Low-abundance antigen detection method based on flow fluorescent single molecule counting

Abstract

The invention provides a low-abundance antigen detection method based on flow-type fluorescent single molecule counting, and belongs to the technical field of biological detection.The method comprises the following steps that after fluorescent encoding microspheres coated with captured antibodies are mixed with a standard sample or a sample to be detected, detection antibodies and nano magnetic beads are added, and the fluorescent encoding microspheres are obtained; or directly adding the nano magnetic beads coated with the detection antibody, reacting to form a double-antibody sandwich compound, separating the compound, and counting by adopting a flow cytometer; and establishing a standard curve of the antigen concentration and the collection number of the fluorescent microspheres, and calculating the concentration of the target antigen in the to-be-detected sample according to the standard curve. The method effectively improves the problem of low sensitivity of a flow fluorescence technology, is low in detection cost and reliable in detection result, and has important significance on low-abundance antigen detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a low-abundance antigen detection method based on flow cytometry fluorescence single molecule counting. Background Art

[0002] Flow cytometry, a clinically applicable high-throughput luminescence detection technology based on coded microspheres and flow cytometry, is also known as liquid chips, suspension arrays, and Luminex xMAP technology. This technology represents a new generation of high-throughput molecular diagnostics, following biochips and chemiluminescence technologies, and is a hot topic in clinical diagnostics and life science research. Combining the technological advantages of colored microspheres, applied fluidics, laser technology, and high-speed digital signal processing, it has been widely applied in research areas such as immunoassays, nucleic acid detection, enzymatic analysis, and receptor and ligand recognition assays.

[0003] Fluorescently coded microspheres are a key technology at the heart of flow cytometry. These uniform 5.6μm polystyrene microspheres can remain permanently suspended in liquid. 100 million activated carboxyl groups on their surface can covalently crosslink specific antibodies or nucleic acid probes, generating a quasi-homogeneous antigen-antibody or hybridization reaction with the target sample components (thus enabling the platform to detect both nucleic acid and immunoassays). The microspheres are dyed and coded with two fluorescent dyes in a 10×10 concentration gradient, resulting in 100 distinct fluorescence profiles. Because the different coded microspheres react independently within the same system without interfering with each other, each microsphere can be covalently crosslinked to any protein or nucleic acid probe, enabling high-throughput combined testing with 100 results in a single test. This also facilitates the development of new assays and the customization of customized test reagents. As each microsphere passes through the instrument, a red laser identifies the microsphere code to determine the assay type, while a green laser reads the analyte signal for quantitative or qualitative analysis.

[0004] Compared to ELISA, flow cytometry offers a wider linear range, up to 3-5 orders of magnitude, and excellent reproducibility. Using a quasi-homogeneous reaction mode, it produces stable results with excellent reproducibility. However, some in the market remain pessimistic about the sensitivity of flow cytometry, and as a result, are also unimpressed with its use for quantitative analyte concentration detection. This is due to the method's relatively narrow signal range (compared to chemiluminescence), which may affect the analytical measurement range for some tests. Compared to typical labeled immunoassays that typically require excessive amounts of raw materials, flow cytometry's analytical sensitivity is also less than optimistic. SimoA technology, a single-molecule immunoassay based on a microarray chip, involves carving (or casting) thousands of micron-sized wells on a millimeter-scale chip, each with a volume of approximately 40 fl. Magnetic immune complex beads are then dispensed into the wells. Fluorescent spots are then counted using a high-resolution fluorescence microscope. Based on Poisson distribution theory, the ratio of the number of wells containing both beads and fluorescent product to the total number of wells containing beads is calculated to determine the protein concentration in the test sample. However, because this method relies on the optical signal intensity in each well, it is not truly absolute quantification. Furthermore, due to the high cost of the equipment, this technology is still in the development and promotion stage. Therefore, there is currently no commercially available method for single-molecule quantification of low-abundance target antigens using flow cytometry fluorescent microsphere counting. Summary of the Invention

[0005] In view of the technical problems existing in the background technology, the present invention provides a method for quantitative detection of low-abundance target antigens based on flow fluorescence single-molecule counting, aiming to solve the technical problems of low sensitivity of flow fluorescence technology and high price of single-molecule immunoassay based on microarray chips.

[0006] The technical solutions of the present invention are as follows: A low-abundance antigen detection method based on flow cytometry fluorescence single-molecule counting comprises the following steps: S1. After mixing the fluorescently encoded microspheres coated with the capture antibody with the standard sample or the sample to be tested, the detection antibody and nanomagnetic beads are added, or the nanomagnetic beads coated with the detection antibody are directly added to form a double antibody sandwich complex after the reaction. The complex is separated and counted using a flow cytometer; S2. Establish a standard curve between antigen concentration and the number of fluorescent microspheres collected, and calculate the concentration of the target antigen in the sample to be tested based on the standard curve.

[0007] In the above-mentioned low-abundance antigen detection method, the fluorescently encoded microspheres are naked microspheres or microspheres whose surfaces are modified with amino groups, carboxyl groups, hydroxyl groups, thiol groups, streptavidin or biotin. The material of the fluorescently encoded microspheres can be polystyrene (PS), polymethyl methacrylate (PMMA) or silica, etc.

[0008] Preferably, in the above-mentioned low-abundance antigen detection method, the fluorescently encoded microspheres are polystyrene microspheres with a particle size of 2-4 μm, and the nanomagnetic beads have a particle size of 20-130 nm. Due to steric hindrance and antigen-antibody reaction kinetics, if the microspheres and magnetic beads are of similar size, uniform and rapid binding will be difficult to occur. The present invention effectively controls the formation of the double-antibody sandwich complex by controlling the particle size of the fluorescently encoded microspheres and nanomagnetic beads, thereby enabling the present invention scheme to have an excellent linear relationship in low-concentration detection.

[0009] In the aforementioned low-abundance antigen detection method, the capture and detection antibody coating methods can be performed with reference to existing techniques. For example, for polystyrene fluorescent microspheres with surface carboxyl groups modified, the carboxyl groups can be activated using activators EDC and NHS. The resulting intermediate ester can then react with an antibody solution for coating, and finally, the surface can be blocked using a protein and / or non-protein blocking agent. For another example, for streptavidin-modified fluorescent microspheres, they can be mixed with a biotinylated antibody and allowed to react fully to obtain the microsphere-antibody coating.

[0010] In the above-mentioned low-abundance antigen detection method, the detection antibody-coated nanomagnetic beads can be directly added, or the detection antibody and nanomagnetic beads can be added separately. For example, in some embodiments of the present invention, the detection antibody is first biotinylated with biotin-NHS reaction, and then formed into a double antibody sandwich complex with the capture antibody-coated fluorescent coding microspheres and antigen, and then indirectly introduced with streptavidin-modified nanomagnetic beads to form an indirect capture complex. In other embodiments, the detection antibody is first coated onto the nanomagnetic beads, and then the nanomagnetic beads coated with the detection antibody are introduced into the mixture of the capture antibody-coated fluorescent coding microspheres and the antigen to form a double antibody sandwich complex.

[0011] Preferably, in the above-described low-abundance antigen detection method, the number of fluorescently encoded microspheres per mL of reaction system in step S1 is greater than 100,000, and the target antigen content is less than 10 pg. Under these conditions, the number of fluorescently encoded microspheres is much higher than the number of target antigen molecules in the sample. Under a quasi-homogeneous reaction mode, one fluorescently encoded microsphere corresponds to one target antigen molecule, thereby achieving flow cytometry single-molecule counting.

[0012] Preferably, in the above-mentioned low-abundance antigen detection method, a MACS separation column is used to separate the double-antibody sandwich complex. Given the correspondence between the fluorescently encoded microspheres and the target antigen molecules, as well as the difference in particle size between the fluorescently encoded microspheres and the nanomagnetic beads, the introduced nanomagnetic beads can bind to a small amount of antigen on the fluorescent microspheres and then be retained by the Miltenyi MACS separation column. The retained fluorescent microspheres can then be absolutely counted. In some embodiments of the present invention, the target antigen is CRP, the capture antibody and the detection antibody are CRP-C1 antibody and CRP-T9 antibody, respectively; experimental data show that the method of the present invention has a very good effect for the detection of low-abundance (0-3 pg / mL) CRP.

[0013] Unlike traditional flow fluorescence technology, the present invention captures antigens in low-abundance samples by coating the non-magnetic fluorescent encoded microspheres with capture antibodies, then coating a nanoscale magnetic bead with a detection antibody, and then mixing the fluorescent microspheres that have captured the antigen with the detection antibody-coated nanomagnetic beads to react to form a double-antibody sandwich complex. The reacted sample is passed through a Miltenyi MACS sorting column. The iron beads in the matrix of the sorting column amplify the magnetic field by 10,000 times. Therefore, the sorting column can be used to retain the fluorescent microspheres that form the sandwich complex in the magnetic field, while the microspheres that have not captured the antigen and cannot form a complex with the nanomagnetic beads cannot remain in the magnetic field. When the sample has completely flowed out of the column, the sorting column is removed from the external magnetic field, and the double-antibody sandwich complex remaining in the column can be eluted by buffer. The eluted product is placed on a flow cytometer for detection, and the target microspheres can be counted, thereby obtaining the concentration of the target antigen in the low-abundance sample.

[0014] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are specifically listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] To more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0016] Figure 1 A flow chart of the low-abundance antigen detection method provided by the present invention; Figure 2 Schematic diagram of using MACS sorting column to capture the double antibody sandwich complex of fluorescent microspheres and nanomagnetic beads in an embodiment of the present invention. DETAILED DESCRIPTION

[0017] The following embodiments of the technical solution of the present invention will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains; the terms used herein are for the purpose of describing specific implementations only and are not intended to limit the present invention; the terms "including" and "having" and any variations thereof herein are intended to cover non-exclusive inclusions.

[0019] To address the low sensitivity of flow fluorescence detection, although existing technologies have developed single-molecule immunoassays based on microarray chips, this technology is not truly absolutely quantitative and is extremely expensive. Therefore, new methods for detecting low-abundance antigens using flow fluorescence technology are still needed. To address this technical problem, the present invention provides a low-abundance antigen detection method based on flow fluorescence single-molecule counting, which can achieve quantitative detection of low-abundance target antigens.

[0020] like Figure 1 As shown, the low-abundance antigen detection method based on flow cytometry single-molecule counting provided by the embodiment of the present invention includes the following steps: (1) After mixing the fluorescently encoded microspheres coated with the capture antibody with the standard sample (containing a specific concentration of the target antigen) or the sample to be tested, the detection antibody and nanomagnetic beads are added, or the nanomagnetic beads coated with the detection antibody are directly added. After the reaction, a double antibody sandwich complex is formed, and the complex is separated and counted using a flow cytometer; (2) Based on the counting results of the standard sample, a standard curve is established between the target antigen concentration and the number of fluorescent microspheres collected; then, the concentration of the target antigen in the sample to be tested is calculated based on the standard curve and the counting results of the sample to be tested.

[0021] Furthermore, in some embodiments, the fluorescently encoded microspheres are polystyrene microspheres with a particle size of 2-4 μm, and the nanomagnetic beads have a particle size of 20-130 nm. Furthermore, per 1 mL of reaction system, the number of fluorescently encoded microspheres reaches over 100,000, and the target antigen content is less than 10 pg. By limiting the particle size of the microspheres and magnetic beads, as well as the number of microspheres relative to the target antigen, a quasi-homogeneous reaction mode is employed, where one fluorescently encoded microsphere corresponds to one target antigen molecule, thereby enabling flow cytometry single-molecule counting.

[0022] Furthermore, in some embodiments, a double antibody sandwich complex formed by fluorescent microspheres and nanomagnetic beads is captured by a MACS sorting column. The principle is as follows: Figure 2 shown.

[0023] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications were used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0024] In the following examples, C-reactive protein (CRP) was used as the target antigen, and the fluorescently encoded microspheres used were all made of polystyrene.

[0025] Example 1

[0026] This embodiment provides a method for quantitatively detecting low-abundance target antigens based on flow cytometry fluorescence single-molecule counting, which specifically includes the following steps: (1) Preparation of CRP antibody-coated carboxyl flow cytometry fluorescence-encoded microspheres.

[0027] 4 μm APC-encoded carboxyl polystyrene microspheres were vortexed and mixed. 1.1e7 microspheres were taken out and placed in a 1.5 mL Eppendorf tube and washed with 900 μL 0.05 M pH 5.5 MES solution. The supernatant was discarded and the washing step was repeated 3 times. The microspheres were resuspended with 0.05 M pH 5.5 MES solution and 20 μL each of 10 mg / mL EDC and NHS were added thereto. The microspheres were shaken for 1000 r and reacted at 25 ° C for 15 min. The microspheres were then washed with 0.05 M pH 6.5 MES and washed twice. After resuspending, 30 μg CRP-C1 antibody solution was added. The microspheres were shaken for 1000 r and reacted at 25 ° C for 3 h. After washing, the microspheres were washed with 0.01 M pH 7.4 PBS buffer containing 1% BSA. The washing was repeated twice and the microspheres were resuspended and blocked. The microspheres were shaken for 1000 r and reacted at 25 ° C for 1 h. Finally, the microspheres were washed with 0.01 M pH 7.4 PBS buffer containing 1% BSA. The washing was repeated twice and the microspheres were resuspended and blocked. The microspheres were shaken for 1000 r and reacted at 25 ° C for 1 h. The cells were washed twice with PBS containing 0.5% BSA, 5% sucrose, and 0.1% Tween and resuspended for storage at 4°C.

[0028] (2) Preparation of biotinylated antibodies.

[0029] CRP-T9 antibody and biotin-PEG12-NHS were mixed and reacted in 0.01M pH 7.4 PBS solution, shaken and mixed for 1000 r, and reacted at 25°C for 1 h. After the reaction, ultrafiltration was performed twice in 0.01M pH 7.4 PBS using an ultrafiltration tube to obtain a biotin-CRP-T9 antibody solution, which was stored at 4°C for later use.

[0030] (3) Generation and separation of double antibody sandwich complexes.

[0031] Take 5e5 APC-encoded carboxyl polystyrene microspheres coated with CRP antibody and add them to the CRP test sample. Then add 1ug / mL biotin-CRP-T9 antibody solution, shake and mix for 1000r, react at 37℃ for 30min, remove the sample, centrifuge at 7000rpm, centrifuge for 10min, remove the supernatant, add 100uL streptavidin-modified 50nm dextran magnetic beads, disperse by pipetting, shake and mix for 1000r, react at 37℃ for 10min, finally dilute the reaction product to 1mL and transfer it to MACS® separation column to intercept the CRP double antibody sandwich complex. After washing twice with 0.01M pH7.4 PBS, remove the separation column from the magnetic field when the liquid stops flowing out, and add buffer to elute the target complex.

[0032] (4) Flow cytometric counting.

[0033] The eluted samples in step (3) are collected and placed in a flow cytometer for counting.

[0034] The concentration of CRP in the CRP test sample used in step (3) was changed, and the counting results at different concentrations were statistically analyzed, as shown in Table 1.

[0035] Table 1 Fluorescent microsphere counting results at different CRP concentrations in Example 1

[0036] Based on the above concentration data relationship, a standard curve was established with the CRP antigen concentration as the y-axis and the number of fluorescent microspheres collected as the x-axis. Specifically, it is: y=2.3E+04x+1.9E+03, R²=0.9995.

[0037] Example 2

[0038] This embodiment provides a method for quantitatively detecting low-abundance target antigens based on flow cytometry fluorescence single-molecule counting, which specifically includes the following steps: (1) Preparation of biotinylated antibody-coated streptavidin fluorescence-encoded microspheres.

[0039] Vortex the 5.6 μm streptavidin fluorescently encoded microspheres to mix thoroughly, remove 1.2e7 microspheres and place them in a 1.5 mL Eppendorf tube. Add 900 μL of 0.01 M pH 7.4 PBS solution for washing, discard the supernatant, repeat the washing step 3 times, and resuspend for later use.

[0040] CRP-C1 antibody and biotin-PEG12-NHS were mixed and reacted in 0.01M pH 7.4 PBS solution, shaken and mixed for 1000 r, and reacted at 25°C for 1 h. After the reaction, ultrafiltration was performed twice in 0.01M pH 7.4 PBS using an ultrafiltration tube to obtain a biotin-CRP-C1 antibody solution, which was stored at 4°C for later use.

[0041] The washed microspheres were mixed with biotin-CRP-C1 antibody, shaken and mixed for 1000 r, and reacted at 25°C for 1 h to obtain CRP-C1 antibody microspheres.

[0042] (2) Preparation of antibody-coated nanomagnetic beads.

[0043] 100 nm dextran magnetic beads were coated with CRP-T9 antibody and stored at 4°C until use.

[0044] (3) Generation and separation of double antibody sandwich complexes.

[0045] Take 5e5 fluorescently encoded microspheres coated with CRP-C1 antibody and add them to the CRP test sample. Then add 100uCRP-T9 antibody-coated nanomagnetic beads, shake and mix for 1000r, and react at 37℃ for 30min. Transfer the reaction product to a MACS® separation column to intercept the CRP double antibody sandwich complex. After washing twice with 0.01M pH7.4 PBS, when the liquid stops flowing out, remove the separation column from the magnetic field and add buffer to elute the target complex.

[0046] (4) Flow cytometric counting.

[0047] The eluted samples in step (3) are collected and placed in a flow cytometer for counting.

[0048] The concentration of CRP in the CRP test sample used in step (3) was changed, and the counting results at different concentrations were statistically analyzed, as shown in Table 2.

[0049] Table 2 Fluorescent microsphere counting results at different CRP concentrations in Example 2

[0050] Based on the above concentration data relationship, a standard curve was established with the CRP antigen concentration as the y-axis and the number of fluorescent microspheres collected as the x-axis. Specifically, it is: y=4.1E+04x+2.1E+02, R²=0.9993.

[0051] Comparative Example 1 Different from the embodiment, the method of this example does not use nanomagnetic beads and performs flow cytometry fluorescence detection on the double antibody sandwich complex, which specifically includes the following steps: (1) Preparation of CRP antibody-coated carboxyl flow cytometry fluorescence-encoded microspheres.

[0052] The 5.6 μm APC-encoded carboxyl polystyrene microspheres were vortexed and mixed. 6.1e12 microspheres were taken out and placed in a 1.5 mL Eppendorf tube and washed with 900 μL 0.05 M pH 5.5 MES solution. The supernatant was discarded and the washing step was repeated 3 times. The microspheres were resuspended with 0.05 M pH 5.5 MES solution and 20 μL each of 10 mg / mL EDC and NHS were added thereto. The microspheres were shaken for 1000 r and reacted at 25 ° C for 15 min. The microspheres were then washed with 0.05 M pH 6.5 MES and washed twice. After resuspending, 30 μg CRP-C1 antibody solution was added. The microspheres were shaken for 1000 r and reacted at 25 ° C for 3 h. After washing, the microspheres were washed with 0.01 M pH 7.4 PBS buffer containing 1% BSA. The washing was repeated twice and the microspheres were resuspended and blocked. The microspheres were shaken for 1000 r and reacted at 25 ° C for 1 h. Finally, the microspheres were washed with 0.01 M pH 7.4 PBS buffer containing 1% BSA. The washing was repeated twice and the microspheres were resuspended and blocked. The microspheres were shaken for 1000 r and reacted at 25 ° C for 1 h. The cells were washed twice with PBS containing 0.5% BSA, 5% sucrose, and 0.1% Tween and resuspended for storage at 4°C.

[0053] (2) Preparation of PE-labeled antibodies.

[0054] CRP-T9 antibody and biotin-PEG12-NHS were mixed and reacted in 0.01M pH 7.4 PBS solution, shaken and mixed for 1000 r, and reacted at 25°C for 1 h. After the reaction, ultrafiltration was performed twice in 0.01M pH 7.4 PBS using an ultrafiltration tube to obtain a biotin-CRP-T9 antibody solution. SA-PE (streptavidin-labeled phycoerythrin) and biotin-CRP-T9 antibody were diluted 1:500 and mixed and stored at 4°C until use.

[0055] (3) Generation of double antibody sandwich complex.

[0056] Take 2e4 fluorescently encoded microspheres coated with CRP-C1 antibody, add them to the CRP test sample, then add 100uL PE-labeled CRP-T9 antibody, shake and mix for 1000r, react at 37℃ for 30min, and finally wash the reaction product twice with 0.01M pH7.4 PBS, and then add buffer to resuspend the sample.

[0057] (4) Flow cytometry detection.

[0058] The resuspended samples were loaded onto a flow cytometer for fluorescence detection.

[0059] The concentration of CRP in the CRP test sample used in step (3) was changed, and the fluorescence detection results at different concentrations were statistically analyzed, as shown in Table 3.

[0060] Table 3 Fluorescent microsphere counting results at different CRP concentrations in Comparative Example 1

[0061] Based on the above concentration data relationship, with the CRP antigen concentration as the y-axis and the average fluorescence value on each microsphere as the x-axis, a standard curve was established: y=606.49x+659.88, R²=0.9448.

[0062] Comparative Example 2 Different from the embodiment, this method uses micron magnetic beads and specifically includes the following steps: (1) Preparation of biotinylated antibody-coated streptavidin fluorescence-encoded microspheres.

[0063] Vortex the 5.6 μm streptavidin fluorescently encoded microspheres to mix thoroughly, take out 1.2e7 microspheres and place them in a 1.5 mL Eppendorf tube, and add 900 μL 0.01 M pH 7.4 PBS solution for washing. Discard the supernatant and repeat the washing step 3 times, and resuspend for later use.

[0064] CRP-C1 antibody and biotin-PEG12-NHS were mixed and reacted in 0.01M pH 7.4 PBS solution, shaken and mixed for 1000 r, and reacted at 25°C for 1 h. After the reaction, ultrafiltration was performed twice in 0.01M pH 7.4 PBS using an ultrafiltration tube to obtain a biotin-CRP-C1 antibody solution, which was stored at 4°C for later use.

[0065] The washed microspheres were mixed with biotin-CRP-C1 antibody, shaken and mixed for 1000 r, and reacted at 25°C for 1 h to obtain CRP-C1 antibody microspheres.

[0066] (2) Preparation of antibody-coated micron magnetic beads.

[0067] 1 mg of 3 μm carboxyl magnetic beads was taken out and placed in a 1.5 mL Eppendorf tube. 900 μL of 0.05 M pH 5.5 MES solution was added for washing. The supernatant was discarded and the washing step was repeated 3 times. The beads were resuspended in 0.05 M pH 5.5 MES solution and 20 μL of 10 mg / mL EDC and NHS were added thereto. The beads were shaken for 1000 r and reacted at 25 ° C for 20 min. The beads were then washed with 0.05 M pH 6.5 MES and washed twice. After resuspending, 20 μg of CRP-T9 antibody solution was added. The beads were shaken for 1000 r and reacted at 25 ° C for 3 h. After washing, 0.01 M pH 7.4 PBS containing 1% BSA was used for washing. The beads were washed twice and then resuspended for blocking. The beads were shaken for 1000 r and reacted at 25 ° C for 1 h. Finally, 0.01 M pH 7.4 The cells were washed twice with PBS containing 0.5% BSA, 5% sucrose, and 0.1% Tween and resuspended for storage at 4°C.

[0068] (3) Generation and separation of double antibody sandwich complexes.

[0069] Take 5e5 fluorescently encoded microspheres coated with CRP-C1 antibodies and add them to the CRP test sample. Then add 100uCRP-T9 coated carboxyl magnetic beads, shake and mix for 1000r, and react at 37℃ for 30min. Finally, wash the reaction product three times with 0.01M pH7.4 PBS on a magnetic stand, remove it from the magnetic pole, add buffer to resuspend the sample in the flow tube, and collect the final double antibody sandwich complex.

[0070] (4) Flow cytometric counting.

[0071] The resuspended samples were counted on a flow cytometer.

[0072] The CRP concentration in the CRP test sample used in step (3) was changed, and the counting results at different concentrations were statistically analyzed, as shown in Table 4.

[0073] Table 4 Fluorescent microsphere counting results at different CRP concentrations in Comparative Example 2

[0074] From the above concentration data relationship, it can be seen that when using micron magnetic beads, the specific reaction of fluorescent microspheres and magnetic beads is poor and low abundance antigen concentrations cannot be distinguished.

[0075] Comparing the data in Tables 1-4, it can be seen that in the low-value test, the sensitivity of fluorescence counting using the method of the present invention is significantly better than that of the methods in Comparative Examples 1 and 2, and the linear relationship in the embodiment is also better than that in Comparative Example 1.

[0076] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the present invention, other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. A low-abundance antigen detection method based on flow cytometry fluorescence single-molecule counting, characterized in that: The following steps are involved: S1. After mixing the fluorescently encoded microspheres coated with the capture antibody with the standard sample or the sample to be tested, the detection antibody and nanomagnetic beads are added, or the nanomagnetic beads coated with the detection antibody are directly added to form a double antibody sandwich complex after the reaction. The complex is separated and counted using a flow cytometer; S2. Establish a standard curve between the target antigen concentration and the number of fluorescent microspheres collected, and calculate the concentration of the target antigen in the sample to be tested based on the standard curve.

2. The low-abundance antigen detection method according to claim 1, characterized in that The fluorescent coding microspheres are bare microspheres or microspheres with surfaces modified with amino groups, carboxyl groups, hydroxyl groups, sulfhydryl groups, streptavidin or biotin. The fluorescent coding microspheres are made of polystyrene, polymethyl methacrylate or silica.

3. The low-abundance antigen detection method according to claim 2, characterized in that: The fluorescent coding microspheres are polystyrene microspheres with a particle size of 2-4 μm, and the particle size of the nanomagnetic beads is 20-130 nm.

4. The low-abundance antigen detection method according to claim 1, characterized in that In each mL of the reaction system in step S1, the number of fluorescently encoded microspheres is more than 100,000, and the content of the target antigen is less than 10 pg.

5. The low-abundance antigen detection method according to claim 1, characterized in that The separation was performed using a MACS column.

6. The low-abundance antigen detection method according to claim 1, characterized in that The target antigen is CRP, and the capture antibody and detection antibody are CRP-C1 antibody and CRP-T9 antibody respectively.

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