma containing brucellosis antibodies; the animal includes any one of pigs, cattle, and sheep; the volume ratio of the initial sample to the diluent is 1:100; and the diluent includes a mixture of 10 mM PBS buffer (pH 7.2~7.4), 0.15 M NaCl, and 0.1% Tween-20.

[0024] In a possible implementation, the amount of the supernatant sample added is 10-50 μL.

[0025] In one possible implementation, the secondary antibody includes Cy3-labeled anti-human IgG and Cy5-labeled anti-animal IgM; the Cy5-labeled anti-animal IgM includes any one of Cy5-labeled anti-sheep IgM, Cy5-labeled anti-bovine IgM and Cy5-labeled anti-pig IgM; the mass ratio of the Cy3-labeled anti-human IgG and the Cy5-labeled anti-animal IgM is (60~80):(20~40).

[0026] In one possible implementation, the first set temperature is 37±1°C, the second set temperature is 4°C; the number of settings is 3 times; the first time is 1~2h, the second time is 2~3h, the third time is 10~15min, the fourth time is 0.5~1.5h, and the fifth time is 40~60min; the first speed is 9000~15000rpm.

[0027] Beneficial technical effects:

[0028] In this application, the first component of the Brucella synergistic composite antigen (S-LPS antigen, BP26 antigen, and VirB12 antigen) and the second component (any one of OMP25, OMP31, OMP2b, OMP16, OMP19, and outer membrane protein multi-epitope chimeric antigen) cover key Brucella antigens such as lipopolysaccharide, conserved proteins, and outer membrane proteins. They can simultaneously recognize host antibodies targeting different structures (such as IgG / IgM), avoiding the limitations of single antigen detection. Furthermore, through IgG / IgM subtype detection, it can effectively distinguish between acute infection (predominantly IgM) and chronic infection (predominantly IgG), providing more accurate diagnosis of infection status for clinical diagnosis. Furthermore, the antigenic epitopes of different components of the composite antigen can simultaneously bind to corresponding antibodies in the sample, forming "multi-target recognition," amplifying the fluorescence signal and improving detection sensitivity and specificity.

[0029] In addition, in the present application, the aldehyde groups on the surface of the solid phase carrier treated with the aldehyde silane coupling agent are connected to the amino groups of the antigen protein. Stable covalent bonds are formed to avoid antigen shedding caused by physical adsorption and improve detection accuracy. Moreover, the spatial conformation of the covalently fixed antigen is stable, which can effectively retain the activity of the antigen epitope. The surface chemical properties of the solid phase carrier used in this application are uniform, which can reduce batch differences and improve chip stability and detection repeatability. In addition, the hydroxylation-aldehyde treatment enhances the hydrophilicity of the solid phase carrier surface, and the uniform coating formed by the silane coupling agent on the solid phase carrier during the pulling and impregnation process can ensure the uniformity of the antigen concentration at the chip site, block the nonspecific binding of impurities in the sample (such as nonspecific proteins), and further improve sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the process of protein chip detection of brucellosis.

[0031] Figure 2 This is a physical picture of the protein chip. DETAILED DESCRIPTION

[0032] In order to make the technical problems, technical solutions, and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below with reference to the following examples. However, this should not be construed as limiting the scope of this application to the following examples. Without departing from the above-mentioned method concepts of this application, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of this application.

[0033] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0034] The following will specifically describe a method for protein chip detection of brucellosis provided by the present application in combination with different embodiments.

[0035] Example 1:

[0036] like Figure 1 As shown, a method for protein chip detection of brucellosis comprises the following steps:

[0037] 1. Soak the aluminum oxynitride sheet in a 1% NaOH solution for 7 minutes, take it out and rinse it with pure water;

[0038] 2. Soak the cleaned aluminum oxynitride sheet in 5% hydrogen peroxide for 15 minutes, take it out and wash it with pure water to obtain a hydroxylated aluminum oxynitride sheet;

[0039] 3. Use an immersion coating machine to cyclically immerse and pull the hydroxylated aluminum oxynitride sheet in an ethanol solution of trimethoxysilyl propionaldehyde; the mass ratio of trimethoxysilyl propionaldehyde to ethanol is 2:98;

[0040] 4. After the cycle is completed, the aluminum oxynitride sheet is dried in a vacuum oven at 115°C for 1 hour to obtain an aluminum oxynitride sheet surface-treated with an aldehyde silane coupling agent;

[0041] 5. Spot 1 μg / mL of Brucella synergistic complex antigen (first component: S-LPS antigen, BP26 antigen, and VirB12 antigen; second component: OMP25) on an aluminum oxynitride sheet treated with an aldehyde silane coupling agent, then dry at 37 ± 1°C for 1 hour and wash three times with detergent.

[0042] 6. Add a mixture of Tris-HCl buffer (0.1 M) at pH 8.0, PEG-20000 (mass concentration 0.5%), and Tween-20 (mass concentration 0.1%), then block at 37 ± 1 °C for 2 h and wash three times with detergent to obtain a protein chip. Figure 2 As shown;

[0043] 7. Collect human serum containing brucellosis antibodies, dilute the sample with a mixture of 10 mM PBS buffer (pH 7.2), 0.15 M NaCl, and 0.1% Tween-20 at a volume ratio of 1:100, and centrifuge at 4°C and 9000 rpm for 10 minutes, and collect the supernatant;

[0044] 8. Add 50 μL of supernatant sample to the protein chip site, incubate with shaking at 37 ± 1 ° C for 0.5 h, and wash three times with detergent. Then add secondary antibody (including Cy3-labeled anti-human IgG and Cy5-labeled anti-goat IgM, where the mass ratio of Cy3-labeled anti-human IgG and Cy5-labeled anti-goat IgM is 75:25), incubate at 37 ± 1 ° C for 40 min, and wash three times;

[0045] 9. Use a fluorescence scanner to read the fluorescence signal, subtract the blank background, judge the positive or negative, and analyze the infection status.

[0046] The detergent used in the above steps is a PBS solution containing 0.075% Tween-20 by mass; the concentration of the PBS solution is 0.1 M and the pH is 7.4.

[0047] Example 2:

[0048] like Figure 1 As shown, a method for protein chip detection of brucellosis comprises the following steps:

[0049] 1. Soak the silicon wafer in a 3% KOH solution for 5 minutes, take it out and rinse it with pure water;

[0050] 2. Soak the cleaned silicon wafer in 10% hydrogen peroxide for 10 minutes, take it out and rinse it with pure water to obtain a hydroxylated silicon wafer;

[0051] 3. Use a dip-coating machine to dip and pull the hydroxylated silicon wafer into an ethanol solution of triethoxysilyl undecanedialdehyde in a cycle; the mass ratio of triethoxysilyl undecanedialdehyde to ethanol is 1:99;

[0052] 4. After the cycle is completed, the silicon wafer is dried in a vacuum oven at 110°C for 1.5 hours to obtain a silicon wafer surface treated with an aldehyde silane coupling agent;

[0053] 5. Spot 2 μg / mL of Brucella synergistic complex antigen (first component: S-LPS antigen, BP26 antigen, and VirB12 antigen; second component: OMP31) on a silicon wafer treated with an aldehyde silane coupling agent, then dry at 37 ± 1°C for 1.5 h and wash three times with detergent.

[0054] 6. Add a mixture of gelatin (mass concentration 1%), glycine (0.1 M), Tris-HCl buffer (pH 7.4) (0.1 M), and Tween-20 (mass concentration 0.05%), then block at 37 ± 1°C for 2.5 h and wash three times with detergent to obtain a protein chip;

[0055] 7. Collect sheep plasma containing brucellosis antibodies, dilute the sample with a mixture of 10 mM PBS buffer (pH 7.3), 0.15 M NaCl, and 0.1% Tween-20 at a volume ratio of 1:100, and centrifuge at 4°C and 10,000 rpm for 12 minutes, and collect the supernatant;

[0056] 8. Add 50 μL of supernatant sample to the protein chip site, incubate with shaking at 37 ± 1 ° C for 0.8 h, and wash three times with detergent. Then add secondary antibody (including Cy3-labeled anti-human IgG and Cy5-labeled anti-goat IgM, where the mass ratio of Cy3-labeled anti-human IgG and Cy5-labeled anti-goat IgM is 70:30), incubate at 37 ± 1 ° C for 50 min, and wash three times;

[0057] 9. Use a fluorescence scanner to read the fluorescence signal, subtract the blank background, judge the positive or negative, and analyze the infection status.

[0058] The detergent used in the above steps is a PBS solution containing 0.075% Tween-20 by mass; the concentration of the PBS solution is 0.1 M and the pH is 7.4.

[0059] Example 3:

[0060] like Figure 1 As shown, a method for protein chip detection of brucellosis comprises the following steps:

[0061] 1. Soak the glass slide in 5% NaOH solution for 10 minutes, take it out and rinse it with pure water;

[0062] 2. Soak the cleaned glass slide in 15% hydrogen peroxide for 20 minutes, take it out and rinse it with pure water to obtain a hydroxylated glass slide;

[0063] 3. Use a dip-and-pull coating machine to cyclically dip and pull the hydroxylated glass sheet in an ethanol solution of triethoxysilyl butyraldehyde; the mass ratio of triethoxysilyl butyraldehyde to ethanol is 5:95;

[0064] 4. After the cycle is completed, the glass sheet is dried in a vacuum oven at 120°C for 2 hours to obtain a glass sheet surface treated with an aldehyde silane coupling agent;

[0065] 5. Spot 5 μg / mL of Brucella synergistic complex antigen (first component: S-LPS antigen, BP26 antigen, and VirB12 antigen; second component: chimeric antigens of OMP25, OMP31, OMP2b, and OMP16) on a glass slide treated with an aldehyde silane coupling agent, then dry at 37 ± 1°C for 2 h and wash three times with detergent.

[0066] 6. Add a mixture of Tris-HCl buffer (0.1 M) at pH 8.0, PEG-20000 (mass concentration 0.5%), and Tween-20 (mass concentration 0.1%), then block at 37 ± 1°C for 2 h and wash three times with detergent to obtain a protein chip;

[0067] 7. Collect bovine serum containing brucellosis antibodies, dilute the sample with a mixture of 10 mM PBS buffer (pH 7.4), 0.15 M NaCl, and 0.1% Tween-20 at a volume ratio of 1:100, and centrifuge at 4°C and 12,000 rpm for 10 minutes, and collect the supernatant;

[0068] 8. Add 50 μL of supernatant sample to the protein chip site, incubate with shaking at 37 ± 1 ° C for 1 hour, and wash with detergent three times. Then add secondary antibody (including Cy3-labeled anti-human IgG and Cy5-labeled anti-bovine IgM, where the mass ratio of Cy3-labeled anti-human IgG and Cy5-labeled anti-bovine IgM is 60:40), incubate at 37 ± 1 ° C for 50 minutes, and wash three times;

[0069] 9. Use a fluorescence scanner to read the fluorescence signal, subtract the blank background, judge the positive or negative, and analyze the infection status.

[0070] The detergent used in the above steps is a PBS solution containing 0.075% Tween-20 by mass; the concentration of the PBS solution is 0.1 M and the pH is 7.4.

[0071] Example 4:

[0072] like Figure 1 As shown, a method for protein chip detection of brucellosis comprises the following steps:

[0073] 1. Soak the aluminum oxynitride sheet in a 2% KOH solution for 8 minutes, take it out and rinse it with pure water;

[0074] 2. Soak the cleaned aluminum oxynitride sheet in 8% hydrogen peroxide for 15 minutes, take it out and wash it with pure water to obtain a hydroxylated aluminum oxynitride sheet;

[0075] 3. Using an immersion coating machine, the hydroxylated aluminum oxynitride sheet was cyclically immersed and pulled in an ethanol solution of triethoxysilyl propionaldehyde; the mass ratio of triethoxysilyl propionaldehyde to ethanol was 3:97;

[0076] 4. After the cycle is completed, the aluminum oxynitride sheet is dried in a vacuum oven at 115°C for 1.5 hours to obtain an aluminum oxynitride sheet surface-treated with an aldehyde silane coupling agent;

[0077] 5. Spot 10 μg / mL of Brucella synergistic composite antigen (first component: S-LPS antigen, BP26 antigen, and VirB12 antigen; second component: chimeric antigens of OMP31, OMP2b, OMP16, and OMP19) on an aluminum oxynitride sheet treated with an aldehyde silane coupling agent, then dry at 37 ± 1°C for 1.5 h and wash three times with detergent.

[0078] 6. Add a mixture of gelatin (mass concentration 1%), glycine (0.1 M), pH 7.4 Tris-HCl buffer (0.1 M), and Tween-20 (mass concentration 0.05%), then block at 37 ± 1°C for 3 h and wash three times with detergent to obtain a protein chip;

[0079] 7. Collect pig plasma containing brucellosis antibodies, dilute the sample with a mixture of 10 mM PBS buffer (pH 7.2), 0.15 M NaCl, and 0.1% Tween-20 at a volume ratio of 1:100, and centrifuge at 4°C and 12,500 rpm for 15 minutes, and collect the supernatant;

[0080] 8. Add 50 μL of supernatant sample to the protein chip site, incubate with shaking at 37 ± 1 ° C for 1.2 hours, and wash three times with detergent. Then add secondary antibody (including Cy3-labeled anti-human IgG and Cy5-labeled anti-swine IgM, where the mass ratio of Cy3-labeled anti-human IgG and Cy5-labeled anti-swine IgM is 65:35), incubate at 37 ± 1 ° C for 50 minutes, and wash three times;

[0081] 9. Use a fluorescence scanner to read the fluorescence signal, subtract the blank background, judge the positive or negative, and analyze the infection status.

[0082] The detergent used in the above steps is a PBS solution containing 0.075% Tween-20 by mass; the concentration of the PBS solution is 0.1 M and the pH is 7.4.

[0083] Example 5:

[0084] like Figure 1 As shown, a method for protein chip detection of brucellosis comprises the following steps:

[0085] 1. Soak the glass slide in 4% NaOH solution for 6 minutes, take it out and rinse it with pure water;

[0086] 2. Soak the cleaned glass piece in 12% hydrogen peroxide for 10 minutes, take it out and rinse it with pure water to obtain a hydroxylated glass piece;

[0087] 3. Using a dip-and-pull coating machine, the hydroxylated glass sheet was cyclically dipped and pulled in an ethanol solution of triethoxysilyl undecanedialdehyde; the mass ratio of triethoxysilyl undecanedialdehyde to ethanol was 2:98;

[0088] 4. After the cycle is completed, the glass sheet is dried in a vacuum oven at 120°C for 1 hour to obtain a glass sheet surface treated with an aldehyde silane coupling agent;

[0089] 5. Spot 15 μg / mL of Brucella synergistic complex antigen (first component: S-LPS antigen, BP26 antigen, and VirB12 antigen; second component: OMP19) on a glass slide treated with an aldehyde silane coupling agent, then dry at 37 ± 1°C for 1 hour and wash three times with detergent.

[0090] 6. Add a mixture of Tris-HCl buffer (0.1 M) at pH 8.0, PEG-20000 (mass concentration 0.5%), and Tween-20 (mass concentration 0.1%), then block at 37 ± 1°C for 2.5 h and wash three times with detergent to obtain a protein chip;

[0091] 7. Collect human plasma containing brucellosis antibodies, dilute the sample with a mixture of 10 mM PBS buffer (pH 7.4), 0.15 M NaCl, and 0.1% Tween-20 at a volume ratio of 1:100, and centrifuge at 4°C and 14,000 rpm for 12 minutes, and collect the supernatant;

[0092] 8. Add 50 μL of supernatant sample to the protein chip site, incubate with shaking at 37 ± 1 ° C for 0.6 h, and wash three times with detergent. Then add secondary antibody (including Cy3-labeled anti-human IgG and Cy5-labeled anti-swine IgM, where the mass ratio of Cy3-labeled anti-human IgG and Cy5-labeled anti-swine IgM is 75:25), incubate at 37 ± 1 ° C for 45 min, and wash three times;

[0093] 9. Use a fluorescence scanner to read the fluorescence signal, subtract the blank background, judge the positive or negative, and analyze the infection status.

[0094] The detergent used in the above steps is a PBS solution containing 0.075% Tween-20 by mass; the concentration of the PBS solution is 0.1 M and the pH is 7.4.

[0095] Example 6:

[0096] like Figure 1 As shown, a method for protein chip detection of brucellosis comprises the following steps:

[0097] 1. Soak the silicon wafer in 2.5% NaOH solution for 7 minutes, take it out and rinse it with pure water;

[0098] 2. Soak the cleaned silicon wafer in 8% hydrogen peroxide for 16 minutes, take it out and rinse it with pure water to obtain a hydroxylated silicon wafer;

[0099] 3. Use a dip-and-pull coating machine to cyclically dip and pull the hydroxylated silicon wafer in an ethanol solution of triethoxysilyl propionaldehyde; the mass ratio of triethoxysilyl propionaldehyde to ethanol is 4:96;

[0100] 4. After the cycle is completed, the silicon wafer is dried in a vacuum oven at 110°C for 2 hours to obtain a silicon wafer surface treated with an aldehyde silane coupling agent;

[0101] 5. Spot 20 μg / mL of Brucella synergistic complex antigens (first component: S-LPS antigen, BP26 antigen, and VirB12 antigen; second component: OMP25, OMP2b, OMP16, and OMP19) on a silicon wafer treated with an aldehyde silane coupling agent, then dry at 37 ± 1°C for 1.5 h and wash three times with detergent.

[0102] 6. Add a mixture of gelatin (mass concentration 1%), glycine (0.1 M), pH 7.4 Tris-HCl buffer (0.1 M), and Tween-20 (mass concentration 0.05%), then block at 37 ± 1°C for 3 h and wash three times with detergent to obtain a protein chip;

[0103] 7. Collect bovine plasma containing brucellosis antibodies, dilute the sample with a mixture of 10 mM PBS buffer (pH 7.4), 0.15 M NaCl, and 0.1% Tween-20 at a volume ratio of 1:100, and centrifuge at 4°C and 15,000 rpm for 14 minutes, and collect the supernatant;

[0104] 8. Add 50 μL of supernatant sample to the protein chip site, incubate with shaking at 37 ± 1 ° C for 1.5 hours, and wash three times with detergent. Then add secondary antibody (including Cy3-labeled anti-human IgG and Cy5-labeled anti-bovine IgM, where the mass ratio of Cy3-labeled anti-human IgG and Cy5-labeled anti-bovine IgM is 70:30), incubate at 37 ± 1 ° C for 60 minutes, and wash three times;

[0105] 9. Use a fluorescence scanner to read the fluorescence signal, subtract the blank background, judge the positive or negative, and analyze the infection status.

[0106] The detergent used in the above steps is a PBS solution containing 0.075% Tween-20 by mass; the concentration of the PBS solution is 0.1 M and the pH is 7.4.

[0107] Comparative Example 1:

[0108] A method for protein chip detection of brucellosis comprises the following steps:

[0109] 1. Spot 1 μg / mL Brucella synergistic complex antigen (first component: S-LPS antigen, BP26 antigen, and VirB12 antigen; second component: OMP25) on aluminum oxynitride slides, then dry at 37 ± 1°C for 1 hour and wash three times with detergent.

[0110] 2. Add a mixture of Tris-HCl buffer (0.1 M) at pH 8.0, PEG-20000 (mass concentration 0.5%), and Tween-20 (mass concentration 0.1%), then block at 37 ± 1°C for 2 h and wash three times with detergent to obtain a protein chip;

[0111] 3. Collect human serum containing brucellosis antibodies, dilute the sample with a mixture of 10 mM PBS buffer (pH 7.2), 0.15 M NaCl, and 0.1% Tween-20 at a volume ratio of 1:100, centrifuge at 9000 rpm for 10 minutes, and collect the supernatant;

[0112] 4. Add 50 μL of supernatant sample to the protein chip site, incubate with shaking at 37 ± 1 ° C for 0.5 h, and wash three times with detergent. Then add secondary antibody (including Cy3-labeled anti-human IgG and Cy5-labeled anti-goat IgM, where the mass ratio of Cy3-labeled anti-human IgG and Cy5-labeled anti-goat IgM is 75:25), incubate at 37 ± 1 ° C for 40 min, and wash three times;

[0113] 5. Use a fluorescence scanner to read the fluorescence signal, subtract the blank background, judge the positive or negative, and analyze the infection status.

[0114] The detergent used in the above steps is a PBS solution containing 0.075% Tween-20 by mass; the concentration of the PBS solution is 0.1 M and the pH is 7.4.

[0115] Comparative Example 2:

[0116] A method for protein chip detection of brucellosis comprises the following steps:

[0117] 1. Soak the glass slide in 5% NaOH solution for 10 minutes, take it out and rinse it with pure water;

[0118] 2. Soak the cleaned glass slide in 15% hydrogen peroxide for 20 minutes, take it out and rinse it with pure water to obtain a hydroxylated glass slide;

[0119] 3. Use a dip-and-pull coating machine to cyclically dip and pull the hydroxylated glass sheet in an ethanol solution of triethoxysilyl butyraldehyde; the mass ratio of triethoxysilyl butyraldehyde to ethanol is 5:95;

[0120] 4. After the cycle is completed, the glass sheet is dried in a vacuum oven at 120°C for 2 hours to obtain a glass sheet surface treated with an aldehyde silane coupling agent;

[0121] 5. Spot 5 μg / mL of S-LPS antigen on a glass slide treated with an aldehyde silane coupling agent, then dry at 37 ± 1°C for 2 h and wash three times with detergent.

[0122] 6. Add a mixture of Tris-HCl buffer (0.1 M) at pH 8.0, PEG-20000 (mass concentration 0.5%), and Tween-20 (mass concentration 0.1%), then block at 37 ± 1°C for 2 h and wash three times with detergent to obtain a protein chip;

[0123] 7. Collect bovine serum containing brucellosis antibodies, dilute the sample with a mixture of 10 mM PBS buffer (pH 7.4), 0.15 M NaCl, and 0.1% Tween-20 at a volume ratio of 1:100, and centrifuge at 4°C and 12,000 rpm for 10 minutes, and collect the supernatant;

[0124] 8. Add 50 μL of supernatant sample to the protein chip site, incubate with shaking at 37 ± 1 ° C for 1 hour, and wash with detergent three times. Then add secondary antibody (including Cy3-labeled anti-human IgG and Cy5-labeled anti-bovine IgM, where the mass ratio of Cy3-labeled anti-human IgG and Cy5-labeled anti-bovine IgM is 60:40), incubate at 37 ± 1 ° C for 50 minutes, and wash three times;

[0125] 9. Use a fluorescence scanner to read the fluorescence signal, subtract the blank background, judge the positive or negative, and analyze the infection status.

[0126] The detergent used in the above steps is a PBS solution containing 0.075% Tween-20 by mass; the concentration of the PBS solution is 0.1 M and the pH is 7.4.

[0127] Comparative Example 3:

[0128] A method for protein chip detection of brucellosis comprises the following steps:

[0129] 1. Spot 20 μg / mL of S-LPS antigen on a silicon wafer, dry it at 37 ± 1°C for 1.5 h, and wash it three times with detergent.

[0130] 2. Add a mixture of gelatin (mass concentration 1%), glycine (0.1 M), pH 7.4 Tris-HCl buffer (0.1 M), and Tween-20 (mass concentration 0.05%), then block at 37 ± 1°C for 3 h and wash three times with detergent to obtain a protein chip;

[0131] 3. Collect bovine plasma containing brucellosis antibodies, dilute the sample with a mixture of 10 mM PBS buffer (pH 7.4), 0.15 M NaCl, and 0.1% Tween-20 at a volume ratio of 1:100, and centrifuge at 4°C and 15,000 rpm for 14 minutes, and collect the supernatant;

[0132] 4. Add 50 μL of supernatant sample to the protein chip site, incubate with shaking at 37 ± 1 ° C for 1.5 hours, and wash three times with detergent. Then add secondary antibody (including Cy3-labeled anti-human IgG and Cy5-labeled anti-bovine IgM, where the mass ratio of Cy3-labeled anti-human IgG and Cy5-labeled anti-bovine IgM is 70:30), incubate at 37 ± 1 ° C for 60 minutes, and wash three times;

[0133] 5. Use a fluorescence scanner to read the fluorescence signal, subtract the blank background, judge the positive or negative, and analyze the infection status.

[0134] The detergent used in the above steps is a PBS solution containing 0.075% Tween-20 by mass; the concentration of the PBS solution is 0.1 M and the pH is 7.4.

[0135] Each sample to be tested, blank sample (buffer only), and negative sample (serum sample without Brucella) were tested ten times using a fluorescence scanner. The test results were statistically analyzed and compared. The mean RFU value ± 3SD of the negative sample was used as the cut-off value. The results are shown in Table 1.

[0136] Table 1 Comparison of test results of samples to be tested, blank samples and negative samples in Examples and Comparative Examples

[0137]

[0138] As shown in Table 1, the RFU mean values ​​of Examples 1 to 6 are all higher than the cut-off value of the negative sample, which indicates that the test sensitivity of this method can cover 1 to 20 μg / mL, with a wide detection range, strong specificity and high sensitivity.

[0139] This is because in this application, the first component of the Brucella collaborative composite antigen (S-LPS antigen, BP26 antigen and VirB12 antigen) and the second component (any one of OMP25, OMP31, OMP2b, OMP16, OMP19 and outer membrane protein multi-epitope chimeric antigen) cover key antigens such as Brucella lipopolysaccharide, conserved protein and outer membrane protein, and can simultaneously identify host antibodies against different structures (such as IgG / IgM), avoiding the limitations of single antigen detection. And through IgG / IgM subtype detection, it can effectively distinguish between acute infection (mainly IgM) and chronic infection (mainly IgG), providing more accurate infection status judgment for clinical diagnosis. Moreover, the antigenic epitopes of different components in the composite antigen can simultaneously bind to the corresponding antibodies in the sample, forming "multi-target recognition", amplifying the fluorescence signal, and improving the detection sensitivity and specificity.

[0140] In this application, the aldehyde groups on the surface of the solid phase carrier treated with the aldehyde silane coupling agent are connected to the amino groups of the antigen protein. Stable covalent bonds are formed to avoid antigen shedding caused by physical adsorption and improve detection sensitivity. Moreover, the spatial conformation of the covalently fixed antigen is stable, which can effectively retain the activity of the antigen epitope, and the surface chemical properties of the solid phase carrier used in this application are uniform, which can reduce batch differences and improve chip stability and detection repeatability. In addition, the hydroxylation-aldehyde treatment enhances the hydrophilicity of the solid phase carrier surface, and the uniform coating formed by the silane coupling agent on the solid phase carrier during the pulling and impregnation process can ensure the uniformity of the antigen concentration at the chip site, block the nonspecific binding of impurities in the sample (such as nonspecific proteins), and further improve sensitivity and specificity.

[0141] In Comparative Examples 1 to 3, at the same concentration of the test sample, the RFU values ​​were significantly reduced, and may be lower than the cut-off value of the negative sample (Comparative Example 1, 1 μg / mL) when the concentration of the test sample is low.

[0142] This is because, in Comparative Example 1, the solid phase carrier was not treated with an aldehyde silane coupling agent, so the surface of the solid phase carrier did not produce aldehyde groups that would interact with the amino groups of the antigen protein. The formation of a stable covalent bond makes it difficult to avoid antigen shedding due to physical adsorption. In addition, in Comparative Example 1, no pulling and dipping was performed, so a uniform coating could not be formed on the solid phase carrier, making it difficult to ensure uniform antigen concentration at all sites on the chip.

[0143] In Comparative Example 2, a single antigen was used instead of a Brucella collaborative composite antigen. Therefore, key antigens such as Brucella lipopolysaccharide, conserved proteins, and outer membrane proteins could not be covered, and host antibodies against different structures (such as IgG / IgM) could not be identified simultaneously. The limitations of single antigen detection were inevitable, and the detection sensitivity and specificity were significantly reduced.

[0144] In Comparative Example 3, neither the surface-treated solid phase carrier nor the cooperative composite antigen was used. Based on the same principle as above, its detection sensitivity and specificity were the lowest.

[0145] The above results show and describe the basic principles and main features of this application as well as the advantages of this application.

[0146] Those skilled in the art should understand that the present application is not limited to the above-described embodiments. The above-described embodiments and descriptions are merely illustrative of the principles of the present application. Various changes and improvements may be made to the present application without departing from the spirit and scope of the present application. Such changes and improvements are intended to fall within the scope of the present application. The scope of protection claimed in the present application is defined by the equivalents of the appended claims.

Claims

1. A protein chip for detecting brucellosis, characterized in that: The preparation method comprises the following steps: Soak the solid phase support in a 1-5% mass concentration NaOH solution or KOH solution for 5-10 minutes, take it out and wash it with pure water; The cleaned solid phase support is immersed in hydrogen peroxide with a mass concentration of 5-15% for 10-20 minutes, taken out and washed with pure water to obtain a hydroxylated solid phase support; The hydroxylated solid phase support is cyclically immersed and pulled in an ethanol solution of an aldehyde silane coupling agent using an immersion and pulling coating machine; After the cycle is completed, the solid phase support is dried in a vacuum oven at 110-120°C for 1-2 hours to obtain a pretreated solid phase support; Spotting Brucella synergistic complex antigens on the pretreated solid phase support, followed by drying and washing with detergent; Add a blocking agent, then block and wash with a detergent to obtain a protein chip; The solid phase carrier comprises any one of an aluminum oxynitride sheet, a glass sheet or a silicon sheet; The aldehyde-based silane coupling agent includes any one of trimethoxysilyl propionaldehyde, triethoxysilyl propionaldehyde, triethoxysilyl butyraldehyde, and triethoxysilyl undecane; in the ethanol solution of the aldehyde-based silane coupling agent, the mass ratio of the aldehyde-based silane coupling agent to ethanol is (1-5): (95-99); The concentration of the Brucella synergistic composite antigen is 1-20 μg / mL, and the composite antigen includes a first component and a second component; the first component includes an S-LPS antigen, a BP26 antigen, and a VirB12 antigen; the second component includes any one of OMP25, OMP31, OMP2b, OMP16, OMP19, and an outer membrane protein multi-epitope chimeric antigen; wherein the outer membrane protein multi-epitope chimeric antigen includes a fusion or chimera of any four of OMP25, OMP31, OMP2b, OMP16, and OMP19.

2. A protein chip for detecting brucellosis according to claim 1, characterized in that: The number of cycles of dipping and pulling is 20 to 50 times; wherein the time of each dipping is 1 to 3 minutes, and the speed of each pulling is 2 to 10 mm / s.

3. A protein chip for detecting brucellosis according to claim 1, characterized in that: The detergent is a PBS solution containing 0.05-0.1% Tween-20; The concentration of the PBS solution is 0.1 M and the pH is 7.

4.

4. A protein chip for detecting brucellosis according to claim 1, characterized in that: The blocking agent includes any one of a first blocking agent and a second blocking agent; wherein the first blocking agent includes a mixture of a pH 8.0, 0.1M Tris-HCl buffer, a mass concentration of 0.5% PEG-20000 and a mass concentration of 0.1% Tween-20; and the second blocking agent includes a mixture of a mass concentration of 1% gelatin, 0.1M glycine, a pH 7.4, 0.1M Tris-HCl buffer and a mass concentration of 0.05% Tween-20.

5. A protein chip for detecting brucellosis according to claim 1, characterized in that: The drying temperature is 37±1° C., and the drying time is 1 to 2 hours; the washing times are set to 3 times; the sealing temperature is 37±1° C., and the sealing time is 2 to 3 hours.

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