A method for detecting autoimmune hemolytic anemia antibodies based on flow cytometry microsphere technology

Abstract

The present invention discloses a method for detecting autoimmune hemolytic anemia antibodies based on flow microsphere technology, which belongs to the field of biomolecular and immunological detection. The specific method is: using polymer microspheres modified with functional functional groups as carriers, coupling the microspheres with detection antigens, reacting with autoimmune hemolytic anemia antibodies and fluorescently labeled secondary antibodies in the sample, forming a complex of "polymer microspheres-detection antigen-antibody-fluorescently labeled secondary antibodies", analyzing the fluorescence intensity of the secondary antibodies on the microspheres by flow cytometry, and then analyzing whether there are antibodies to autoimmune hemolytic anemia in the sample to be tested and the concentration of the antibodies. The present invention adopts flow microsphere detection technology, which can improve detection efficiency, and can also be prepared into a test kit, which is simple and convenient, and provides a basis for clinical diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomolecular detection and immunological detection, and is specifically a method for detecting autoimmune hemolytic anemia antibodies based on flow microsphere technology. Technical Background

[0002] Autoimmune hemolytic anemia (AIHA) refers to a disorder in the regulation of the body's immune function. The serum of patients with autoimmune hemolytic anemia contains antibodies and / or complement against their own red blood cells adsorbed on the surface of red blood cells, which accelerates the destruction of red blood cells through antigen-antibody reaction, causing a type of hemolytic anemia.

[0003] Flow cytometry is a biological technique used to count and sort tiny particles suspended in a fluid. This technique can be used to continuously analyze multiple parameters of individual cells flowing through an optical or electronic detector. Flow cytometry is a technique that detects fluorescent signals of markers in single cells or other biological particles in suspension to achieve high-speed, one-by-one quantitative analysis and sorting of cells. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting autoimmune hemolytic anemia antibodies based on flow microsphere technology.

[0005] The technical solution of the present invention is:

[0006] Using polymer microspheres modified with functional groups as carriers, the microspheres are first coupled to the detection antigen, and then react with the autoimmune hemolytic anemia antibodies and fluorescently labeled secondary antibodies in the sample to form a "polymer microsphere-detection antigen-antibody-fluorescently labeled secondary antibody" complex. The fluorescence intensity of the secondary antibody on the microspheres is analyzed by flow cytometry, and then the presence of autoimmune hemolytic anemia antibodies in the sample to be tested and the concentration of the antibodies are analyzed.

[0007] The polymer microspheres used are carboxyl-modified polystyrene microspheres with a particle size of micrometer order.

[0008] The fluorescently labeled secondary antibody used was goat anti-human immunoglobulin IgG, and the fluorescent group carried by the secondary antibody was fluorescein isothiocyanate FITC.

[0009] The specific preparation process and detection method are as follows:

[0010] (1) Polymer microspheres coupled to detection antigens: 1.0×10 6Place carboxyl polystyrene microspheres produced in the same batch in a centrifuge tube, rinse the microspheres with washing solution, then add activation buffer to activate the microspheres, shake at room temperature in the dark for 0.5-1 h, add detection antigen to the activated microspheres, mix well and shake at room temperature in the dark, wash three times with washing solution after shaking, finally add blocking solution and shake for 2 h, and store the "polymer microsphere-detection antigen" complex in storage solution at 4°C in the dark for later use;

[0011] (2) Take a certain amount of "polymer microsphere-detection antigen" complex and put it into a centrifuge tube, add the sample to be tested, mix well and shake at room temperature in the dark for 1 hour. After the reaction, centrifuge and wash to remove unreacted antibodies to form a "polymer microsphere-detection antigen-antibody" complex;

[0012] (3) Add FITC fluorescently labeled secondary antibody to the "polymer microsphere-detection antigen-antibody" complex, mix well, and react in the dark at room temperature for 1 h. After the reaction, centrifuge and wash to remove unreacted secondary antibody to form a "polymer microsphere-detection antigen-antibody-fluorescently labeled secondary antibody" complex;

[0013] (4) The "polymer microsphere-detection antigen-antibody-fluorescently labeled secondary antibody" complex is diluted in diluent, and the fluorescence intensity is detected by flow cytometry to detect the presence and concentration of the antibody.

[0014] Preferably, the polymer microspheres used are carboxyl-treated polystyrene fluorescent microspheres.

[0015] Preferably, the fluorescently labeled secondary antibody is goat anti-human immunoglobulin IgG.

[0016] Preferably, the fluorescent group used in the fluorescently labeled secondary antibody is fluorescein isothiocyanate FITC.

[0017] Preferably, the activation buffer is NaH 2 PO 4 Solution.

[0018] Preferably, the blocking solution is 1% BSA in PBS.

[0019] In this field, as well as western, immunohistochemistry, ELISA, etc., there is a blocking step, and the type, concentration and time of the blocking solution have a great impact on the results. However, proteins are not continuous, there are many gaps, and for example, there are gaps between sample wells, and antibodies are also proteins, and they will be adsorbed in the empty holes. In this way, there will be many non-specific signals, but if a protein-rich blocking solution is used with polymer microspheres, all the holes will be filled with BSA or other proteins. However, through a large number of experiments, it was found that using only 1% BSA in PBS solution is not the best effect. There will still be a small number of holes in the polymer microspheres that are not completely blocked, and the secondary antibody will be adsorbed on the microspheres, affecting the accuracy of the subsequent signals.

[0020] The present invention provides a blocking solution combination, preferably, the blocking solution also includes carbohydrates, hydroxyethyl cellulose, and sorbitol. Further, the blocking solution specifically includes the following components in mass percentage: 5% carbohydrates, 0.1% hydroxyethyl cellulose, 1% BSA, 0.5% sorbitol, and the rest is PBS buffer.

[0021] Preferably, the sugar substance is any one of guar gum, trehalose, sucrose and glucose.

[0022] The present invention is a little bit in that:

[0023] The present invention uses polymer microspheres modified with functional groups as carriers, first couples the microspheres with detection antigens, then couples with autoimmune hemolytic anemia antibodies in the sample, centrifuges and washes them, and then reacts with fluorescently labeled secondary antibodies to form a "polymer microsphere-detection antigen-antibody-fluorescently labeled secondary antibody" complex, analyzes the fluorescence intensity of the secondary antibody on the microspheres by flow cytometry, and then analyzes the concentration of the antibody in the sample to be tested. The advantages of the present invention are that the flow microsphere detection technology is used to improve the detection efficiency, and it can also be prepared into a kit, which is simple and convenient, and provides a basis for clinical diagnosis and treatment.

[0024] The present invention uses flow cytometry for detection. This detection technology is mature, has high detection sensitivity, and short detection time, which can improve detection efficiency. The fluorescent group has multiple selectivities, and the types of secondary antibodies also have multiple selectivities. It can be prepared into a kit, which is simple and convenient, and provides a basis for clinical diagnosis and treatment.

[0025] At the same time, the present invention also provides a blocking liquid composition, which can reduce the number of antibodies directly combined with polymer microspheres and improve detection accuracy. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with specific implementation modes.

[0027] Embodiment 1:

[0028] 1. Polymer microspheres coupled with detection antigen: 1.0×10 6 Carboxyl polystyrene microspheres produced in the same batch were placed in a 1.5 mL centrifuge tube and a certain amount of phosphate buffer solution (PBS solution, Na 2 HPO 4 and KH 2 PO 4 composition, 0.1 mol / L, pH 7.4) to rinse the microspheres, and then add NaH 2 PO 4 (0.1 mol / L, pH 6.2) to activate the microspheres, shake them at room temperature in the dark for 0.5-1 h, add 100 μL of detection antigen to the activated microspheres, mix well and shake them at room temperature in the dark, wash them three times with PBS solution after shaking, finally add PBS solution containing 1% BSA and shake for 2 h, store the "polymer microsphere-detection antigen" complex in storage solution at 4°C in the dark for later use.

[0029] 2. Take a certain amount of "polymer microsphere-detection antigen" complex in a 1.5mL centrifuge tube, add the sample to be tested, mix evenly, and react at room temperature in the dark for 1 hour. After the reaction, centrifuge at 3000 rpm for 3 min, and wash 3 times with 500 μL PBS solution to remove unreacted antibodies to form a "polymer microsphere-detection antigen-antibody" complex.

[0030] 3. Take the "polymer microsphere-detection antigen-antibody" complex in a 1.5 mL centrifuge tube, add FITC fluorescently labeled secondary antibody, mix well, and react at room temperature in the dark for 1 h. After the reaction, centrifuge at 3000 rpm for 3 min, wash and remove unreacted secondary antibody to form a "polymer microsphere-detection antigen-antibody-fluorescently labeled secondary antibody" complex.

[0031] 4. Add the "polymer microsphere-detection antigen-antibody-fluorescently labeled secondary antibody" complex into the diluent for dilution, and use flow cytometry to detect the fluorescence intensity to detect the presence and concentration of the antibody.

[0032] Embodiment 2:

[0033] 1. Polymer microspheres coupled with detection antigen: 1.0×10 6 Carboxyl polystyrene microspheres produced in the same batch were placed in a 1.5 mL centrifuge tube and a certain amount of phosphate buffer solution (PBS solution, Na 2 HPO 4 and KH 2 PO 4 composition, 0.1 mol / L, pH 7.4) to rinse the microspheres, and then add NaH2 PO 4 (0.1 mol / L, pH 6.2) to activate the microspheres, shake them at room temperature in the dark for 0.5-1 h, add 100 μL of detection antigen to the activated microspheres, mix well and shake at room temperature in the dark, wash three times with PBS solution after shaking, finally add PBS solution containing 1% BSA, 5% guar gum, 0.1% hydroxyethyl cellulose, and 0.5% sorbitol and shake for 2 h, store the "polymer microsphere-detection antigen" complex in preservation solution at 4°C in the dark for later use.

[0034] 2. Take a certain amount of "polymer microsphere-detection antigen" complex in a 1.5mL centrifuge tube, add the sample to be tested, mix evenly, and react at room temperature in the dark for 1 hour. After the reaction, centrifuge at 3000 rpm for 3 min, and wash 3 times with 500 μL PBS solution to remove unreacted antibodies to form a "polymer microsphere-detection antigen-antibody" complex.

[0035] 3. Take the "polymer microsphere-detection antigen-antibody" complex in a 1.5 mL centrifuge tube, add FITC fluorescently labeled secondary antibody, mix well, and react at room temperature in the dark for 1 h. After the reaction, centrifuge at 3000 rpm for 3 min, wash and remove unreacted secondary antibody to form a "polymer microsphere-detection antigen-antibody-fluorescently labeled secondary antibody" complex.

[0036] 4. Add the "polymer microsphere-detection antigen-antibody-fluorescently labeled secondary antibody" complex into the diluent for dilution, and use flow cytometry to detect the fluorescence intensity to detect the presence and concentration of the antibody.

[0037] Example 3: Comparison of accuracy between Example 1 and Example 2

[0038] 10 μg / ML of antibody was accurately weighed and tested according to the methods of Example 1 and Example 2, respectively. The results were:

[0039] The final measured result of Example 1 is 11.3 μg / ML, and the measured result of Example 2 is 9.6 μg / ML.

[0040] It can be seen that the result of Example 1 is higher than the actual antibody concentration. The reason is that after the secondary antibody is added, a small amount of unenclosed space in the microspheres is adsorbed by the secondary antibody, resulting in a false positive and causing the test result to be higher.

[0041] The above embodiments are only used to help those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. Any equivalent replacement made in the present invention belongs to the protection scope of the present invention.

Claims

1. A method for detecting autoimmune hemolytic anemia antibodies based on flow cytometry microsphere technology, which is a method for non-disease diagnosis purposes, characterized in that: The specific steps are as follows: Polymer microspheres coupled with detection antigen: 1.0×10 6 Place carboxyl polystyrene microspheres produced in the same batch in a centrifuge tube, rinse the microspheres with washing solution, then add activation buffer to activate the microspheres, shake at room temperature in the dark for 0.5-1 h, add detection antigen to the activated microspheres, mix well and shake at room temperature in the dark, wash three times with washing solution after shaking, finally add blocking solution and shake for 2 h, and store the "polymer microsphere-detection antigen" complex in storage solution at 4°C in the dark for later use; The activation buffer is a NaH2PO4 solution; the blocking solution specifically includes the following components in percentage by mass: 5% guar gum, 0.1% hydroxyethyl cellulose, 1% BSA, 0.5% sorbitol, and the rest is PBS buffer; Take a certain amount of "polymer microsphere-detection antigen" complex in a centrifuge tube, add the sample to be tested, mix evenly, and react at room temperature in the dark for 1 hour. After the reaction, centrifuge and wash to remove unreacted antibodies to form a "polymer microsphere-detection antigen-antibody" complex; Add FITC fluorescently labeled secondary antibody to the "polymer microsphere-detection antigen-antibody" complex, mix well, and react at room temperature in the dark for 1 h. After the reaction, centrifuge and wash to remove unreacted secondary antibody to form a "polymer microsphere-detection antigen-antibody-fluorescently labeled secondary antibody" complex; The "polymer microsphere-detection antigen-antibody-fluorescently labeled secondary antibody" complex is diluted in diluent, and the fluorescence intensity is detected by flow cytometry to detect the presence and concentration of the antibody.

2. The method for detecting autoimmune hemolytic anemia antibodies based on flow cytometry technology according to claim 1, characterized in that: The polymer microspheres used are carboxyl-treated polystyrene fluorescent microspheres.

3. The method for detecting autoimmune hemolytic anemia antibodies based on flow cytometry technology according to claim 1, characterized in that: The fluorescently labeled secondary antibody was goat anti-human immunoglobulin IgG.