Colorectal cancer detection kit as well as preparation method and use method thereof

Abstract

The invention relates to the field of medical detection and discloses a colorectal cancer detection kit as well as a preparation method and a use method thereof. The colorectal cancer detection kit comprises a fluorescence labeling material and a lateral chromatography test strip, the fluorescent labeling material comprises fluorescent microspheres, an antibody corresponding to the colorectal cancer exosome marker and a primary antibody of an independent C line, wherein the antibody and the primary antibody are connected with the fluorescent microspheres; the lateral chromatography test strip comprises a composite pad, a detection membrane and a water absorption pad; one end of the detection film is lapped with the composite pad, and the other end is lapped with the water absorption pad; a T line close to the composite pad and a C line close to the water absorption pad are arranged on the detection film; the T line is coated with an exosome marker protein corresponding antibody; the C line is coated with a secondary antibody of an independent C line; the fluorescence labeling material and the lateral chromatography test strip are independently stored, or the fluorescence labeling material is attached to a composite pad of the lateral chromatography test strip. The colorectal cancer detection kit can realize detection of colorectal cancer marker protein, and further realizes detection of colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of medical testing, specifically to a colorectal cancer detection kit and its preparation and usage methods. Background Technology

[0002] Colorectal cancer is one of the most common cancers worldwide, with 1.926 million new cases and 904,000 deaths globally in 2022, ranking among the top cancers. Currently, colonoscopy is the most effective method for detecting colorectal cancer. However, due to the large population, scarce and unevenly distributed colonoscopy resources, it is not suitable for large-scale population screening. Furthermore, colonoscopy is highly invasive, leading to poor patient compliance and hindering its widespread adoption. In addition, fecal occult blood testing (FIT), multi-target fecal FIT-DNA detection, sigmoidoscopy, and colonic CT imaging techniques can also detect colorectal cancer, but these methods suffer from low sensitivity, high false positive rates, high cost, and radiation hazards. Therefore, developing simple and sensitive methods to detect specific biomarkers for colorectal cancer holds promise for solving these problems and providing new insights into the diagnosis of colorectal cancer.

[0003] Exosomes are phospholipid bilayer vesicles secreted by cells, carrying important molecules such as specific membrane proteins, nucleic acids, and lipids specific to the mother cell. In colorectal cancer, colorectal cancer cells exhibit different molecular biological behaviors than normal intestinal epithelial cells, and the exosomes they secrete also possess different molecular "fingerprints." It has been reported that CD147, A33, and CD147&A33 can effectively distinguish between colorectal cancer patients and healthy donors, achieving a clinical sensitivity of 89%, while the sensitivity of carcinoembryonic antigen (CEA) is only 40%. Therefore, CD147 and A33 can serve as specific biomarkers for colorectal cancer, and developing detection methods for CD147 and / or A33 proteins holds promise for improving the specificity and sensitivity of colorectal cancer detection. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of poor convenience, high false positive rate and high cost of colorectal cancer detection in the prior art, and to provide a colorectal cancer detection kit and its preparation and usage methods.

[0005] To achieve the above objectives, the present invention provides a colorectal cancer detection kit, which includes a fluorescent labeling material and a lateral chromatography test strip;

[0006] The fluorescent labeling material includes fluorescent microspheres and primary antibodies corresponding to colorectal cancer exosome markers and independent C-lines connected to the fluorescent microspheres;

[0007] The lateral chromatography test strip includes a composite pad, a detection membrane, and an absorbent pad; one end of the detection membrane overlaps with the composite pad, and the other end overlaps with the absorbent pad; the detection membrane has a T-line near the composite pad and a C-line near the absorbent pad;

[0008] The T line is coated with an antibody corresponding to the exosome marker protein, and the C line is coated with a secondary antibody that separates the C line.

[0009] The antibodies corresponding to the colorectal cancer exosome markers are anti-CD147 antibody and / or anti-A33 antibody;

[0010] The fluorescent labeling material and the lateral chromatography test strip are stored independently, or the fluorescent labeling material is attached to the composite pad of the lateral chromatography test strip.

[0011] Preferably, the diameter of the fluorescent microspheres is 50-400 nm; and / or

[0012] The fluorescent microspheres are selected from quantum dot fluorescent microspheres and / or fluorescent polystyrene microspheres.

[0013] Preferably, the antibody corresponding to the exosome marker protein is at least one of anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody.

[0014] Preferably, the primary antibody for the independent C-line is selected from chicken IgY antibody or DNP-BSA; the secondary antibody for the independent C-line is selected from goat anti-chicken IgY or mouse anti-DNP antibody.

[0015] Preferably, the lateral chromatography test strip further includes a base plate located below the composite pad, the detection membrane, and the absorbent pad.

[0016] Preferably, one end of the composite pad near the absorbent pad extends above the detection membrane;

[0017] Preferably, the end of the absorbent pad near the composite pad extends above the detection membrane.

[0018] A second aspect of the present invention provides a method for the colorectal cancer detection kit described above, the method comprising the following steps:

[0019] (1) Spray T-line solution and C-line solution onto the T-line and C-line of the detection membrane respectively, and then dry them;

[0020] (2) Prepare fluorescent labeling material; optionally, spray the fluorescent labeling material onto the composite pad;

[0021] (3) Overlap one end of the dried detection membrane near the T line in step (1) with the composite pad and the other end with the absorbent pad;

[0022] The T-line solution contains antibodies corresponding to exosome marker proteins, and the C-line solution contains secondary antibodies for independent C-lines.

[0023] Preferably, the process of preparing the fluorescently labeled material includes: activating the fluorescent microspheres with an activator, and then reacting them with the primary antibody corresponding to the colorectal cancer exosome marker and the independent C line;

[0024] Preferably, the activator is selected from EDC / NHS activator or EDC / Sulfo-NHS activator.

[0025] A third aspect of the present invention provides a colorectal cancer detection system, which includes the colorectal cancer detection kit and optical detection instrument described above.

[0026] This invention provides a method of using the colorectal cancer detection kit described above, the method comprising:

[0027] Add the sample to be tested onto the composite pad;

[0028] The sample to be tested is brought into contact with the fluorescently labeled material.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The colorectal cancer detection kit of the present invention combines fluorescence technology with lateral chromatography test strip technology. The fluorescent labeling material can identify and bind to colorectal cancer exosome-specific markers in the sample. The exosome marker protein antibody attached to the T line can trap the exosomes on the T line. After detection by a fluorescence detection instrument, the colorectal cancer marker protein can be detected, thereby enabling the detection of colorectal cancer.

[0031] 2. The colorectal cancer detection kit of the present invention can not only qualitatively observe the bands to determine whether they contain colorectal cancer markers, but also quantitatively detect colorectal cancer markers based on the fluorescence intensity. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the structure of a lateral chromatography test strip according to some preferred embodiments of the present invention;

[0033] Figure 2 This is a schematic diagram illustrating how the test results are interpreted when using a colorectal cancer detection kit.

[0034] Figure 3 This is a graph showing the results of testing normal cells and colorectal cancer cells using a colorectal cancer detection kit in a test case.

[0035] Explanation of reference numerals in the attached figures

[0036] 1 composite pad 2 T line

[0037] 3C line 4 detection membrane

[0038] 5 absorbent pads 6 base plates Detailed Implementation

[0039] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0040] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0041] In one aspect, the present invention provides a colorectal cancer detection kit, the colorectal cancer detection kit comprising a fluorescent labeling material and a lateral chromatography test strip;

[0042] The fluorescent labeling material includes fluorescent microspheres and primary antibodies corresponding to colorectal cancer exosome markers and independent C-lines connected to the fluorescent microspheres;

[0043] The lateral chromatography test strip includes a composite pad 1, a detection membrane 4, and an absorbent pad 5; one end of the detection membrane 4 overlaps with the composite pad 1, and the other end overlaps with the absorbent pad 5; the detection membrane 4 is provided with a T line (detection line) 2 near the composite pad 1 and a C line (control line) 3 near the absorbent pad 5.

[0044] The T-line 2 is coated with an antibody corresponding to the exosome marker protein, and the C-line 3 is coated with a secondary antibody that separates the C-line.

[0045] The antibodies corresponding to the colorectal cancer exosome markers are anti-CD147 antibody and / or anti-A33 antibody;

[0046] The fluorescent labeling material and the lateral chromatography test strip are stored independently, or the fluorescent labeling material is attached to the composite pad 1 of the lateral chromatography test strip.

[0047] In this invention, the colorectal cancer detection kit utilizes fluorescence to detect colorectal cancer exosome markers (CD147 antigen and / or A33 antigen), thereby enabling the detection of colorectal cancer. The fluorescent labeling material and lateral chromatography strip in the colorectal cancer detection kit can be stored independently, or the fluorescent labeling material can be attached to the composite pad of the lateral chromatography strip (preferably the end near the detection membrane). When the fluorescent labeling material and the lateral chromatography strip are stored independently, the sample to be tested, the fluorescent labeling material, and the chromatography solution can be premixed before use, and then the mixture can be dropped onto the composite pad 1 of the lateral chromatography strip. When the fluorescent labeling material is attached to the composite pad 1 of the lateral chromatography strip near the detection membrane 4, the sample to be tested and the chromatography solution can be mixed first, and then the mixture can be dropped onto the end of the composite pad 1 away from the detection membrane 4 (i.e., the area without attached fluorescent detection material).

[0048] In this invention, the fluorescent microspheres can be selected from fluorescent microspheres used in the field of fluorescence immunoassay for protein or antibody detection. By linking the fluorescent microspheres to antibodies corresponding to colorectal cancer exosome markers, not only can the colorectal cancer exosome markers be bound via antigen-antibody interaction, but they also possess luminescent properties, allowing for the determination of whether the sample contains colorectal cancer exosome markers using ultraviolet or fluorescence detection instruments. The fluorescent microspheres can be commercially available products or laboratory-made. In a preferred embodiment, the fluorescent microspheres are selected from quantum dot fluorescent microspheres and / or fluorescent polystyrene microspheres.

[0049] In a preferred embodiment, the quantum dot fluorescent microspheres may be carboxylated CdTe / ZnSe quantum dot fluorescent microspheres with a size of 90-120 nm and a maximum emission wavelength of 530±5 nm or 625±5 nm.

[0050] In a preferred embodiment, the fluorescent polystyrene microspheres can be selected from high-brightness fluorescent dye-type fluorescent polystyrene microspheres or rare-earth-type fluorescent polystyrene microspheres. The high-brightness fluorescent dye-type fluorescent polystyrene microspheres are typically monodisperse polystyrene microspheres containing high-brightness fluorescent dyes. The fluorescent dye is embedded inside the microspheres, effectively preventing fluorescence quenching and reducing the impact of environmental factors on fluorescence intensity, while maintaining the original functional groups on the microsphere surface. The rare-earth-type fluorescent polystyrene microspheres typically comprise polystyrene microspheres and rare earth elements; for example, they can be obtained by staining carboxylated polystyrene microspheres with europium, a rare-earth lanthanide chelate.

[0051] According to some preferred embodiments of the present invention, the diameter of the fluorescent microspheres is 50-400 nm, more preferably 80-200 nm.

[0052] In this invention, when the antibodies corresponding to the colorectal cancer exosome markers are anti-CD147 antibody and anti-A33 antibody, in a preferred case, the weight ratio of the anti-CD147 antibody to the anti-A33 antibody is 0.2-5:1; specifically, it can be 0.2:1, 0.5:1, 1:1, 2:1, 3:1, 4:1 or 5:1.

[0053] In this invention, an independent C-line system (composed of a primary antibody and a secondary antibody with specific binding capabilities) is employed. This allows the secondary antibody coated on the C-line to specifically bind to the primary antibody in the fluorescently labeled material, forming a detectable signal, unaffected by competition from the antigen captured at the T-line position, resulting in more accurate and stable detection results. The primary and secondary antibodies described in this invention can be selected from antibody pairs conventionally used in independent C-line systems in the art. Preferably, the primary antibody for the independent C-line is selected from chicken IgY antibody or DNP-BSA, and the secondary antibody for the independent C-line is selected from goat anti-chicken IgY antibody or mouse anti-DNP antibody. Specifically, when the primary antibody is chicken IgY antibody, the corresponding secondary antibody is goat anti-chicken IgY antibody; when the primary antibody is DNP-BSA, the corresponding secondary antibody is mouse anti-DNP antibody.

[0054] In a preferred embodiment, the antibody corresponding to the exosome marker protein is at least one of anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody. More preferably, the antibody corresponding to the exosome marker protein is a combination of anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody. Specifically, the weight ratio of anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody can be 1:1:1.

[0055] According to some preferred embodiments of the present invention, the lateral chromatography test strip further includes a base plate 6, which is located below the composite pad 1, the detection membrane 4, and the absorbent pad 5. Specifically, the composite pad 1, the detection membrane 4, and the absorbent pad 5 are sequentially arranged on the base plate 6 along its length. Specifically, the end of the composite pad 1 away from the detection membrane 4, the end of the detection membrane 4, and the end of the absorbent pad 5 away from the detection membrane 4 are all attached to the base plate 6. There are no special requirements for the material of the base plate; it can be any material conventionally used in the art, such as PVC (polyvinyl chloride).

[0056] In a preferred embodiment, the composite pad is made of glass cellulose or polyester film.

[0057] In a preferred embodiment, the detection membrane 4 can be a nitrocellulose membrane. For example, it can be a nitrocellulose membrane of type CN140 or CN95. The length of the detection membrane is preferably 3.5-4.5 cm.

[0058] In a preferred embodiment, the absorbent pad 5 is made of absorbent paper. There are no special requirements regarding the type and source of the absorbent paper; it can be any type of absorbent paper commonly used in the art. The length of the absorbent pad is preferably 1.5-2 cm.

[0059] According to the present invention, when the fluorescent labeling material and the lateral chromatography test strip are stored independently, the test solution is premixed with the fluorescent labeling material and the chromatography solvent to achieve contact between the exosomes and the fluorescent material. Therefore, the premixed solution can be added dropwise to any area of ​​the composite pad 1. In a preferred embodiment, when the fluorescent labeling material is attached to the end of the composite pad 1 near the detection membrane 4, the test solution is first mixed with the chromatography solvent, and then dropped onto the end of the composite pad 1 away from the detection membrane (i.e., without the fluorescent labeling material attached). As the solution flows, contact between the exosomes and the fluorescent labeling material in the solution is achieved.

[0060] In a preferred embodiment, the T-line 2 and the C-line 3 are arranged parallel to each other in the detection membrane 4, and the distance between them can be 4-8 mm.

[0061] In this invention, when the composite pad 1 overlaps with the detection membrane 4, one end of the composite pad 1 near the absorbent pad 5 extends above the detection membrane 4. Preferably, the overlap width (i.e., the width of the overlapping portion) between the composite pad 1 and the detection membrane 4 is 1-2 mm.

[0062] In this invention, when the absorbent pad 5 overlaps with the detection membrane 4, one end of the absorbent pad 5 near the composite pad 1 extends above the detection membrane 4. Preferably, the overlap width (i.e., the width of the overlapping portion) between the absorbent pad 5 and the detection membrane 4 is 1-2 mm.

[0063] In a preferred embodiment, the width of the composite pad 1, the detection membrane 4, and the absorbent pad 5 is the same as the width of the base plate 6.

[0064] In a preferred embodiment, the colorectal cancer detection kit further includes a chromatography buffer. The chromatography buffer is a common type of chromatography buffer in the art, such as a PBS buffer containing 0.1 wt% BSA and 0.01 wt% Tween 20.

[0065] In this invention, there are no special requirements for the size of the lateral chromatography test strip, and it can be a size commonly used in the art.

[0066] A second aspect of the present invention provides a method for the colorectal cancer detection kit described above, the method comprising the following steps:

[0067] (1) Spray T-line solution and C-line solution onto the T-line and C-line of the detection membrane respectively, and then dry them;

[0068] (2) Prepare fluorescent labeling material; optionally, spray the fluorescent labeling material onto the composite pad;

[0069] (3) Overlap one end of the dried detection membrane near the T line in step (1) with the composite pad and the other end with the absorbent pad;

[0070] The T-line solution contains antibodies corresponding to exosome marker proteins, and the C-line solution contains secondary antibodies for independent C-lines.

[0071] In a preferred embodiment, the concentration of the antibody corresponding to the exosome marker protein in the T-line solution is 0.1-5 mg / mL, and the concentration of the secondary antibody for the independent C-line in the C-line solution is 0.1-5 mg / mL.

[0072] In the method described in this invention, in step (1), the T-line solution and the C-line solution can be sprayed onto the detection membrane using a gold spraying device, and the spraying parameters can be set to 0.8-1.2 μL / cm.

[0073] In the method described in this invention, there are no special requirements for the drying conditions in step (1), and the drying can be carried out according to the conventional conditions in the art.

[0074] In this invention, the fluorescent labeling material comprises fluorescent microspheres and primary antibodies corresponding to colorectal cancer exosome markers and independent C-lines connected to the fluorescent microspheres. The preparation process of the fluorescent labeling material can be performed in accordance with conventional methods in the art. According to some preferred embodiments of the present invention, the process of preparing the fluorescent labeling material includes: activating the fluorescent microspheres with an activator, and then reacting them with primary antibodies corresponding to colorectal cancer exosome markers and independent C-lines.

[0075] More preferably, the activator is selected from EDC / NHS activator or EDC / Sulfo-NHS activator.

[0076] In the method described in this invention, when the lateral chromatography test strip includes a base plate, the specific process of step (1) includes: attaching the detection membrane to the base plate, then spraying the T-line solution and C-line solution onto the T-line and C-line of the detection membrane respectively, and then drying.

[0077] In the method described in this invention, the fluorescent labeling material can be sprayed onto the composite pad using a spectrophotometer. Specifically, the fluorescent labeling material solution can be sprayed onto the composite pad and then dried to adhere the fluorescent labeling material to the composite pad.

[0078] In this invention, when the fluorescent labeling material and the lateral chromatography test strip in the kit are stored independently, the method for preparing the colorectal cancer detection kit includes the following steps:

[0079] (1) Attach the detection membrane to the substrate, then spray the T-line solution and C-line solution onto the T-line and C-line of the detection membrane respectively, and then dry it;

[0080] (2) Preparation of fluorescent labeling materials;

[0081] (3) Overlap one end of the dried detection membrane near the T line in step (1) with the composite pad and the other end with the absorbent pad;

[0082] The T-line solution contains antibodies corresponding to exosome marker proteins, and the C-line solution contains secondary antibodies for independent C-lines.

[0083] In this invention, the method for preparing a colorectal cancer detection kit includes the following steps when the fluorescent labeling material in the kit adheres to the composite pad 1 of the lateral chromatography test strip:

[0084] (1) Attach the detection membrane to the substrate, then spray the T-line solution and C-line solution onto the T-line and C-line of the detection membrane respectively, and then dry it;

[0085] (2) Preparation of fluorescent labeling material; spray the fluorescent labeling material solution onto the composite pad and then dry it;

[0086] (3) Overlap one end of the dried detection membrane near the T line in step (1) with the composite pad obtained in step (2), and overlap the other end with the absorbent pad;

[0087] The T-line solution contains antibodies corresponding to exosome marker proteins, and the C-line solution contains secondary antibodies for independent C-lines.

[0088] A third aspect of the present invention provides a colorectal cancer detection system, which includes the colorectal cancer detection kit and optical detection instrument described above.

[0089] In this invention, the optical detection instrument can be selected from instruments commonly used in the art, such as ultraviolet lamps, gel imaging systems, and dry immunofluorescence analyzers. By using fluorescence instruments to photograph or observe the T-line and C-line, the detection result can be determined by whether the C-line and T-line are displayed.

[0090] A fourth aspect of the present invention provides a method for using the colorectal cancer detection kit described above, the method comprising:

[0091] Add the sample to be tested onto the composite pad;

[0092] The sample to be tested is brought into contact with the fluorescently labeled material.

[0093] In this invention, when the fluorescent labeling material and the lateral chromatography test strip are stored independently, during use, the sample to be tested, the fluorescent labeling material, and the chromatography solution are mixed and contacted, and then dropped onto the composite pad 1 of the lateral chromatography test strip. Through capillary action, the liquid passes through the composite pad 1 - detection membrane 4 (T line 2 first, then C line 3) - absorbent pad 5. The colorectal cancer exosome markers (CD147 antigen and / or A33 antigen) in the sample to be tested first bind to the corresponding antibodies of the colorectal cancer exosome markers on the fluorescent labeling material. Then, the antibodies corresponding to the exosome marker proteins on the T line can bind to the exosome marker proteins in the sample to be tested, thereby partially retaining the fluorescent labeling material on the T line 2. As the solution flows on the test strip, the secondary antibody on the C line 3 can bind to the primary antibody in the fluorescent labeling material, thereby retaining another part of the fluorescent labeling material and making the C line appear.

[0094] According to some specific embodiments of the present invention, when the fluorescent labeling material and the lateral chromatography test strip are stored independently, the method of using the colorectal cancer detection kit includes the following steps:

[0095] A1: Premix the sample to be tested, the chromatography solvent and the fluorescent labeling material to obtain a premix solution;

[0096] A2; Drop the premixed solution onto the composite pad of the lateral chromatography test strip;

[0097] A3: After a preset reaction time, the detection result is obtained based on the color development of the T line and the C line.

[0098] In a preferred embodiment, in step A3, the preset reaction time is 5-20 min, more preferably 10 min.

[0099] In this invention, when the fluorescent labeling material is attached to the composite pad 1, during use, the sample to be tested is mixed with the chromatography solution and then dropped onto the composite pad 1 of the lateral chromatography test strip (when the fluorescent labeling material is attached to the end of the composite pad near the detection membrane, it is preferably dropped onto the end of the composite pad 1 away from the detection membrane). Through capillary action, the solution travels through the composite pad - detection membrane (T line first, then C line) - absorbent pad. The colorectal cancer exosome markers (CD147 antigen and / or A33 antigen) in the sample to be tested first bind to the corresponding antibodies of the colorectal cancer exosome markers on the fluorescent labeling material on the composite pad. Then, the antibodies corresponding to the exosome marker proteins on the T line can bind to the exosome marker proteins in the sample to be tested, thereby partially trapping the fluorescent labeling material on the T line. As the solution flows on the test strip, the secondary antibody on the C line can bind to the primary antibody in the fluorescent labeling material, thereby trapping another portion of the fluorescent labeling material on the C line for color development.

[0100] According to some specific embodiments of the present invention, when the fluorescently labeled material is attached to the composite pad, the method of using the colorectal cancer detection kit includes the following steps:

[0101] B1: Mix the sample to be tested and the chromatography solvent to obtain a mixed chromatography solvent;

[0102] B2; Drop the mixed chromatography solution onto the composite pad of the lateral chromatography test strip;

[0103] B3: After a preset reaction time, the detection result is obtained based on the color development of the T line and the C line.

[0104] In a preferred embodiment, in step B3, the preset reaction time is 5-20 min, more preferably 10 min.

[0105] In this invention, in conjunction with reference to Figure 2 When using the aforementioned colorectal cancer detection kit, the test results are interpreted as follows: if both the T line and C line show color, the result is positive; if only the C line shows color, the result is negative. If the C line does not show color, the result is invalid regardless of whether the T line shows color.

[0106] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0107] In the following embodiments, the quantum dot fluorescent microspheres are quantum dot fluorescent nanospheres with the brand name JYFNS purchased from Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd.; the high-brightness fluorescent dye-type fluorescent polystyrene microspheres are green fluorescent polystyrene microspheres with the brand name LFGNC020 purchased from Suzhou Nano-Micro Technology Co., Ltd.; and the rare earth fluorescent polystyrene microspheres are time-resolved fluorescent microspheres with the brand name LFTNC020 purchased from Suzhou Nano-Micro Technology Co., Ltd.

[0108] Example 1

[0109] In the kit of this embodiment, the antibody corresponding to the colorectal cancer exosome marker is anti-CD147 antibody, the fluorescent microspheres are quantum dot fluorescent microspheres, the antibodies corresponding to the exosome marker proteins are anti-CD63 antibody, anti-CD9 antibody and anti-CD81 antibody, the primary antibody for the independent C line is chicken IgY antibody, and the secondary antibody for the independent C line is goat anti-chicken IgY antibody.

[0110] The specific preparation process is as follows:

[0111] Preparation of fluorescently labeled materials:

[0112] After the quantum dot fluorescent microsphere suspension was ultrasonically dispersed evenly, it was washed once with PBS buffer containing 25 mM 4-morpholinoethanesulfonic acid, centrifuged, and then 400 μL of PBS buffer containing 25 mM 4-morpholinoethanesulfonic acid was added and ultrasonically dispersed evenly. 50 μL of 10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and 50 μL of 10 mg / mL N-hydroxysuccinimide (NHS) solution were added and activated at room temperature (25℃) for 30 min. After activation, the supernatant was discarded by centrifugation, and 400 μL of buffer containing 25 mM 4-morpholinoethanesulfonic acid was added and ultrasonically dispersed evenly for later use.

[0113] Add 40 μg of anti-CD147 antibody and 40 μg of chicken IgY antibody, react at room temperature (25℃) for 2 h, then add 80 μL of 10 wt% BSA solution for blocking for 30 min; centrifuge and discard the supernatant, add 800 μL of 10 mM PBS buffer (pH 7.4), sonicate to disperse evenly and centrifuge; discard the supernatant and add PBS buffer (0.01 M concentration) containing 1 wt% BSA, and store at 4℃ for later use.

[0114] Preparation of lateral chromatography test strips:

[0115] a. Spraying composite pad: Apply 1 mL of fluorescent labeling material solution slowly and evenly to one side of the composite pad (made of glass cellulose) using a gold spraying machine, and then dry it to allow the fluorescent labeling material to adhere to the composite pad.

[0116] b. Scrubbing: Place the detection membrane (made of nitrocellulose membrane) in the center of the base plate (made of PVC) and scrub the membrane using a scrubber. First, clean the tubing and aspirate 10 μL of air. Then, aspirate the T-line solution (concentrations of anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody are all 1 mg / mL) and the C-line solution (concentration of goat anti-chicken IgY antibody is 2 mg / mL) into the tubing. Set the scrubber parameters to 1 μL / cm, with a 6 mm gap between the T-line and C-line. Fix the base plate with the detection membrane in the slot and begin scrubbing. After scrubbing, place the scrubbed membrane in a 37°C oven to dry for 2 hours.

[0117] c. Assemble and cut strips: Cut the composite pad and absorbent pad. Attach the composite pad to the base plate, overlapping the side of the composite pad with the fluorescent marker material onto the detection membrane near the T-line with an overlap width of 1mm. Attach the conjugate pad to the base plate, overlapping a portion of the conjugate pad onto the detection membrane near the C-line with an overlap width of 1mm. Use a high-speed strip cutter to cut the assembled test strips, ensuring that the width of the composite pad, detection membrane, and absorbent pad is the same as the width of the base plate. After cutting, place the strips in a waterproof bag and dry for later use.

[0118] Example 2

[0119] The method described in Example 1 was implemented, except that the antibody corresponding to the colorectal cancer exosome marker was an anti-A33 antibody.

[0120] Example 3

[0121] The method described in Example 1 was followed, except that the antibody corresponding to the colorectal cancer exosome marker was an anti-CD147 antibody and an anti-A33 antibody in a weight ratio of 3:1. Specifically, in the preparation of the fluorescent labeling material, 40 μg of anti-CD147 antibody was replaced with a combination of 30 μg of anti-CD147 antibody and 10 μg of anti-A33 antibody.

[0122] Example 4

[0123] The method described in Example 1 was implemented, except that the independent C-line system was adjusted to have a primary antibody of DNP-BSA and a secondary antibody of mouse anti-DNP antibody.

[0124] Example 5

[0125] The method described in Example 1 was followed, except that the antibodies corresponding to the colorectal cancer exosome markers were anti-CD147 antibody and anti-A33 antibody in a 1:1 weight ratio, the fluorescent microspheres were high-brightness dye-type fluorescent polystyrene microspheres (green), and the preparation process of the fluorescent labeling materials was different. A schematic diagram of the prepared colorectal cancer detection kit is shown below. Figure 1 As shown.

[0126] Specifically, the preparation process of fluorescently labeled materials is as follows:

[0127] Add 50 μL of fluorescent polystyrene microspheres to a 2 mL conical centrifuge tube containing 1 mL of LMES buffer (pH 5.0), sonicate to mix, and centrifuge to remove the supernatant. Add EDC solution (10 mg / mL) and Sulfo-NHS solution (10 mg / mL) according to the carboxyl content, and sonicate to mix. Activate in a rotary reactor at 37°C in the dark for 0.5 h, and centrifuge to remove the supernatant. Add 0.75 mL of LMES buffer (pH 5.0), sonicate to mix, and then add 10 μg of anti-CD147 antibody, 1... 0 μg anti-A33 antibody and 10 μg chicken IgY antibody were added to the activated microsphere solution and vortexed to mix. The microspheres were coupled in a rotary reactor at 37°C in the dark for 2 h. The supernatant was removed by centrifugation, and the microspheres were blocked overnight with blocking buffer (HEPES, pH 7.4 + 1% BSA). The blocking buffer was removed by centrifugation, and 0.5 mL of stock solution (HEPES, pH 7.4 + 1% BSA (w / v) + 0.05% Proclin 300 (w / v)) was added and sonicated to mix. After sonication, the microspheres were stored at 4°C in the dark for later use.

[0128] Example 6

[0129] The method described in Example 1 was implemented, except that the colorectal cancer exosome marker antibodies were anti-CD147 antibody and anti-A33 antibody in a weight ratio of 1:1, the fluorescent microspheres were rare earth fluorescent polystyrene microspheres, and the preparation process of the fluorescent labeling materials was different.

[0130] Specifically, the preparation process of fluorescently labeled materials is as follows:

[0131] Add 50 μL of fluorescent polystyrene microspheres to a 2 mL conical centrifuge tube containing 1 mL of MES buffer (pH 5.0), sonicate to mix, and centrifuge to remove the supernatant. Add EDC solution (10 mg / mL) and Sulfo-NHS solution (10 mg / mL) according to the carboxyl content, and sonicate to mix. Activate in a rotary reactor at 37°C in the dark for 0.5 h, and centrifuge to remove the supernatant. Add 0.75 mL of MES buffer (pH 5.0), sonicate to mix, and then add 10 μg of anti-CD147 antibody and 10 μg of anti-A33 antibody. The inoculum and 10 chicken IgY antibody were added to the activated microsphere solution and vortexed to mix. The microspheres were coupled in a rotary reactor at 37°C in the dark for 2 hours. The supernatant was removed by centrifugation, and the microspheres were blocked overnight with blocking buffer (HEPES, pH 7.4 + 1% BSA). The blocking buffer was removed by centrifugation, and 0.5 mL of stock solution (HEPES, pH 7.4 + 1% BSA (w / v) + 0.05% Proclin 300 (w / v)) was added. The microspheres were sonicated to mix and stored in the dark at 4°C for later use.

[0132] Test case

[0133] The kit prepared in Example 5 was used to detect exosome samples from normal cells and exosome samples from colorectal cancer cells, respectively. The detection method was as follows:

[0134] The sample to be tested and the chromatography buffer (PBS buffer containing 0.1 wt% BSA and 0.01 wt% Tween 20) are mixed to obtain a mixed chromatography buffer; the mixed chromatography buffer is dropped into the sample area of ​​the lateral chromatography test strip; after 10 minutes, the detection result is obtained according to the color development of the T line and C line.

[0135] like Figure 3 As shown, the left side represents the test results for normal cells, and the right side represents the results for colorectal cancer cells. The test results are: the normal cell group showed negative results, while the colorectal cancer cell group showed positive results.

[0136] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A colorectal cancer detection kit, characterized in that, The colorectal cancer detection kit includes fluorescently labeled materials and lateral chromatography test strips; The fluorescent labeling material includes fluorescent microspheres and primary antibodies corresponding to colorectal cancer exosome markers and independent C-lines connected to the fluorescent microspheres; The lateral chromatography test strip includes a composite pad (1), a detection membrane (4), and an absorbent pad (5); one end of the detection membrane (4) overlaps with the composite pad (1), and the other end overlaps with the absorbent pad (5); the detection membrane (4) is provided with a T line (2) near the composite pad (1) and a C line (3) near the absorbent pad (5); The T line (2) is coated with an antibody corresponding to the exosome marker protein; the C line (3) is coated with a secondary antibody that separates the C line. The antibodies corresponding to the colorectal cancer exosome markers are anti-CD147 antibody and / or anti-A33 antibody; The fluorescent labeling material and the lateral chromatography test strip are stored independently, or the fluorescent labeling material is attached to the composite pad (1) of the lateral chromatography test strip.

2. The colorectal cancer detection kit according to claim 1, characterized in that, The fluorescent microspheres have a diameter of 50-400 nm; and / or The fluorescent microspheres are selected from quantum dot fluorescent microspheres and / or fluorescent polystyrene microspheres.

3. The colorectal cancer detection kit according to claim 1 or 2, characterized in that, The antibody corresponding to the exosome marker protein is at least one of anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody.

4. The colorectal cancer detection kit according to any one of claims 1-3, characterized in that, The primary antibody for the independent C-line is selected from chicken IgY antibody or DNP-BSA; the secondary antibody for the independent C-line is selected from goat anti-chicken IgY antibody or mouse anti-DNP antibody.

5. The colorectal cancer detection kit according to claim 1, characterized in that, The lateral chromatography test strip also includes a base plate (6), which is located below the composite pad (1), the detection membrane (4) and the absorbent pad (5).

6. The colorectal cancer detection kit according to any one of claims 1-5, characterized in that, The composite pad (1) extends from one end near the absorbent pad (5) to above the detection membrane (4); Preferably, the absorbent pad (5) extends from one end near the composite pad (1) to above the detection membrane (4).

7. A method for preparing a colorectal cancer detection kit according to any one of claims 1-6, characterized in that, The method includes the following steps: (1) Spray T-line solution and C-line solution onto the T-line and C-line of the detection membrane respectively, and then dry them; (2) Prepare fluorescent labeling material; optionally, spray the fluorescent labeling material onto the composite pad; (3) Overlap one end of the dried detection membrane near the T line in step (1) with the composite pad and the other end with the absorbent pad; The T-line solution contains antibodies corresponding to exosome marker proteins, and the C-line solution contains secondary antibodies for independent C-lines.

8. The method according to claim 7, characterized in that, The process of preparing fluorescently labeled materials includes: activating fluorescent microspheres with an activator, and then reacting them with antibodies corresponding to colorectal cancer exosome markers and primary antibodies against independent C-lines; Preferably, the activator is selected from EDC / NHS activator or EDC / Sulfo-NHS activator.

9. A colorectal cancer detection system, characterized in that, The system includes the colorectal cancer detection kit and optical detection instrument as described in any one of claims 1-6.

10. A method of using the colorectal cancer detection kit according to any one of claims 1-6, characterized in that, The method includes: Add the sample to be tested onto the composite pad; The sample to be tested is brought into contact with the fluorescently labeled material.

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