A sample pretreatment method and application

Abstract

The application discloses a sample pretreatment method and application, and the sample pretreatment method comprises the following steps: activating microspheres, coupling capture antibodies, mixing the microspheres coupled with the capture antibodies with a sample to be detected, centrifuging and discarding supernatant after sufficient reaction, and obtaining a concentrated sample to be detected. The sample pretreatment method of some examples of the application can effectively enrich and concentrate large-volume samples, improve the detection sensitivity, and solve the problem that the existing sample enrichment cannot be realized by using magnetic beads when the sample is more than 10ml. Compared with the conventional capture antibody labeled magnetic bead enrichment technology, the microspheres used in the sample detection method have a smaller particle size and a larger specific surface area, can more effectively capture target objects in the sample, and require a smaller amount of microspheres for capturing the same amount of target objects, so that the mass ratio of the microspheres to the capture antibodies is only 10: (0.5-2), the amount of the microspheres is reduced, and the detection sensitivity is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of detection, specifically relating to a sample preprocessing method and its application. Background Technology

[0002] In the field of in vitro diagnostics, especially point-of-care testing (POCT), obtaining accurate test results simply, quickly, and at low cost is a pursuit of medical testing institutions and medical device manufacturers. This has led to the emergence of many excellent products, and manufacturers are constantly updating and iterating their offerings. The working principle typically employs direct competition, indirect competition, or double-antibody sandwich methods. The key technology involves using labeled materials to conjugate antibodies, causing the antibodies to develop color and thus recognize antigens. However, when improving and optimizing product performance, manufacturers often only optimize the sample testing reagents, neglecting the sample pretreatment steps before testing.

[0003] In practical applications, the effective target analytes in samples are often present in very low concentrations, or even not enough are collected. This results in the failure to detect low-abundance antigens in clinical testing, leading to common problems such as low sensitivity and high false negatives. Furthermore, the small sample volume often results in low content and purity of the target analytes, limiting the detection capacity and testing scenarios.

[0004] One way to solve the above problem is to concentrate a large volume of sample and then take a small volume of the concentrated sample for testing. However, when the sample exceeds 10 ml, magnetic separation cannot be achieved when using the currently common magnetic beads for sample enrichment and concentration. Therefore, it is necessary to explore new enrichment and concentration methods.

[0005] Developing a sample pretreatment method that can effectively achieve concentration and enrichment is of great significance for improving the sensitivity and accuracy of sample detection. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a sample preprocessing method and its application.

[0007] The technical solution adopted in this invention is:

[0008] The first aspect of the present invention provides:

[0009] A sample preprocessing method includes the following steps:

[0010] Microsphere activation: Microspheres are added to an activation solution for activation to obtain pretreatment solution A, wherein the microspheres include latex microspheres or fluorescent microspheres;

[0011] Microspheres were coupled with capture antibodies: the capture antibodies were added to pretreatment solution A, the mass ratio of microspheres to capture antibodies was 10:(0.5~2), and the mixture was stirred to couple the microspheres to obtain pretreatment solution B.

[0012] Pretreatment solution B is mixed with the sample to be tested to obtain the sample pretreatment solution;

[0013] Centrifuge the pretreatment solution of the sample to be tested and discard the supernatant to obtain the concentrated sample to be tested.

[0014] In some sample pretreatment methods, the diameter of the microspheres ranges from 0.2 to 1.5 μm. Experimental data show that microspheres of this diameter can achieve better detection results.

[0015] In some examples of sample pretreatment methods, during microsphere activation, the activation solution comprises carbodiimide and N-hydroxysuccinimide, wherein the mass ratio of carbodiimide to N-hydroxysuccinimide is 10:((2-4).

[0016] In some examples of sample pretreatment methods, after adding the capture antibody to pretreatment solution A for conjugation, the following steps are also taken: adding TBST + 1% BSA and mixing and shaking at 2000 rpm / min, separating the supernatant after shaking, and resuspending to obtain pretreatment solution B.

[0017] In some examples of sample pretreatment methods, before mixing the pretreatment solution B with the sample to be tested, a first conjugate is added to the pretreatment solution B, the first conjugate comprising fluorescein isothiocyanate (FITC) or biotin.

[0018] In some examples of sample pretreatment methods, the diameter of the microspheres is 0.2–1.5 μm, and the activation solution for activating the microspheres includes carbodiimide and N-hydroxysuccinimide, wherein the mass ratio of carbodiimide to N-hydroxysuccinimide is 10:((2–4).

[0019] In some examples of sample pretreatment methods, the diameter of the microspheres is 0.2–1.5 μm. When the microspheres are activated, the activation solution includes carbodiimide and N-hydroxysuccinimide, and the mass ratio of carbodiimide to N-hydroxysuccinimide is 10:((2–4). After adding the capture antibody to the pretreatment solution A for coupling, the method further includes adding TBST + 1% BSA and mixing and shaking at 2000 rpm / min. After shaking, the supernatant is removed and the mixture is resuspended to obtain pretreatment solution B.

[0020] In some examples of sample pretreatment methods, the diameter of the microspheres is 0.2–1.5 μm. When the microspheres are activated, the activation solution includes carbodiimide and N-hydroxysuccinimide, and the mass ratio of carbodiimide to N-hydroxysuccinimide is 10:((2–4). After adding the capture antibody to the pretreatment solution A for conjugation, the method further includes adding TBST + 1% BSA and mixing and shaking at 2000 rpm / min. After shaking, the supernatant is separated and resuspended to obtain pretreatment solution B. Before mixing pretreatment solution B with the sample to be tested, the method further includes adding a first conjugate to pretreatment solution B. The first conjugate includes fluorescein isothiocyanate (FITC) or biotin.

[0021] In some examples of sample pretreatment methods, the mass ratio of microspheres to carbodiimide during microsphere activation is 20:(0.5-2).

[0022] In some examples of sample pretreatment methods, after adding the first conjugate to pretreatment solution B, the sample is washed, resuspended in a buffer solution with the following composition: 5-500 mM Tris-HCl, 15-1500 mM NaCl, 0.01-1% Tween 20, 0.1-10% BSA, and 0.05-0.09% Procline 300.

[0023] Some examples of sample preprocessing methods include the following steps:

[0024] Microsphere activation: Microspheres with a diameter of 0.2–1.5 μm are added to an activation solution for activation. The activation solution includes carbodiimide and N-hydroxysuccinimide, wherein the mass ratio of carbodiimide to N-hydroxysuccinimide is 10:((2–4), and the mass ratio of microspheres to carbodiimide is 20:(0.5–2), to obtain pretreatment solution A. The microspheres include latex microspheres or fluorescent microspheres.

[0025] Microspheres were coupled with capture antibodies: the capture antibodies were added to pretreatment solution A, the mass ratio of microspheres to capture antibodies was 10:(0.5~2), and the mixture was stirred to couple the microspheres to obtain pretreatment solution B.

[0026] After adding the first conjugate to pretreatment solution B, washing is performed after coupling is complete.

[0027] The pretreatment solution B of the first coupling conjugate is mixed with the sample to be tested to obtain the sample pretreatment solution.

[0028] Centrifuge the pretreatment solution of the sample to be tested and discard the supernatant to obtain the concentrated sample to be tested.

[0029] A second aspect of the present invention provides:

[0030] An enrichment sample kit comprising a pretreatment solution B obtained by the sample pretreatment method described in the first aspect of the present invention.

[0031] In some examples of enrichment sample kits, a first conjugate is coupled to the pretreatment solution B.

[0032] In some examples of enrichment sample kits, after the pretreatment solution B is coupled with the first conjugate, it is washed, separated into solid and liquid components, and resuspended in a buffer solution for storage. The buffer solution consists of: 5-500 mM Tris-HCl, 15-1500 mM NaCl, 0.01-1% Tween 20, 0.1-10% BSA, and 0.05-0.09% Procline 300.

[0033] A third aspect of the present invention provides:

[0034] A sample detection method, wherein the microspheres in the concentrated sample to be tested are latex microspheres, includes the following steps:

[0035] According to the sample pretreatment method described in the first aspect of the present invention, a concentrated sample to be tested is obtained;

[0036] Add labeled magnetic beads that can bind to the sample to be tested, react, mix thoroughly, and then separate the magnetic beads;

[0037] The sample to be tested is eluted from the separated magnetic beads and then detected.

[0038] In some examples of sample detection methods, a first conjugate is added and coupled to the pretreatment solution B before mixing with the test sample; after the test sample is eluted, a labeled luminescent material is added to allow for a complete reaction, followed by the addition of an excitation solution to activate the reaction and perform detection. The labeled luminescent material can bind to the separated magnetic beads, wherein:

[0039] The labeled magnetic beads are coupled with a second conjugate, and the labeled luminescent material is either a luminescent material labeled with the antibody of the test sample or the labeled magnetic beads are labeled with the antibody of the test sample, and the labeled luminescent material is a luminescent material labeled with the second conjugate; the first conjugate is mutually compatible with the second conjugate under detection conditions.

[0040] In some examples of sample detection methods, the luminescent material is selected from acridine esters, oxalates, and ferric luminol.

[0041] In some examples of sample detection methods, the first conjugate is fluorescein isothiocyanate (FITC), and the corresponding second conjugate is an anti-FITC antibody.

[0042] In some examples of sample detection methods, the first conjugate is biotin, and the corresponding second conjugate is streptavidin.

[0043] In some examples of sample detection methods, the luminescent material is selected from acridine ester, oxalate ester, and ferric luminol, the first conjugate is fluorescein isothiocyanate (FITC), and the corresponding second conjugate is an anti-FITC antibody.

[0044] In some examples of sample detection methods, the luminescent material is selected from acridine ester, oxalate ester, and ferric luminol, the first conjugate is biotin, and the corresponding second conjugate is streptavidin.

[0045] A fourth aspect of the present invention provides:

[0046] A sample detection method, wherein the microspheres in the concentrated sample to be tested are fluorescent microspheres, and the detection method is fluorescence immunochromatography, comprising:

[0047] According to the sample pretreatment method described in the first aspect of the present invention, a concentrated sample to be tested is obtained;

[0048] The concentrated sample to be tested is mixed with the fluorescent immunochromatographic assay reagent and then detected.

[0049] Fluorescent immunochromatographic assay reagents can be [specific types of reagents].

[0050] A fifth aspect of the present invention provides:

[0051] A test kit, comprising:

[0052] 1) Pretreatment liquid B of the first aspect of the present invention; and

[0053] 2) Fluorescent immunochromatographic assay reagents or chemiluminescent assay reagents.

[0054] The beneficial effects of this invention are:

[0055] The sample pretreatment methods of some examples of the present invention can effectively enrich and concentrate large-volume samples, improve detection sensitivity, and solve the problem that existing methods cannot enrich samples with magnetic beads when the sample volume exceeds 10 ml, and can only process samples by magnetic separation.

[0056] The sample detection method of some examples of the present invention uses microspheres to conjugate capture antibodies. Compared with conventional capture antibody-labeled magnetic bead enrichment technology, the microspheres have smaller particle size and larger specific surface area, which can more effectively capture target substances in the sample. The amount of microspheres required to capture the same amount of target substances is less than that of magnetic beads. The mass ratio of microspheres to capture antibodies is only 10:(0.5~2), which helps to reduce the amount of microspheres used and improves detection sensitivity. Detailed Implementation

[0057] The abbreviations used in this invention have specific meanings commonly used in the art. The specific meanings of some of the abbreviations are as follows:

[0058] AE: acridine ester

[0059] Bio: Biotin

[0060] BSA: Bovine serum albumin

[0061] EDC: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide

[0062] FITC: Fluorescein isothiocyanate

[0063] LAM: Lipoarabinomannan

[0064] MES: Morpholin taurine buffer

[0065] NHS: N-hydroxysuccinimide

[0066] SA: Streptavidin.

[0067] The technical solution of the present invention will be further illustrated below with examples and experimental data. In the following examples, unless otherwise specified, the diameter of the microspheres used is 0.2 to 1.5 μm.

[0068] In practical applications, the effective target analytes in samples are often present in very low concentrations, or even not enough are collected, resulting in the failure to detect low-abundance antigens in clinical testing. This leads to common problems such as low sensitivity and high false negatives. Furthermore, the small sample volume often results in low content and purity of the target analytes, limiting the detection capacity and testing scenarios.

[0069] Therefore, it is necessary to concentrate large-volume samples and then take a small volume of the concentrated sample for testing. This is one way to solve the above problems. However, when the sample exceeds 10 ml, the current method of enriching and concentrating the sample using magnetic beads is not feasible because the sample volume is too large to be separated by magnetic force. Therefore, it is necessary to explore new enrichment and concentration methods.

[0070] Therefore, this invention proposes a sample preprocessing method that improves the sensitivity of sample detection by preprocessing, concentrating and enriching the sample.

[0071] Example 1:

[0072] (1) Take 2 mg of latex microspheres and dilute them with MES to 2 mg / ml. Add 10 μL of EDC (10 mg / ml) and 2.5 μL of NHS (10 mg / ml). Incubate at room temperature and 30 rpm for 30 min. Centrifuge at 20000 g for 10 min and discard the supernatant. Resuspend the microspheres with 1.0 ml of MES to obtain pretreatment solution A.

[0073] (2) Add 200ug of 2109 (capture antibody), mix well, and sonicate; incubate at room temperature and 30rpm for 60min.

[0074] (3) Add 0.2 ml of 10% BSA and mix well. Sonicate. Incubate at room temperature and 30 rpm for 60 min.

[0075] (4) Centrifuge at 20000g for 10min, discard the supernatant, and resuspend the latex microspheres in 1.0ml of 0.1M sodium carbonate (pH 9.0) to obtain pretreatment solution B.

[0076] (5) Add 40ug of bio and mix well, then sonicate; incubate overnight at 2-8℃.

[0077] (6) Add 1ml TBST + 1% BSA and wash 3 times;

[0078] (7) Resuspend in buffer solution, sonicate, and store at 2–8°C for later use. The concentration of bio-2109-latex microspheres is 1 mg / ml. Buffer solution: 250 mM Tris-HCl, 1000 mM NaCl, 0.5% Tween 20, 5% BSA, 0.07% Procline 300;

[0079] (8) Take 50ul bio-2109-latex microspheres (1mg / ml) and add 10ml LAM standard, place them on a turntable mixer and mix at room temperature for 30min; to obtain the pretreatment solution for the sample to be tested;

[0080] (9) Centrifuge at 20000g for 10 min, and discard the supernatant after centrifugation;

[0081] (10) Add 500ul buffer solution to resuspend the sample to be tested and use it for chemiluminescence analysis.

[0082] (11) On a fully automated chemiluminescence immunoassay analyzer, take 50 μL of the above sample and add 50 μL of SA-magnetic beads. React at 37°C for 10 min. Wash 4 times.

[0083] (12) Add 50 μL of 2107 (detection antibody)-AE, react at 37°C for 10 min; wash 4 times;

[0084] (13) Add 100 μL of pre-activation solution and 100 μL of activation solution; react for 2 min;

[0085] (14) Obtain the luminous value.

[0086] Example 2:

[0087] The only difference from Example 1 is that the mass ratio of latex microspheres to EDC is 20:0.3.

[0088] Example 3:

[0089] The only difference from Example 1 is that the mass ratio of latex microspheres to EDC is 20:0.5.

[0090] Example 4:

[0091] The only difference from Example 1 is that the mass ratio of latex microspheres to EDC is 20:2.

[0092] Example 5:

[0093] The only difference from Example 1 is that the mass ratio of latex microspheres to EDC is 20:4.

[0094] Example 6:

[0095] The only difference from Example 1 is that the mass ratio of carbodiimide to N-hydroxysuccinimide is 10:1.

[0096] Example 7:

[0097] The only difference from Example 1 is that the mass ratio of carbodiimide to N-hydroxysuccinimide is 10:2.

[0098] Example 8:

[0099] The only difference from Example 1 is that the mass ratio of carbodiimide to N-hydroxysuccinimide is 10:4.

[0100] Example 9:

[0101] The only difference from Example 1 is that the mass ratio of carbodiimide to N-hydroxysuccinimide is 10:5.

[0102] Example 10:

[0103] The only difference from Example 1 is that the mass ratio of latex microspheres to capture antibodies is 10:0.3.

[0104] Example 11:

[0105] The only difference from Example 1 is that the mass ratio of latex microspheres to capture antibodies is 10:0.5.

[0106] Example 12:

[0107] The only difference from Example 1 is that the mass ratio of latex microspheres to capture antibodies is 10:2.

[0108] Example 13:

[0109] The only difference from Example 1 is that the mass ratio of latex microspheres to capture antibodies is 10:3.

[0110] Example 14:

[0111] The difference from Example 1 is that the buffer solution consists of 5 mM Tris-HCl, 15 mM NaCl, 0.01% Tween 20, 0.1% BSA, and 0.05% Procline 300.

[0112] Example 15:

[0113] The difference from Example 1 is that the buffer solution consists of 500mM Tris-HCl, 1500mM NaCl, 1% Tween 20, 5% BSA, and 0.09% Procline 300.

[0114] Example 16:

[0115] The difference from Example 1 is that the Tween20 content in the buffer solution is 0.5%.

[0116] Example 17:

[0117] The difference from Example 1 is that the BSA content in the buffer is 5% and the Procline 300 content is 0.07%.

[0118] After pretreatment of LAM samples using Examples 1-17 above, measurements were performed on a fully automated chemiluminescence immunoassay analyzer SMART6500S. LAM test samples included blank samples and LAM standards at different concentrations: 10 pg / ml, 100 pg / ml, and 1000 pg / ml.

[0119] Table 1 summarizes the signal values ​​obtained from detecting different concentrations of LAM standards at different latex microsphere to EDC mass ratios (Examples 1-5).

[0120] Table 1. Comparison of test results for different latex microsphere to EDC mass ratios

[0121] LAM concentration (pg / ml) Example 1 Example 2 Example 3 Example 4 Example 5 0 655 589 641 789 1200 10 3521 3020 3411 3780 3950 100 23501 20235 21011 23541 26340 1000 185395 102356 125416 195011 195977

[0122] Table 1 shows that different mass ratios of latex microspheres to EDC resulted in varying sensitivities after sample pretreatment (Example 2 had a latex microsphere to EDC mass ratio of 20:0.3), leading to low luminescence values ​​in the calibration curve and low reagent sensitivity. As the mass of EDC increased, the reagent signal also increased, but simultaneously, the background signal (0 pg / ml) also increased, causing the signal-to-noise ratio to decrease. Therefore, the optimal mass ratio of latex microspheres to EDC is 20:(0.5–2); a further optimized mass ratio of latex microspheres to EDC is 20:1.

[0123] Table 2 summarizes the signal values ​​obtained from detecting LAM standards at different concentrations with different EDC:NHS mass ratios (Examples 1, 6-9).

[0124] Table 2. Comparison of test results for different EDC:NHS quality ratios

[0125] LAM concentration (pg / ml) Example 1 Example 6 Example 7 Example 8 Example 9 0 655 504 624 805 1086 10 3521 2030 3650 3878 3361 100 23501 20560 24560 27893 24501 1000 185395 91265 155394 195300 205021

[0126] As shown in Table 2, the sensitivity of samples with different EDC:NHS mass ratios after preprocessing varies. When the EDC:NHS mass ratio changes from 10:1 to 10:5, the signal-to-noise ratio first increases and then decreases. It can be seen that the optimal range for the EDC:NHS mass ratio is 10:(2-4); the optimal EDC:NHS mass ratio is 10:2.5.

[0127] Table 3 summarizes the signal values ​​obtained from detecting different concentrations of LAM standards with different latex microsphere and capture antibody mass ratios (Examples 1, 10-13).

[0128] Table 3. Comparison of detection results for different latex microsphere and capture antibody mass ratios

[0129] LAM concentration (pg / ml) Example 1 Example 10 Example 11 Example 12 Example 13 0 655 544 614 806 1100 10 3521 2891 3045 3620 3752 100 23501 19564 22014 24540 25501 1000 185395 95463 164865 190395 215395

[0130] As shown in Table 3, different mass ratios of latex microspheres and capture antibodies have different sensitivities after sample pretreatment. When the mass ratio of latex microspheres to capture antibodies changes from 10:0.3 to 10:3, the signal-to-noise ratio first increases and then decreases. It can be seen that the optimal range for the mass ratio of latex microspheres to capture antibodies is 10:(0.5~2); the optimal mass ratio of latex microspheres to capture antibodies is 10:1.

[0131] Accelerated stability test:

[0132] The microsphere-capture antibody was stored in different buffer solutions (Examples 1, 14-17) and subjected to accelerated destruction at 37°C. After storage for 3, 7, and 14 days, the signal values ​​of each level of the LAM standard were measured and compared with the initial signal values ​​to obtain the recovery rate. The average recovery rate was calculated, and the results are shown in Table 4.

[0133] Table 4. Results of experiments on accelerated destruction of microspheres and captured antibodies using different buffer solutions.

[0134] serial number 3-day recovery rate / % 7-day recovery rate / % 14-day recovery rate / % Example 1 97 96 94 Example 14 96 94 92 Example 15 97 93 90 Example 16 93 77 61 Example 17 96 83 66

[0135] As shown in Table 4, the buffer solutions of Examples 1, 14, and 15 have good stability and high recovery rates. Therefore, the buffer composition ratio of Example 1 is further optimized.

[0136] Preparation of capture antibody-latex microspheres-FITC:

[0137] 1) Take 2 mg of latex microspheres (particle size 0.2-1.5 μm) and dilute with MES to 2 mg / ml. Add 10 μl of EDC (10 mg / ml) and 2.5 μl of NHS (10 mg / ml). Incubate at room temperature for 30 min at 30 rpm. Centrifuge and discard the supernatant. Resuspend the latex microspheres in 1 ml of buffer.

[0138] 2) Add 200ug of LAM capture antibody 2109, mix well, and sonicate; incubate at room temperature on a turntable at 30rpm for 60min;

[0139] 3) Add 0.2 ml of 10% BSA and mix well. Sonicate. Incubate at room temperature for 60 min at 30 rpm.

[0140] 4) Centrifuge and discard the supernatant, then resuspend the microspheres in 1.0 ml of 0.1 M sodium carbonate (pH 9.0);

[0141] 5) Add 40ug FITC, mix well, and sonicate; incubate overnight at 2-8℃.

[0142] 6) Add 1ml TBST + 1% BSA and wash 3 times;

[0143] 7) Resuspend the microspheres in 2 ml TBST + 1% BSA and sonicate. Store at 2-8℃ for later use. Label as 2109-Latex Microspheres-FITC.

[0144] Chemiluminescence immunoassay

[0145] Method 1.1)

[0146] 2109-latex microspheres-FITC were mixed and incubated with LAM samples to form a (FITC-latex microspheres-2109-LAM) complex, which was then mixed and incubated with anti-FITC-magnetic beads to form a (magnetic bead-anti-FITC-FITC-latex microspheres-2109-LAM) complex. Finally, 2107-AE (AE-labeled LAM detection antibody) was added, and the luminescence value was detected. The specific operation is as follows:

[0147] 1) Take 500ul of LAM standard and add 50ul of 2109-latex microspheres-FITC (1mg / ml). Place the mixture on a rotary mixer and mix at room temperature for 30min.

[0148] 2) In a fully automated chemiluminescence immunoassay, take 50 μL of the above sample, add 50 μL of anti-FITC magnetic beads, react at 37°C for 10 min; wash 4 times.

[0149] 3) Add 50 μL of 2107-AE, react at 37°C for 10 min; wash 4 times;

[0150] 4) Add 100 μL of pre-activation solution and 100 μL of activation solution, and react for 2 min;

[0151] 5) Obtain the luminance value.

[0152] Method 1.2)

[0153] 2109-latex microspheres-FITC were mixed and incubated with LAM samples to form a (FITC-latex microspheres-2109-LAM) complex, which was then mixed and incubated with 2107-magnetic beads to form a (magnetic bead-2107 (antibody)-LAM-2109 (antibody)-latex microspheres-FITC) complex. Anti-FITC-AE was then added, and the luminescence value was detected. The specific operation is as follows:

[0154] 1) Take 500ul of LAM standard and add 50ul of 2109-latex microspheres-FITC (1mg / ml). Place the mixture on a rotary mixer and mix at room temperature for 30min.

[0155] 2) In a fully automated chemiluminescence immunoassay, take 50 μL of the above sample, add 50 μL of 2107-magnetic beads, react at 37°C for 10 min; wash 4 times.

[0156] 3) Add 50 μL of anti-FITC-AE, react at 37°C for 10 min; wash 4 times;

[0157] 4) Add 100 μL of pre-activation solution and 100 μL of activation solution; react for 2 min;

[0158] 5) Obtain the luminance value.

[0159] The pre-activation solution and activation solution are commercially available products.

[0160] Experimental group 1: 50ul 2109-latex microspheres-FITC (1mg / ml) + 500ul LAM standard, anti-FITC-magnetic bead capture, 2107-AE detection.

[0161] Experimental group 2: 50ul 2109-latex microspheres-FITC (1mg / ml) + 500ul LAM standard, 2107-magnetic bead capture, anti-FITC-AE detection.

[0162] Experimental group 3: 25ul 2109-latex microspheres-FITC (1mg / ml) + 500ul LAM standard, anti-FITC-magnetic bead capture, 2107-AE detection.

[0163] Control group 1: 50ul 2109-magnetic beads (1mg / ml) + 500ul LAM standard, 2107-AE detection.

[0164] 1) In a fully automated chemiluminescence immunoassay analyzer, take 50 μL of LAM standard and add 50 μL of 2109-magnetic beads, react at 37°C for 10 min; wash 4 times;

[0165] 2) Add 50 μL of 2107-AE, react at 37°C for 10 min; wash 4 times;

[0166] 3) Add 100 μL of pre-activation solution and 100 μL of activation solution; react for 2 min;

[0167] 4) Obtain the luminance value.

[0168] Control group 2: 25ul 2109-magnetic beads (1mg / ml) + 500ul LAM standard, 2107-AE detection.

[0169] 1) In a fully automated chemiluminescence immunoassay analyzer, take 50 μL of LAM standard and add 25 μL of 2109-magnetic beads, react at 37°C for 10 min; wash 4 times;

[0170] 2) Add 50 μL of 2107-AE, react at 37°C for 10 min; wash 4 times;

[0171] 3) Add 100 μL of pre-activation solution and 100 μL of activation solution; react for 2 min;

[0172] 4) Obtain the luminance value.

[0173] Signal-to-noise ratio = average luminous value / average background luminous value, where background luminous value is the luminous value without the addition of LAM standard.

[0174] The results of experimental groups 1-3 and control groups 1-2 are shown in Table 5.

[0175] Table 5. Luminescence values ​​of different groups at different LAM concentrations

[0176]

[0177] As shown in Table 5, compared with control group 1, the LAM detection sensitivity of experimental group 1 was increased by 2 to 3.8 times, and the LAM detection sensitivity of experimental group 2 was increased by 1.1 to 3.6 times. Compared with control group 2, the LAM detection sensitivity of experimental group 3 was increased by 1.1 to 2.3 times, but weaker than that of experimental group 1.

[0178] Preparation of Bio-2109-Latex Microspheres:

[0179] 1) Take 2 mg of latex microspheres, dilute them with MES to 2 mg / ml, add 10 μL of EDC (10 mg / ml) and 2.5 μL of NHS (10 mg / ml), incubate at room temperature and 30 rpm for 30 min; centrifuge and discard the supernatant; resuspend the microspheres with 1 ml of MES;

[0180] 2) Add 0.2 ml of 10% BSA, sonicate to disperse, incubate overnight at room temperature on a rotating plate at 30 rpm; centrifuge and discard the supernatant; resuspend the microspheres in 1 ml of 0.1 M sodium carbonate solution (pH 9.0);

[0181] 3) Centrifuge and discard the supernatant; resuspend the microspheres in 1 ml of PBS;

[0182] 4) Add 40ug of Bio-2109, incubate at room temperature on a rotating plate at 30rpm for 30min; centrifuge and discard the supernatant;

[0183] 5) Add 1ml TBST + 1% BSA and wash 3 times;

[0184] 6) Resuspend the sample in 2ml TBST + 1% BSA and sonicate. Store at 2-8℃ until use.

[0185] Detection method:

[0186] Method 2.1) Bio-2109 latex microspheres were mixed and incubated with LAM samples to form a (bio-2109-latex microsphere-LAM) complex, which was then mixed and incubated with SA-magnetic beads to form a (magnetic bead-SA-bio-2109-latex microsphere-LAM) complex. 2107-AE was then added, and the luminescence value was detected. Specifically, this includes:

[0187] 1) Take 500ul of LAM standard and add 50ul of bio-2109-latex microspheres (1mg / ml). Place the mixture on a rotary mixer and mix at room temperature for 30min.

[0188] 2) In a fully automated chemiluminescence immunoassay, take 50 μL of the above sample, add 50 μL of anti-bio-magnetic beads, react at 37°C for 10 min; wash 4 times.

[0189] 3) Add 50 μL of 2107-AE, react at 37°C for 10 min; wash 4 times;

[0190] 4) Add 100 μL of pre-activation solution and 100 μL of activation solution; react for 2 min;

[0191] 5) Obtain the luminance value.

[0192] Method 2.2) Bio-2109 latex microspheres were mixed and incubated with LAM samples to form a (bio-2109-latex microsphere-LAM) complex, which was then mixed and incubated with 2107 magnetic beads to form a (magnetic bead-2107 (antibody)-LAM-2109 (antibody)-latex microsphere-bio) complex. SA-AE (SA-labeled acridine ester) was then added, and the luminescence value was detected. Specifically, this includes:

[0193] 1) Take 500ul of LAM standard and add 50ul of bio-2109-latex microspheres (1mg / ml), mix well;

[0194] 2) In a fully automated chemiluminescence immunoassay, take 50 μL of the above sample, add 50 μL of 2107-magnetic beads, react at 37°C for 10 min; wash 4 times.

[0195] 3) Add 50 μL SA-AE, react at 37°C for 10 min; wash 4 times;

[0196] 4) Add 100 μL of pre-activation solution and 100 μL of activation solution; react for 2 min;

[0197] 5) Obtain the luminance value.

[0198] Experimental group 4: 50ul bio-2109-latex microspheres (1mg / ml) + 500ul LAM standard, SA-magnetic bead capture, 2107-AE detection.

[0199] Experimental group 5: 50ul bio-2109-latex microspheres (1mg / ml) + 500ul LAM standard, 2107-magnetic bead capture, SA-AE detection.

[0200] Control group 3: 50ul 2109-magnetic beads (1mg / ml) + 500ul LAM standard, 2107-AE detection.

[0201] The test results of experimental groups 4-5 and control group 3 are shown in Table 6.

[0202] Table 6. Emission values ​​of different groups at different LAM concentrations

[0203]

[0204] As shown in Table 6, compared with the control group 3, the detection sensitivity of experimental group 4 was increased by about 4 times, and the detection sensitivity of experimental group 5 was increased by about 4.5 times.

[0205] Experimental group 6: 50ul bio-2109-latex microspheres (1mg / ml) + 10ml LAM standard, SA-magnetic bead capture, 2107-AE detection.

[0206] 1) Add 10 ml of LAM standard to 50 μl of bio-2109-latex microspheres (1 mg / ml), place on a rotary mixer, and mix at room temperature for 30 min.

[0207] 2) Centrifuge and discard the supernatant;

[0208] 3) Add 500 μL of sample diluent to resuspend the latex microspheres for chemiluminescence assay.

[0209] 4) In a fully automated chemiluminescence immunoassay, take 50 μL of the above sample, add 50 μL of SA-magnetic beads, react at 37°C for 10 min; wash 4 times.

[0210] 5) Add 50 μL of 2107-AE, react at 37°C for 10 min; wash 4 times;

[0211] 6) Add 100 μL of pre-activation solution and 100 μL of activation solution; react for 2 min;

[0212] 7) Obtain the luminance value.

[0213] Experimental group 7: 50ul bio-2109-latex microspheres (1mg / ml) + 15ml LAM standard, SA-magnetic bead capture, 2107-AE detection.

[0214] 1) Add 15 ml of LAM standard to 50 μl of bio-2109-latex microspheres (1 mg / ml), place on a rotary mixer, and mix at room temperature for 30 min.

[0215] The remaining steps are the same as in experimental group 6.

[0216] Experimental group 8: 50ul bio-2109-latex microspheres (1mg / ml) + 20ml LAM standard, SA-magnetic bead capture, 2107-AE detection.

[0217] 1) Add 50ul of bio-2109-latex microspheres (1mg / ml) to 20ml of LAM standard, place on a rotary mixer, and mix at room temperature for 30min.

[0218] The remaining steps are the same as in experimental group 6.

[0219] Experimental group 9: 50ul bio-2109-latex microspheres (1mg / ml) + 40ml LAM standard, SA-magnetic bead capture, 2107-AE detection.

[0220] 1) Add 50ul of bio-2109-latex microspheres (1mg / ml) to 40ml of LAM standard, place on a rotary mixer, and mix at room temperature for 30min.

[0221] The remaining steps are the same as in experimental group 6.

[0222] Experimental group 10: 50ul 2109-latex microspheres-FITC (1mg / ml) + 10ml LAM standard, anti-FITC-magnetic bead capture, 2107-AE detection.

[0223] 1) Add 50ul of 2109-latex microspheres-FITC (1mg / ml) and 10ml of LAM standard to a rotary mixer and mix at room temperature for 30min.

[0224] 2) Centrifuge and discard the supernatant;

[0225] 3) Add 500 μL of sample diluent to resuspend the latex microspheres for chemiluminescence assay.

[0226] 4) In a fully automated chemiluminescence immunoassay, take 50 μL of the above sample, add 50 μL of anti-FITC magnetic beads, react at 37°C for 10 min; wash 4 times.

[0227] 5) Add 50 μL of 2107-AE, react at 37°C for 10 min; wash 4 times;

[0228] 6) Add 100 μL of pre-activation solution and 100 μL of activation solution; react for 2 min;

[0229] 7) Obtain the luminance value.

[0230] Experimental group 11: 50ul 2109-latex microspheres-FITC (1mg / ml) + 15ml LAM standard, anti-FITC-magnetic bead capture, 2107-AE detection.

[0231] 1) Add 50ul of 2109-latex microspheres-FITC (1mg / ml) and 15ml of LAM standard to a rotary mixer and mix at room temperature for 30min.

[0232] 2) The remaining steps are the same as in experimental group 10.

[0233] Experimental group 12: 50ul 2109-latex microspheres-FITC (1mg / ml) + 20ml LAM standard, anti-FITC-magnetic bead capture, 2107-AE detection.

[0234] 1) Add 50ul of 2109-latex microspheres-FITC (1mg / ml) and 20ml of LAM standard to a rotary mixer and mix at room temperature for 30min.

[0235] 2) The remaining steps are the same as in experimental group 10.

[0236] Experimental group 13: 50ul 2109-latex microspheres-FITC (1mg / ml) + 40ml LAM standard, anti-FITC-magnetic bead capture, 2107-AE detection.

[0237] 1) Add 50ul of 2109-latex microspheres-FITC (1mg / ml) to 40ml of LAM standard, place on a rotary mixer, and mix at room temperature for 30min.

[0238] 2) The remaining steps are the same as in experimental group 10.

[0239] Control group 1: 50ul 2109-magnetic beads (1mg / ml) + 50ul LAM standard, 2107-AE detection.

[0240] 1) In a fully automated chemiluminescence immunoassay analyzer, take 50 μL of LAM standard and add 50 μL of 2109-magnetic beads, react at 37°C for 10 min; wash 4 times;

[0241] 2) Add 50 μL of 2107-AE, react at 37°C for 10 min; wash 4 times;

[0242] 3) Add 100 μL of pre-activation solution and 100 μL of activation solution; react for 2 min;

[0243] 4) Obtain the luminance value.

[0244] Example 1: The test results of experimental groups 6-13 are shown in Table 7.

[0245] Table 7. Luminescence values ​​under different detection conditions

[0246]

[0247] The results in Table 7 show that:

[0248] 1) This method can effectively enrich LAM in samples, enabling the enrichment and concentration of large-volume samples and improving detection sensitivity. It solves the problem that existing methods cannot enrich samples using magnetic beads when the sample volume exceeds 10 ml, and instead rely on magnetic separation for sample processing.

[0249] 2) The latex microspheres used in this method have a large specific surface area. The larger specific surface area allows the latex microspheres to couple with more capture antibodies compared to existing magnetic beads, thereby capturing more LAM in the sample. For example, within a certain sample volume range (10ml-40ml), 50ul bio-2109-latex microspheres (1mg / ml) capture more LAM as the sample volume increases, thus increasing the sensitivity.

[0250] 3) Compared with Example 1, latex microspheres have a larger specific surface area, which allows them to capture LAM in samples more effectively. They have the advantages of high efficiency and speed, and can bring more significant detection signals, thus achieving an enhancement effect.

[0251] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A sample preprocessing method, comprising the following steps: 1) Take 2 mg of latex microspheres and dilute them with MES to 2 mg / ml. Add 10 μL of 10 mg / ml EDC and 2.5 μL of 10 mg / ml NHS. Incubate at room temperature and 30 rpm for 30 min. Centrifuge at 20000g for 10 min and discard the supernatant. Resuspend the microspheres in 1.0 ml of MES to obtain pretreatment solution A. 2) Add 200ug of capture antibody 2109, mix well, and sonicate; incubate at room temperature on a turntable at 30rpm for 60min; 3) Add 0.2 ml of 10% BSA and mix well. Sonicate. Incubate at room temperature and 30 rpm for 60 min. 4) Centrifuge at 20000g for 10 min, discard the supernatant, and resuspend the latex microspheres in 1.0 ml of 0.1 M sodium carbonate; 5) Add 40 μg of the first conjugate biotin, mix well, and sonicate; incubate overnight at 2–8 °C to obtain bio-2109-latex microspheres. 6) Add bio-2109-latex microspheres to 1ml TBST + 1% BSA and wash 3 times; 7) Resuspend bio-2109-latex microspheres in buffer solution, sonicate, and store at 2-8℃ for later use. The storage concentration of bio-2109-latex microspheres is 1 mg / ml. Buffer solution: 250 mM Tris-HCl, 1000 mM NaCl, 0.5% Tween 20, 5% BSA, 0.07% Procline 300. 8) Take 50 μL of bio-2109-latex microspheres with a concentration of 1 mg / mL and add 10 mL of the sample to be tested. Place the sample on a rotary mixer and mix at room temperature for 30 min to obtain the sample pretreatment solution. 9) Centrifuge at 20000g for 10 minutes, discard the supernatant after centrifugation, and obtain the concentrated sample to be tested.

2. A sample detection method, wherein the microspheres in the concentrated sample to be tested are latex microspheres, comprising the following steps: The concentrated sample to be tested is obtained by the sample pretreatment method according to claim 1; Add labeled magnetic beads that can bind to the sample to be tested, react, mix thoroughly, and then separate the magnetic beads; The sample to be tested is eluted from the separated magnetic beads and then detected.

3. The sample detection method according to claim 2, characterized in that, After elution of the sample, a labeled luminescent material is added for a full reaction. Then, an excitation solution is added to activate the reaction and for detection. The labeled luminescent material can bind to the separated magnetic beads, wherein: The labeled magnetic beads are coupled with a second conjugate streptavidin, and the labeled luminescent material is a luminescent material labeled with the antibody used to detect the sample to be tested; or the labeled magnetic beads are labeled with the antibody used to detect the sample to be tested, and the labeled luminescent material is a luminescent material labeled with the second conjugate; the first conjugate is mutually compatible with the second conjugate under detection conditions.

4. The sample detection method according to claim 3, characterized in that, The luminescent material is selected from one of acridine ester, oxalate ester, and ferric luminol.

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